Université De Strasbourg

Changwei Yu

Thèse Dirigée

M László
email: laszlo@igbmc.fr

M Stephane Vincent
email: vincent@igbmc.fr

M Ferenc Mueller

M Antoine

H F M Peters

Mme Petra Hajkova

M Irwin Davidson

Elena Torres-Padilla

Dr Robert Schneider

Dr Dinah S Singer

Dr Maria Elena Torres-Padilla

Dr Gavin Kelsey

Emese Gazdag

Matthieu Jung

Mathilde Joint

Luc Negroni

Stéphane D Vincent

Lászlo Tora

Sarina Ravens

Tao Ye

Matthieu Stierle

Laszlo Tora

Analysis of the composition and the function of oocyte-specific TBP2-containing transcription machinery during mouse oogenesis

Keywords: TFIID, TBP, RNA polymerase II, TFIIA, TBP-associated factors (TAFs), transcription elements), oocytes, primary follicles, secondary follicles, mass spectrometry, RNA-seq

Analysis of the composition and the function of oocyte-specific TBP2-containing transcription machinery during mouse oogenesis

Changwei

Introduction

Chez les mammifères, la maturation des ovocytes passe par plusieurs étapes successives de différenciation. Ce processus implique la synthèse et l'accumulation d'ARN et de protéines nécessaires à la fertilité et au développement embryonnaire précoce.

Bien que de nombreux gènes nécessaires à ce programme de développement ont déjà été indentifiés, peu de choses sont encore connues sur la nature et la fonction de la machinerie transcriptionnelle présente au cours de l'ovogenèse.

La régulation de l'initiation de la transcription par l'ARN polymérase II (Pol II) est cruciale pour le développement embryonnaire. Lors de l'initiation de la transcription, la Pol II et les facteurs généraux de la transcription (GTF) TFIIA,-B,-D,-E,-F,-H forment le complexe de préinitiation (PIC) au niveau des promoteurs. Dans les cellules somatiques, le facteur général TFIID est le premier à initier l'assemblage du PIC au niveau des promoteurs pour le recrutement de la Pol II. Chez les métazoaires, TFIID est composé de la protéine TATA box Binding Protein (TBP) associé à 13 TBP-Associated Factors (TAFs) (Tora, 2002;Muller et al., 2004). Il existe trois protéines paralogues de TBP chez les métazoaires. TRF1 est spécifique des insectes. La protéine TBP-like factor (TLF/TBPL1/TRF2/TRP) a été identifiée chez plusieurs espèces de métazoaires, et peut interagir avec TFIIA et TFIIB mais ne peut pas lier la boite TATA. Enfin, TLF est essentielle à la spermatogenèse chez la souris (Zhang et al., 2001b). La dernière est la protéine TBP2 (aussi appelée TRF3 ou TBPL2) qui est spécifique des vertébrés.

Dans leurs parties C terminales contenant le domaine de liaison à l'ADN, TBP et TBP2 partagent 92% de similarités au niveau de la séquence protéique. TBP2 est capable de lier la boite TATA et interagit avec TFIIA et TFIIB. De plus, TBP2 est capable de médier la transcription in vitro [START_REF] Bartfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Jallow et al., 2004;[START_REF] Deato | MyoD Targets TAF3/TRF3 to Activate Myogenin Transcription[END_REF].

Des études antérieures menées dans le laboratoire, ont montré que TBP est remplacée par TBP2 au cours de la croissance ovocytaire et que la délétion de Tbp2 bloque le Résumé xii développement des ovocytes à leur terme, entrainant la stérilité des femelles (Gazdag et al., 2007). Ces données suggèrent donc que TBP2 joue un rôle crucial dans la régulation transcriptionnelle dans les ovocytes, contrôlant ainsi leur croissance et leur maturation.

Dans cette étude, nous avons caractérisé la machinerie transcriptionnelle contenant TBP2 puis nous avons analysé sa fonction dans la régulation transcriptionnelle au cours de l'ovogenèse.

Objectifs, stratégie et résultats

Le but de ce projet est de comprendre pourquoi spécifiquement dans les ovocytes, TBP est remplacée par TBP2. Ce projet s'intéresse en particulier au rôle et à la fonction de TBP2 et de la machinerie transcriptionnelle associée pour la transcription dans les ovocytes. Des analyses similaires dans les ovocytes, avec l'optimisation du protocole d'immunoprécipitation (microIP) à partir de 500 fois moins de matériel de départ, ont montré que TBP2 interagit avec TFIIA mais avec aucune sous-unité de TFIID. L'interaction entre TBP2 et TFIIA est maintenue même après déplétion de toute sous-unité de TFIID, par immunoprécipitation de TAF7 et de TAF10. Cela suggère donc que TBP2 et TFIIA forment également un complexe stable in vivo. ii. DamID-seq et Cut&Run pour TBP2

Les techniques de DamID-seq et de ChIP-seq pour l'analyse du profil d'enrichissement de TBP2 dans les cellules surexprimant TBP2 ont montré que TBP2 lie les séquences appartenant à plus de 4000 gènes. Grâce à la collaboration avec Màté

Résumé xiv

Borsos du laboratoire de Maria Elena Torres Padilla, nous avons procédé à l'injection de l'ARNm codant la protéine fusion Dam-TBP2 dans des ovocytes collectés à partir de femelles âgées de 7 ou 14 jours. L'analyse des résultats sont en cours, tout comme ceux obtenus avec la technique de Cut&Run. Table 1-

Table of Figures

Figures of introduction

Table of Figures

Table of Figures

Table of Tables

Table of Tables

Introduction

In eukaryotic cells, the nuclear genome is organized into chromatin which comprises of DNA, RNA and associated proteins [START_REF] Mondal | Characterization of the RNA content of chromatin[END_REF][START_REF] Yadav | Chromatin plasticity: A versatile landscape that underlies cell fate and identity[END_REF]. Transcription is the process of decoding the genetic information from DNA into RNA by RNA polymerase (RNA Pol) enzymes. Typically, the eukaryotic nuclear genome is transcribed by three ubiquitous multisubunit complexes: RNA Pol I, II and III (except in higher plants also by two extra non-essential RNA polymerases, Pol IVa and Pol IVb [START_REF] Pikaard | Roles of RNA polymerase IV in gene silencing[END_REF]).

The three polymerases were first described after their chromatographic separation [START_REF] Roeder | Multiple forms of DNA-dependent RNA polymerase in eukaryotic organisms[END_REF][START_REF] Roeder | Specific nucleolar and nucleoplasmic RNA polymerases[END_REF]. Subsequent studies of their sensitivity to the toxin α-amanitin revealed that each RNA Pol is responsible for the synthesis of different classes of RNA [START_REF] Seifart | Alpha-amanitin, a specific inhibitor of transcription by mammalian RNA-polymerase[END_REF][START_REF] Kedinger | Alpha-amanitin: a specific inhibitor of one of two DNA-pendent RNA polymerase activities from calf thymus[END_REF][START_REF] Lindell | Specific inhibition of nuclear RNA polymerase II by alpha-amanitin[END_REF][START_REF] Zylber | Products of RNA polymerases in HeLa cell nuclei[END_REF][START_REF] Weinmann | Role of DNA-dependent RNA polymerase 3 in the transcription of the tRNA and 5S RNA genes[END_REF]. RNA Pol I synthesizes the 47S ribosomal RNA (rRNA) precursor, which could be processed into three mature rRNAs (28S, 18S and 5.8S), while 5S rRNA, transfer RNAs (tRNAs), 7SL RNA, U6 small nuclear RNA (snRNA) and a few other small stable RNAs are synthesized by RNA Pol III. Together the activities of RNA Pol I and Pol III transcription dominate over 80% of total RNA synthesis in growing cells [START_REF] Paule | Survey and summary: transcription by RNA polymerases I and III[END_REF]. Finally, RNA

Pol II synthesizes precursors of messenger RNAs (mRNAs), most snRNAs, microRNAs and long noncoding RNAs.

Gene expression is regulated primarily at the level of transcription and underlies all life processes. How transcription is regulated in a time-precise and cell type-specific manner is thus a central question in biology. Due to the fact that all protein-coding genes are transcribed by RNA Pol II, regulation of RNA Pol II transcription is one of the most important steps in controlling of cell identity, growth, differentiation, development, homeostasis and pathologies.

The focus of my introduction mainly concerns the mechanisms of RNA Pol II-Mediated transcription. In the first chapter, I will describe chromatin organization, plasticity, dynamics during development and their impacts on transcription. Secondly, I will detail how RNA Pol II transcription is tightly controlled at different levels with special emphasis on the formation of the pre-initiation complex (PIC). Thirdly, I will explore the cell-type-specific transcription machinery, and particularly I will highlight the diversification of the basal transcription machinery. Finally, I will present advances in transcription regulation during oogenesis.

Chromatin dynamics and transcription regulation 1.1 The hierarchy of chromatin organization

Eukaryotic cells compact their nuclear DNA in the nucleus through hierarchical levels of chromatin organization, which has profound effects on gene regulation and activity, by modulating the accessibility of DNA to the regulatory factors and elements.

The basic unit of chromatin is the nucleosome, which is composed of a core particle with 147 base pairs (bp) of DNA wrapped around a histone octamer and a segment of 20 to 90 bp linker DNA that can bind to histone H1 [START_REF] Olins | Chromatin history: our view from the bridge[END_REF][START_REF] Richmond | The structure of DNA in the nucleosome core[END_REF][START_REF] Szerlong | Nucleosome distribution and linker DNA: connecting nuclear function to dynamic chromatin structure[END_REF]. The histone octamer consists of two copies of histones H2A, H2B, H3 and H4.

Non-condensed nucleosomes can form "beads-on-a-string" structure [START_REF] Olins | Ultrastructural features of chromatin nu bodies[END_REF]. The chromatin filaments further coil to reach higher-level structures (Figure 1-1). into the nucleus. From [START_REF] Tonna | Metabolic memory and diabetic nephropathy: potential role for epigenetic mechanisms[END_REF].

The long-standing textbook model of chromatin compaction is that 11 manometer (nm) nucleosome chains fold into 30nm chromatin fibers, subsequently into 300 nm to 700 nm fibers, and ultimately mitotic chromosomes [START_REF] Woodcock | Chromatin higher-order structure and dynamics[END_REF][START_REF] Tonna | Metabolic memory and diabetic nephropathy: potential role for epigenetic mechanisms[END_REF] (Figure 1-1).

However, this hierarchical model has been challenged by the findings that chromatin is a flexible and disordered 5 to 24 nm-diameter chain that is packed together at different 3D concentration densities in interphase nuclei and mitotic chromosomes [START_REF] Fussner | Open and closed domains in the mouse genome are configured as 10-nm chromatin fibres[END_REF][START_REF] Ou | ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF].

Traditionally, chromatin is divided into two structurally and functionally distinguishable territories: heterochromatin and euchromatin [START_REF] Babu | Chromosome structure: euchromatin and heterochromatin[END_REF][START_REF] Huisinga | The contradictory definitions of heterochromatin: transcription and silencing[END_REF][START_REF] Tamaru | Confining euchromatin/heterochromatin territory: jumonji crosses the line[END_REF]. Heterochromatin was originally defined as the chromatin that remains condensed and deeply stained at interphase. It typically refers to the highly condensed, gene-poor and less transcriptionally active regions of chromatin, and it has been further subdivided into two subsets, the permanently condensed constitutive heterochromatin and facultative heterochromatin, which may change its state of condensation during development [START_REF] Babu | Chromosome structure: euchromatin and heterochromatin[END_REF][START_REF] Wegel | Gene activation and deactivation related changes in the three-dimensional structure of chromatin[END_REF]. The euchromatin is less condensed, gene-rich, and transcription-prone. The chromatin can also be classified into five principal types defined by unique combinations of proteins [START_REF] Filion | Systematic protein location mapping reveals five principal chromatin types in Drosophila cells[END_REF].

Further refinement of genomic approaches for mapping chromatin properties and chromatin interactions facilitated the generation of numerous high-resolution genome-wide maps, which have provided us a better understanding of chromatin spatial organization (Figure 1-2) with chromatin loops, topologically associating domains (TADs), A/B compartments and chromosome territories [START_REF] Bickmore | Genome architecture: domain organization of interphase chromosomes[END_REF][START_REF] Gibcus | The hierarchy of the 3D genome[END_REF][START_REF] Ea | Contribution of Topological Domains and Loop Formation to 3D Chromatin Organization[END_REF][START_REF] Pombo | Three-dimensional genome architecture: players and mechanisms[END_REF][START_REF] Sexton | The role of chromosome domains in shaping the functional genome[END_REF][START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Schmitt | Genome-wide mapping and analysis of chromosome architecture[END_REF][START_REF] Dekker | The 4D nucleome project[END_REF].

Introduction

Chromatin dynamics and transcription regulation

Transcription-relevant epigenetic regulations

Epigenetics was defined as "a stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence" [START_REF] Berger | An operational definition of epigenetics[END_REF]. Epigenetic regulations, including DNA methylation, histone post-translational modifications (PTMs), chromatin remodeling and histone variants can alter or even reshape the chromatin landscape locally at the individual gene level as well as globally across the epigenome, therefore modulating the genome accessibility and activity.

DNA methylation

Many eukaryotic genomes contain DNA methylation such as 5-methylcytosine (5mC) and N6-methyladenine (6mA) at different abundance levels [START_REF] Li | DNA methylation in mammals[END_REF][START_REF] Luo | DNA N(6)-methyladenine in metazoans: functional epigenetic mark or bystander?[END_REF]. In mammals, the major form is 5mC, which is mainly found in CpG dinucleotides and generally associated with transcriptional silencing [START_REF] Zemach | Genome-wide evolutionary analysis of eukaryotic DNA methylation[END_REF][START_REF] Schubeler | Function and information content of DNA methylation[END_REF]. 5mC can change the functional state of regulatory regions and thus play important roles in various biological processes including genomic imprinting, X chromosome inactivation, repetitive elements silencing and transcription regulation [START_REF] Bird | DNA methylation patterns and epigenetic memory[END_REF][START_REF] Jones | Functions of DNA methylation: islands, start sites, gene bodies and beyond[END_REF][START_REF] Neri | Intragenic DNA methylation prevents spurious transcription initiation[END_REF].

The mammalian DNA methylation pattern is established during embryonic development by de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B, in combination with the cofactor DNMT3L [START_REF] Okano | Cloning and characterization of a family of novel mammalian DNA (cytosine-5) methyltransferases[END_REF][START_REF] Okano | DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development[END_REF][START_REF] Bourc'his | Dnmt3L and the establishment of maternal genomic imprints[END_REF]. Of note, a rodent DNMT3C methylates the promoters of young retrotransposon during spermatogenesis [START_REF] Barau | The DNA methyltransferase DNMT3C protects male germ cells from transposon activity[END_REF]. 5mC at the CpG dinucleotides can be maintained during cell division by DNMT1, which recognizes and completes hemi-methylated CpG sites with its functional partner [START_REF] Hermann | The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites[END_REF][START_REF] Bostick | UHRF1 plays a role in maintaining DNA methylation in mammalian cells[END_REF][START_REF] Sharif | The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA[END_REF][START_REF] Song | Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation[END_REF].

5mC is reversible (Figure 1-3), and it can be removed either by passive demethylation through imperfect maintenance (Chen et al., 2003a) or by active DNA demethylation through ten-eleven translocation (TET) family of proteins [START_REF] Tahiliani | Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1[END_REF][START_REF] Wu | TET-mediated active DNA demethylation: mechanism, function and beyond[END_REF]. DNMTs convert cytosine to 5mC. TET proteins convert 5mC back to cytosine by iterative oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). 5fC or 5caC can be efficiently removed by thymine DNA glycosylase (TDG) coupled with base excision repair (BER). From [START_REF] Wu | TET-mediated active DNA demethylation: mechanism, function and beyond[END_REF].

Whole-genome analysis of DNA methylation have revealed the dynamics of 5mC at different gene regulatory regions as well as in different cell types and developmental stages, and this indicates that 5mC is more than a repressive regulator of promoter activity, and that it has broader regulatory roles in development and disease [START_REF] Ziller | Charting a dynamic DNA methylation landscape of the human genome[END_REF][START_REF] Schultz | Human body epigenome maps reveal noncanonical DNA methylation variation[END_REF][START_REF] Luo | Dynamic DNA methylation: In the right place at the right time[END_REF].

Histone modifications

Both the protruding N-terminal tails and the globular domains of histones can carry post-translational modifications (PTMs) that include acetylation, methylation, phosphorylation, ubiquitinylation, citrullination, SUMOylation, ADP ribosylation, deamination, propionylation and butyrylation [START_REF] Kouzarides | Chromatin modifications and their function[END_REF][START_REF] Kebede | Novel types and sites of histone modifications emerge as players in the transcriptional regulation contest[END_REF].

Histone PTMs can affect the chromatin structure and function as platforms for the recruitment of specific effector proteins [START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF]. Thus, histone PTMs play important roles at different levels of transcriptional regulation, from chromatin architecture to specific loci regulation through the recruitment of transcriptional regulators. The PTMs that have been reported to be involved in transcription regulation are listed in (Table 1-1).

Table 1-1. Overview of the transcription related histone PTMs. Adapted from [START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF].

Histone Modifications Roles References

H2A

Histone Tail H2AK4/5ac Mitosis; chromatin assembly [START_REF] Barber | The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved[END_REF] H2AK4/5ac Transcriptional activation [START_REF] Fusauchi | Tetrahymena histone H2A. Acetylation in the N-terminal sequence and phosphorylation in the C-terminal sequence[END_REF] H2AK7ac

Transcriptional activation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF] H2AK119P Spermatogenesis [START_REF] Baarends | Increased phosphorylation and dimethylation of XY body histones in the Hr6b-knockout mouse is associated with derepression of the X chromosome[END_REF]

H2AK119ub

Transcriptional repression [START_REF] Wang | Role of histone H2A ubiquitination in Polycomb silencing[END_REF] Globular Domain H2AQ105

Enriched over RNA Pol I transcribed genes [START_REF] Tessarz | Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification[END_REF] H2AK119ub

Linked to Polycomb-mediated gene silencing [START_REF] Wang | Role of histone H2A ubiquitination in Polycomb silencing[END_REF] H2B Histone Tail H2BS33P Transcriptional activation [START_REF] Maile | TAF1 activates transcription by phosphorylation of serine 33 in histone H2B[END_REF]

H2BK5ac

Transcriptional activation [START_REF] Golebiowski | Inhibition of core histones acetylation by carcinogenic nickel(II)[END_REF] H2BK11/12ac Transcriptional activation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF] H2BK15/16ac Transcriptional activation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF]

H2BK20ac

Transcriptional activation [START_REF] Golebiowski | Inhibition of core histones acetylation by carcinogenic nickel(II)[END_REF] H2BK120ub Spermatogenesis / meiosis [START_REF] Baarends | Increased phosphorylation and dimethylation of XY body histones in the Hr6b-knockout mouse is associated with derepression of the X chromosome[END_REF]

Introduction

Chromatin dynamics and transcription regulation 10

H3

Histone Tail H3K4me2 Linked to active transcription [START_REF] Liang | Distinct localization of histone H3 acetylation and H3-K4 methylation to the transcription start sites in the human genome[END_REF]

H3K4me3

Transcription elongation; active chromatin [START_REF] Santos-Rosa | Active genes are tri-methylated at K4 of histone H3[END_REF] H3K9me1/2/3 Transcriptional repression (Wang et al., 2008) H3R17me1/2 Transcriptional activation [START_REF] Bauer | Methylation at arginine 17 of histone H3 is linked to gene activation[END_REF] H3K27me2

Transcriptional repression (Wang et al., 2008)

H3K27me3

Transcriptional silencing; bivalent genes (Bernstein et al., 2006) H3K36me3

Transcriptional elongation [START_REF] Bell | Localized H3K36 methylation states define histone H4K16 acetylation during transcriptional elongation in Drosophila[END_REF]

H3K4ac

Transcriptional activation [START_REF] Strahl | Methylation of histone H3 at lysine 4 is highly conserved and correlates with transcriptionally active nuclei in Tetrahymena[END_REF] H3K9ac

Histone deposition; transcriptional activation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF] H3K14ac

Transcriptional activation [START_REF] Agalioti | Deciphering the transcriptional histone acetylation code for a human gene[END_REF]

H1K18ac

Transcriptional activation; DNA repair / replication (Wang et al., 2008)

H3K23ac

Transcriptional activation [START_REF] Tsai | TRIM24 links a non-canonical histone signature to breast cancer[END_REF]

H3K27ac

Active enhancer mark [START_REF] Calo | Modification of enhancer chromatin: what, how, and why?[END_REF]

H3S10P

Mitosis; meiosis; transcriptional activation [START_REF] Lee | Basal c-Jun N-terminal kinases promote mitotic progression through histone H3 phosphorylation[END_REF] H3S28P Mitosis; transcriptional activation [START_REF] Lau | Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing[END_REF]

Globular

Domain

H3Y41

Prevents HP1α binding to chromatin [START_REF] Dawson | JAK2 phosphorylates histone H3Y41 and excludes HP1alpha from chromatin[END_REF]

H3R42me2a

Positive transcriptional effects [START_REF] Casadio | H3R42me2a is a histone modification with positive transcriptional effects[END_REF] H3K56me3 Conserved heterochromatin mark [START_REF] Jack | H3K56me3 is a novel, conserved heterochromatic mark that largely but not completely overlaps with H3K9me3 in both regulation and localization[END_REF]

H3K64ac

Facilitates transcription [START_REF] Di Cerbo | Acetylation of histone H3 at lysine 64 regulates nucleosome dynamics and facilitates transcription[END_REF]

H3K64me3

Enriched at pericentric heterochromatin [START_REF] Daujat | H3K64 trimethylation marks heterochromatin and is dynamically remodeled during developmental reprogramming[END_REF] H3K79me1/2/3 Active chromatin; Telomeric silencing [START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF]

H3T118

Transcriptional activation; Nucleosome remodeling [START_REF] North | Phosphorylation of histone H3(T118) alters nucleosome dynamics and remodeling[END_REF]

H3K122

Transcriptional activation; Nucleosome eviction [START_REF] Tropberger | Regulation of transcription through acetylation of H3K122 on the lateral surface of the histone octamer[END_REF])

H4

Histone

Tail

H4R3me

Transcriptional activation (Wang et al., 2001a)

H4K20me1

Transcriptional silencing [START_REF] Nishioka | PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin[END_REF] H4K20me3 Heterochromatin [START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF]

H4K5ac

Histone deposition; transcriptional activation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF]

H4K8ac

Transcriptional activation and elongation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF]

H4K12ac

Telomeric silencing; transcriptional activation [START_REF] Suka | Highly specific antibodies determine histone acetylation site usage in yeast heterochromatin and euchromatin[END_REF]

H4K16ac

Transcriptional activation; DNA repair [START_REF] Akhtar | Activation of transcription through histone H4 acetylation by MOF, an acetyltransferase essential for dosage compensation in Drosophila[END_REF] Globular Domain H4S47P H3.3 deposition regulation [START_REF] Zhang | PP1alpha, PP1beta and Wip-1 regulate H4S47 phosphorylation and deposition of histone H3 variant H3.3[END_REF])

H4K91ac

Enriched at TSS of active and poised genes (Wang et al., 2008) Histone PTMs can be reversible by histone-modifying enzymes including a rich repertoire of writers, readers and erasers [START_REF] Allis | The molecular hallmarks of epigenetic control[END_REF]. For instance, histone acetyltransferases [START_REF] Marmorstein | Writers and readers of histone acetylation: structure, mechanism, and inhibition[END_REF] and deacetylases [START_REF] Seto | Erasers of histone acetylation: the histone deacetylase enzymes[END_REF] for histone acetylation, and histone methyltransferases and demethylases for histone methylation [START_REF] Greer | Histone methylation: a dynamic mark in health, disease and inheritance[END_REF][START_REF] Hyun | Writing, erasing and reading histone lysine methylations[END_REF]. Thus the dynamics of histone PTMs adds another layer of regulation for accurate gene expression.

Chromatin remodelers

ATP-dependent chromatin-remodeling complexes (remodelers) modulate the chromatin structure and accessibility through loading, moving, destabilizing, ejecting or restructuring the nucleosomes. They are thereby essential regulators of all chromosomal processes, including transcription.

Phylogenetically, transcription-relevant chromatin remodelers can be classified into four subfamilies: imitation switch (ISWI), chromodomain helicase DNA-binding (CHD), switch/sucrose non-fermentable (SWI/SNF) and INO80. Functionally, chromatin remodelers can be classified as assembly remodelers, access remodelers and editing remodelers, according to their specialty or preference to conduct function of nucleosome assembly and organization, chromatin access or nucleosome editing (histone exchange), respectively [START_REF] Flaus | Identification of multiple distinct Snf2 subfamilies with conserved structural motifs[END_REF][START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF][START_REF] Hargreaves | ATP-dependent chromatin remodeling: genetics, genomics and mechanisms[END_REF][START_REF] Clapier | Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes[END_REF].

Most ISWI subfamily chromatin remodelers assemble nucleosomes and ensure the proper density and spacing of nucleosomes to limit chromatin accessibility, thus contribute to gene repression, except the NURF complex, which has accessory subunits to promote transcription [START_REF] Xiao | Dual functions of largest NURF subunit NURF301 in nucleosome sliding and transcription factor interactions[END_REF][START_REF] Clapier | Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes[END_REF]. CHD subfamily remodelers are involved in all three general remodeling functions [START_REF] Lusser | Distinct activities of CHD1 and ACF in ATP-dependent chromatin assembly[END_REF][START_REF] Murawska | CHD chromatin remodelers and the transcription cycle[END_REF][START_REF] Konev | CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo[END_REF]. SWI/SNF subfamily remodelers typically facilitate chromatin access as they can slide nucleosomes, evict histone dimers or eject octamers, which usually promote gene expression by exposing the binding sites for transcription factors [START_REF] Boeger | Removal of promoter nucleosomes by disassembly rather than sliding in vivo[END_REF]. INO80 subfamily remodelers mainly carry out nucleosome editing through replication-independent nucleosome histone exchange to remove a particular histone and replace it with either a canonical or a variant histone [START_REF] Mizuguchi | ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex[END_REF][START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF].

Together, chromatin remodelers ensure the proper density and spacing of nucleosomes on the chromatin, while still ensure rapid access to regulatory factors to specific loci for other genome activities. Chromatin remodelers provide an important mechanism for chromatin structure and DNA accessibility modulation, thus adding another layer of control for transcription regulation.

Histone variants and chaperones

Histone variants are non-allelic isoforms of their corresponding canonical histones counterparts with differences in their primary sequence, expression level and expression timing.

In human, eight H2A variants, two testis-specific H2B variants and six H3 variants have been identified (Figure 1-4) [START_REF] Buschbeck | Variants of core histones and their roles in cell fate decisions, development and cancer[END_REF]. The variants show different genomic distribution and physiological roles. H2A.Z.1, H3.3 and CENP-A are essential for mouse embryonic development as knockout mice are embryonic lethal, while macroH2A and H2A.X are not essential for development but KO mice display defects (Table 12. testis-specific variants and alternative splice isoforms, respectively. From [START_REF] Buschbeck | Variants of core histones and their roles in cell fate decisions, development and cancer[END_REF].

Histone chaperones are a group of proteins that bind to histones and regulate nucleosome assembly [START_REF] Burgess | Histone chaperones in nucleosome assembly and human disease[END_REF]. They escort histones throughout their cellular life and play a crucial role in histones folding, transport, oligomerization, assembly into nucleosome and turnover on the chromatin [START_REF] Gurard-Levin | Histone chaperones: assisting histone traffic and nucleosome dynamics[END_REF][START_REF] Hammond | Histone chaperone networks shaping chromatin function[END_REF].

Thus histone chaperones are important actors in all chromosomal process, including transcription.

In fact, the deposition and function of histone variants are strongly linked to specialized chaperones (Table1-3) and remodelers [START_REF] Clapier | Mechanisms of action and regulation of ATP-dependent chromatin-remodelling complexes[END_REF]. For example, H3.3 turnover is governed by the histone regulator A (HIRA) complex in transcribed regions and promoters, as well as by DAXX-ATRX in heterochromatic regions [START_REF] Drane | The death-associated protein DAXX is a novel histone chaperone involved in the replication-independent deposition of H3.3[END_REF][START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF].

HIRA facilitates H3.3-H4 deposition at promoters and in gene bodies by interacting with transcription factors and RNA Pol II, respectively [START_REF] Banaszynski | Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells[END_REF][START_REF] Pchelintsev | Placing the HIRA histone chaperone complex in the chromatin landscape[END_REF][START_REF] Soni | Transcription factor EKLF (KLF1) recruitment of the histone chaperone HIRA is essential for beta-globin gene expression[END_REF].

Table 1-2: Function of histone variants. Adapted from [START_REF] Buschbeck | Variants of core histones and their roles in cell fate decisions, development and cancer[END_REF].

Histone

Variant Distribution Function

H2A.X

Genome-wide DNA damage response [START_REF] Rogakou | DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139[END_REF], knockout (KO) mice are viable but show male infertility [START_REF] Celeste | Genomic instability in mice lacking histone H2AX[END_REF].

H2A.Z.1

Regulatory regions (promoters and enhancers) and heterochromatin Facilitates the binding of regulatory complexes and increases nucleosome dynamics (Hu et al., 2013a), KO mice are embryonic lethal [START_REF] Faast | Histone variant H2A.Z is required for early mammalian development[END_REF].

H2A.Z.2

Facilitates the binding of regulatory complexes [START_REF] Vardabasso | Histone Variant H2A.Z.2 Mediates Proliferation and Drug Sensitivity of Malignant Melanoma[END_REF].

MacroH2A1

Facultative and constitutive heterochromatin Gene repression and signal-induced gene activation [START_REF] Changolkar | Developmental changes in histone macroH2A1-mediated gene regulation[END_REF], KO mice are viable but show metabolic defects [START_REF] Boulard | Histone variant macroH2A1 deletion in mice causes female-specific steatosis[END_REF].

MacroH2A2

Gene repression and signal-induced gene activation, macroH2A KO mice show reduced growth [START_REF] Pehrson | Mice without macroH2A histone variants[END_REF];

MacroH2A2 KO zebrafishes show malformations [START_REF] Buschbeck | The histone variant macroH2A is an epigenetic regulator of key developmental genes[END_REF].

H2A.Bbd

Euchromatin (In testis and brain)

Associates with sites of active transcription and replication and increases nucleosome dynamics [START_REF] Tolstorukov | Histone variant H2A.Bbd is associated with active transcription and mRNA processing in human cells[END_REF][START_REF] Sansoni | The histone variant H2A.Bbd is enriched at sites of DNA synthesis[END_REF].

TH2A and

TH2B

(Testis and oocytes) Paternal genome activation in zygote [START_REF] Shinagawa | Histone variants enriched in oocytes enhance reprogramming to induced pluripotent stem cells[END_REF].

H3.3 cis-regulatory

elements, gene bodies and repetitive, heterochromatic sequences Gene activation, retroviral element silencing, genome integrity and the establishment of heterochromatin [START_REF] Goldberg | Distinct factors control histone variant H3.3 localization at specific genomic regions[END_REF][START_REF] Banaszynski | Hira-dependent histone H3.3 deposition facilitates PRC2 recruitment at developmental loci in ES cells[END_REF][START_REF] Elsasser | Histone H3.3 is required for endogenous retroviral element silencing in embryonic stem cells[END_REF][START_REF] Jang | Histone H3.3 maintains genome integrity during mammalian development[END_REF][START_REF] Nashun | Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis[END_REF], double KO of H3f3a and H3f3b are early embryonic lethal, H3f3a knockouts are viable but show male subfertility, H3f3b knockouts are growth-deficient and died at birth.

H3f3b heterozygotes also show males infertility (Tang et al., 2015a). [START_REF] Tyler | The RCAF complex mediates chromatin assembly during DNA replication and repair[END_REF][START_REF] Munakata | A human homologue of yeast anti-silencing factor has histone chaperone activity[END_REF] CHAF1A / p150 H3.1-H4 CAF1 (RBAP48 can also form into HDAC, NuRF, NuRD, and PRC2)

Deposition factor coupled to DNA synthesis: replication, DNA repair [START_REF] Smith | Purification and characterization of CAF-I, a human cell factor required for chromatin assembly during DNA replication in vitro[END_REF][START_REF] Volk | The role of the chromatin assembly complex (CAF-1) and its p60 subunit (CHAF1b) in homeostasis and disease[END_REF] CHAF1B [START_REF] Bortvin | Evidence that Spt6p controls chromatin structure by a direct interaction with histones[END_REF][START_REF] Dronamraju | Spt6 Association with RNA Polymerase II Directs mRNA Turnover During Transcription[END_REF] SPT16 H2A-H2B, H3-H4 FACT Replication-coupled and-independent histone deposition, H2A-H2B eviction during transcription [START_REF] Belotserkovskaya | FACT facilitates transcription-dependent nucleosome alteration[END_REF][START_REF] Kemble | FACT Disrupts Nucleosome Structure by Binding H2A-H2B with Conserved Peptide Motifs[END_REF][START_REF] Tsunaka | Integrated molecular mechanism directing nucleosome reorganization by human FACT[END_REF][START_REF] Yang | The Histone Chaperone FACT Contributes to DNA Replication-Coupled Nucleosome Assembly[END_REF]) SSRP1 H2A-H2B, H3-H4 ANP32E H2A.Z-H2B P400-TIP60

H2A.Z-H2B eviction [START_REF] Mao | Anp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A[END_REF][START_REF] Obri | ANP32E is a histone chaperone that removes H2A.Z from chromatin[END_REF] YL1

Chromatin dynamics during mouse germ cell development

In metazoan, germ cells represents a continuous cellular link that ensure the perpetuation of the genetic and epigenetic information across generations. After primordial germ cells (PGCs) specification and migration, germ cells acquire the competencies to create totipotency during spermatogenesis and oogenesis through elaborate transcription regulation, along with morphology changes and chromatin dynamics. Here I mainly focus on cytological aspects of germ cell development and the chromatin dynamics.

Germ cell development

PGCs specification and migration

In mammals, PGCs, the precursors for both sperm and oocytes, are specified from a small population of proximal-posterior epiblast cells during embryonic development [START_REF] Hayashi | Germ cell specification in mice[END_REF]. The specification of PGCs is induced by signals from extra-embryonic tissues (Figure 123456), other than asymmetric inheritance of maternal cytoplasmic factors [START_REF] Lawson | Clonal analysis of the origin of primordial germ cells in the mouse[END_REF][START_REF] Saitou | Specification of germ cell fate in mice[END_REF][START_REF] Ohinata | A signaling principle for the specification of the germ cell lineage in mice[END_REF]. [START_REF] Aramaki | A mesodermal factor, T, specifies mouse germ cell fate by directly activating germline determinants[END_REF]. Adapted from [START_REF] Saitou | Gametogenesis from Pluripotent Stem Cells[END_REF] In response to bone morphogenetic protein (BMP) and WNT signals, the earliest known marker of PGC precursors, PR domain containing protein 1 (Prdm1/Blimp1) [START_REF] Vincent | The zinc finger transcriptional repressor Blimp1/Prdm1 is dispensable for early axis formation but is required for specification of primordial germ cells in the mouse[END_REF], is initially expressed in approximately six cells in the proximal-posterior epiblast at embryonic day 6.25 (E6.25) [START_REF] Ohinata | Blimp1 is a critical determinant of the germ cell lineage in mice[END_REF]. Expression of another key regulators for PGC specification, Prdm14, is also induced by the signals and can be detected at ∼ E6.5 in PRDM1-positive cells (Of note, Prdm14 is transiently expressed in the inner cell mass (ICM) cells of blastocysts and silenced by E5.5 [START_REF] Yamaji | Critical function of Prdm14 for the establishment of the germ cell lineage in mice[END_REF]). PRDM1 and PRDM14 further induce the expression of AP2γ at around E7.25 [START_REF] Weber | Critical function of AP-2 gamma/TCFAP2C in mouse embryonic germ cell maintenance[END_REF][START_REF] Magnusdottir | A tripartite transcription factor network regulates primordial germ cell specification in mice[END_REF].

Together, these three transcription factors form a core specification network that is necessary and sufficient for PGC induction [START_REF] Magnusdottir | A tripartite transcription factor network regulates primordial germ cell specification in mice[END_REF][START_REF] Tang | Specification and epigenetic programming of the human germ line[END_REF].

PRDM1-positive PGC precursors form a cluster of ∼20 cells at E6.75 [START_REF] Lawson | Clonal analysis of the origin of primordial germ cells in the mouse[END_REF][START_REF] Kurimoto | Complex genome-wide transcription dynamics orchestrated by Blimp1 for the specification of the germ cell lineage in mice[END_REF]. These cells first become identifiable as PGCs as a cluster of ∼40 alkaline phosphatase (AP)-positive cells at the base of incipient allantois at ∼E7.25, and expression of Dppa3/Stella also marks the establishment of PGCs (Figure 1234567) [START_REF] Ginsburg | Primordial germ cells in the mouse embryo during gastrulation[END_REF][START_REF] Saitou | A molecular programme for the specification of germ cell fate in mice[END_REF][START_REF] Sato | Identification of PGC7, a new gene expressed specifically in preimplantation embryos and germ cells[END_REF]Saitou et al., 2012b). PGCs migrate to the developing hindgut endoderm at ∼E7.75, and the majority of migrating PGCs are arrested at the G2 phase between E7.75 and E8.75. They continue to migrate through the hindgut endoderm and mesentery individually while keep proliferating, and finally colonize the genital ridges at ∼E10.5 with ∼1,000 cells [START_REF] Tam | Proliferation and migration of primordial germ cells during compensatory growth in mouse embryos[END_REF][START_REF] Molyneaux | Time-lapse analysis of living mouse germ cell migration[END_REF][START_REF] Seki | Cellular dynamics associated with the genome-wide epigenetic reprogramming in migrating primordial germ cells in mice[END_REF][START_REF] Saitou | Gametogenesis from Pluripotent Stem Cells[END_REF].

PGCs exit migratory state and initiate sexual differentiation while continue to proliferate between E10.5 and E 12.5 [START_REF] Lesch | Genetics of germ cell development[END_REF]. In the male genital ridge, the PGCs are committed to spermatogenesis at ∼E12.5 and gradually enter into mitotic arrest at ∼E13.5 until after birth [START_REF] Mclaren | Primordial germ cells in the mouse[END_REF][START_REF] Western | Dynamic regulation of mitotic arrest in fetal male germ cells[END_REF]. Whereas, ∼12,000 female PGCs are committed to oogenesis at ∼E13.5 and subsequently enter the first meiotic prophase in the mouse [START_REF] Pepling | Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles[END_REF][START_REF] Mclaren | Primordial germ cells in the mouse[END_REF]. This marks the end of PGC stage of germ cell development. During PGC specification and migration, genome-wide epigenetic reprogramming occurs, including DNA demethylation and histone modification changes, which will be described later together with spermatogenesis and oogenesis (see section 1.3.2).

Spermatogenesis

Spermatogenesis is the developmental process during which diploid spermatogonia generate haploid spermatozoa through mitotic expansions, meiotic reduction divisions and spermiogenesis (Figure 1-8) [START_REF] Griswold | Spermatogenesis: The Commitment to Meiosis[END_REF][START_REF] Chen | Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis[END_REF]. In mouse embryonic testis, mitotically arrested germ cells lost their alkaline phosphatase expression at ∼14.5 and differentiate into gonocytes (also called pro-spermatogonia) [START_REF] Culty | Gonocytes, the forgotten cells of the germ cell lineage[END_REF]. At around postnatal day (P) 5, gonocytes resume active proliferation, and some of them are recruited as spermatogonial stem cells (SSCs), which are the singly isolated predominant stem cells during spermatogenesis [START_REF] Yoshida | Stem cells in mammalian spermatogenesis[END_REF]Saitou et al., 2012b). The SSCs can divide to form a pair of cells connected by an intercellular bridge termed A paired spermatogonia (Apr), and Apr can further divide to form A aligned cell syncytia of 4, 8 or 16 cells. This pool of undifferentiated spermatogonia differentiate into A1 differentiating spermatogonia without division and subsequently undergo 5 divisions to form B spermatogonia, which gives rise to two preleptotene spermatocytes through another mitosis [START_REF] Spradling | Germline stem cells[END_REF][START_REF] Griswold | Spermatogenesis: The Commitment to Meiosis[END_REF].

Meiosis starts in preleptotene spermatocytes. Following DNA replication, synapsis and crossing over occur in the prolonged meiotic prophase I, which can be subdivided into leptotene, zygotene, pachytene and diplotene stages. After meiotic prophase I, spermatocytes undergo two rounds of chromosome segregation to produce haploid round spermatids. These round spermatids further undergo dramatic morphological and cytological changes to form mature spermatozoa through the process of spermiogenesis [START_REF] Jan | Molecular control of rodent spermatogenesis[END_REF][START_REF] Chen | Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis[END_REF].

Concomitant with cytological changes, epigenetic changes including paternal imprint acquisition, histones replacement by histone variants, transition proteins and protamines, also occur at different stages of spermatogenesis.

Oogenesis

Oogenesis begins when female PGCs colonize the genital ridge, proliferate as oogonia and form germline cysts from ∼E10.5 [START_REF] Pepling | From primordial germ cell to primordial follicle: mammalian female germ cell development[END_REF]. At ∼E13.5, unlike male PGCs entering mitotic arrest, most female germ cells within germline cysts begin to enter meiosis and subsequently arrest in the diplotene stage of meiotic prophase I gradually from ∼E17.5, and it takes several days until all oocytes are in diplotene arrest (Figure 123456789) [START_REF] Pepling | Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles[END_REF][START_REF] Dutta | Arrest at the diplotene stage of meiotic prophase I is delayed by progesterone but is not required for primordial follicle formation in mice[END_REF]. [START_REF] Pepling | From primordial germ cell to primordial follicle: mammalian female germ cell development[END_REF][START_REF] Racki | CPEB controls oocyte growth and follicle development in the mouse[END_REF][START_REF] Sasaki | Epigenetic events in mammalian germ-cell development: reprogramming and beyond[END_REF][START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF].

After birth, germline cysts break apart as individual oocyte enclosed with several pre-granulosa cells to form dormant primordial follicles, and this process is accompanied by extensive germ cell loss, of which only the minority undergo apoptosis [START_REF] Pepling | Mouse ovarian germ cell cysts undergo programmed breakdown to form primordial follicles[END_REF]. It is generally thought that this pool of oocytes in primordial follicles are finite and non-renewable after birth [START_REF] Telfer | On regenerating the ovary and generating controversy[END_REF][START_REF] Albertini | A detour in the quest for oogonial stem cells: methods matter[END_REF], although this theory has been challenged by studies showing the existence of oogonial stem cells (OSCs) in adult ovary [START_REF] Johnson | Germline stem cells and follicular renewal in the postnatal mammalian ovary[END_REF][START_REF] White | Oocyte formation by mitotically active germ cells purified from ovaries of reproductive-age women[END_REF].

The majority of oocytes enclosed in primordial follicles are maintained in dormancy during reproductive life [START_REF] Kim | Control of ovarian primordial follicle activation[END_REF]. These resting oocytes surrounded by flat granulosa cells in primordial follicles are small in size, with a diameter of 12-20 μm, and can be routinely obtained from ovaries of P1 to P3 mice (Pedersen et al., 1968;[START_REF] Mangia | Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities[END_REF].

Upon induction, a limited number of dormant primordial follicles are recruited into the growing pool and undergo further development progressively in an asynchronous manner.

Activated primordial follicles first develop into primary follicles, in which granulosa cells increase in number and form single-layered cuboidal granulosa cells, followed by dramatic growth of oocytes and the initiation of zona pellucida formation. Growing oocytes in primary follicles with a diameter of 30 to 60 μm can be obtained from P5 to P7 [START_REF] Mangia | Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities[END_REF][START_REF] Sanchez | Molecular control of oogenesis[END_REF].

Secondary follicle formation proceeds with the generation of multiple layers of granulosa cells around the oocyte. In secondary follicles, the zona pellucida is completely formed, and the diameter of the oocytes is around 60 to 75 μm, and can be obtained at P12 to P14 [START_REF] Mangia | Biochemical studies of growing mouse oocytes: preparation of oocytes and analysis of glucose-6-phosphate dehydrogenase and lactate dehydrogenase activities[END_REF]. Theca cell layer forms during the transition from the preantral to early antral stage [START_REF] Orisaka | Oocyte-granulosa-theca cell interactions during preantral follicular development[END_REF], followed by the formation of antrum and cumulus oophorus in antral stage. In the preovulatory follicle, germinal vesicle (GV) oocyte and cumulus cells reside in the fluid-filled antrum [START_REF] Racki | CPEB controls oocyte growth and follicle development in the mouse[END_REF][START_REF] Paulini | Ultrastructural changes in oocytes during folliculogenesis in domestic mammals[END_REF].

Upon hormonal signaling, oocyte enter the maturation stage with the germinal vesicle breakdown (GVBD), followed by meiosis I spindle assembly and chromosome migration.

Subsequently, meiosis II starts and the mature oocyte arrests in metaphase II, which will be bypassed after fertilization [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF].

Chromatin dynamic during germ cell development

Global epigenetic reprogramming occurs during germ cell development (Figure 1-10), including changes in DNA methylation, histone modifications, histone variants incorporation and chromatin organization.

Chromatin dynamics in PGCs

Mouse PGCs are specified from epiblast, whose DNA is hypermethylated. PGCs undergo genome-wide DNA demethylation as they migrate and colonize the genital ridge, which results in the erasure of genomic imprinting and X-chromosome reactivation. By ∼E13.5, CpG methylation levels drop from ∼70% in the epiblast to 14% and 7% in male and female PGCs, respectively [START_REF] Seisenberger | The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells[END_REF][START_REF] Kobayashi | High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice[END_REF]. Of note, some repetitive elements, such as IAPs and LTR-ERV1, show resistance to this global methylation erasure [START_REF] Hajkova | Epigenetic reprogramming in mouse primordial germ cells[END_REF][START_REF] Guibert | Global profiling of DNA methylation erasure in mouse primordial germ cells[END_REF].

Along with DNA methylation, reorganization of repressive histone marks also occurs.

Apart from widespread depletion of H3K9me2, H3K27me3 and H2A/H4R3me2s are enriched in PGCs, whereas H3K9me3 is retained relatively constant at centromeric heterochromatin [START_REF] Seki | Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice[END_REF][START_REF] Ancelin | Blimp1 associates with Prmt5 and directs histone arginine methylation in mouse germ cells[END_REF]. Moreover, a non-canonical form of H3K27me3 with broad distribution is acquired in PGCs [START_REF] Sachs | Bivalent chromatin marks developmental regulatory genes in the mouse embryonic germline in vivo[END_REF][START_REF] Ng | In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures[END_REF][START_REF] Hammoud | Chromatin and transcription transitions of mammalian adult germline stem cells and spermatogenesis[END_REF][START_REF] Zheng | Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals[END_REF]Hanna et al., 2018a).

Subsequently, DNA methylation re-establishment and chromatin changes occurred in a sex-specific manner. from (Saitou et al., 2012a;[START_REF] Burton | Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis[END_REF][START_REF] Saitou | Gametogenesis from Pluripotent Stem Cells[END_REF][START_REF] Tang | Specification and epigenetic programming of the human germ line[END_REF]Hanna et al., 2018a;[START_REF] Xu | Epigenome in Early Mammalian Development: Inheritance, Reprogramming and Establishment[END_REF].

Chromatin dynamics during spermatogenesis

During spermatogenesis, DNA remethylation starts after E13.5 in gonocytes, by E16.5, global methylation levels already increase to ∼50%, and the methylation patterns are fully established at birth [START_REF] Seisenberger | The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells[END_REF][START_REF] Stewart | Establishment and functions of DNA methylation in the germline[END_REF].

Following meiotic divisions, spermatids undergo extensive nuclear and morphological changes including the histone-to-protamine transition, during which the majority of core histones are replaced sequentially, first by histone variants and transition proteins and subsequently by protamines [START_REF] Bao | Epigenetic regulation of the histone-to-protamine transition during spermiogenesis[END_REF]. As a result, only 1% residual histones is retained in mature sperm.

Residual nucleosomes are largely composed of the H3.3 histone variant and H3K4me3 [START_REF] Erkek | Molecular determinants of nucleosome retention at CpG-rich sequences in mouse spermatozoa[END_REF], and they (or at least a subset of them) are enriched at CpG-rich sequences with low DNA methylation, although it has been reported that the majority of residual nucleosomes locate at gene-poor regions [START_REF] Carone | High-resolution mapping of chromatin packaging in mouse embryonic stem cells and sperm[END_REF]. Residual histones in sperm provide other ways for epigenetic inheritance through the male germline without changes of DNA methylation [START_REF] Gill | Parental epigenetic control of embryogenesis: a balance between inheritance and reprogramming?[END_REF][START_REF] Siklenka | Disruption of histone methylation in developing sperm impairs offspring health transgenerationally[END_REF].

Chromatin dynamics during oogenesis

During oogenesis, de novo methylation only occurs after birth during oocyte growth, in a transcription-dependent manner, and is largely completed in GV oocyte at ∼P21 [START_REF] Hiura | Oocyte growth-dependent progression of maternal imprinting in mice[END_REF][START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Stewart | Establishment and functions of DNA methylation in the germline[END_REF]. Oocyte acquires lower global methylation than sperm, but with non-canonical genome-wide distribution over transcribed gene bodies [START_REF] Kobayashi | High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice[END_REF][START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF]. However, loss of DNA methylation through deletion of Dnmt3a or Dnmt3L has no effect on oogenesis [START_REF] Bourc'his | Dnmt3L and the establishment of maternal genomic imprints[END_REF][START_REF] Kaneda | Essential role for de novo DNA methyltransferase Dnmt3a in paternal and maternal imprinting[END_REF].

Intriguingly, a non-canonical pattern of H3K4me3 (ncH3K4me3) with broad peaks is formed during oocyte growth, and overlaps almost exclusively with partially methylated DNA domains [START_REF] Zhang | Allelic reprogramming of the histone modification H3K4me3 in early mammalian development[END_REF][START_REF] Dahl | Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition[END_REF][START_REF] Liu | Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos[END_REF]Hanna et al., 2018b).

Formation of ncH3K4me3 is through the recruitment of MLL2 to unmethylated CpG-rich regions in a transcription-independent manner (Hanna et al., 2018b).

The non-canonical form of H3K27me3 that is gained in PGCs, with weak promoter enrichment and relatively high enrichment at non-promoter /distal regions, is present broadly at unmethylated genomic regions throughout oogenesis [START_REF] Zheng | Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals[END_REF].

Growing oocytes have relatively high levels of histone acetylation, and undergo abrupt de-acetylation during meiotic resumption [START_REF] Kim | Changes in histone acetylation during mouse oocyte meiosis[END_REF]. Double deletion of Hdac1 and Hdac2 impairs transcription and oocyte growth, leading to female sterility as follicle development arrests at the secondary follicle stage [START_REF] Ma | Compensatory functions of histone deacetylase 1 (HDAC1) and HDAC2 regulate transcription and apoptosis during mouse oocyte development[END_REF][START_REF] Ma | HDAC1 and HDAC2 in mouse oocytes and preimplantation embryos: Specificity versus compensation[END_REF].

Moreover, histone variant H3.3 is continuously deposited on the chromatin of growing oocyte by HIRA, which is essential for transcription regulation and de novo DNA methylation during oogenesis [START_REF] Nashun | Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis[END_REF]. Besides, growing oocyte chromatin also contains histone variant macroH2A, which remains associated with maternal chromatin following fertilization [START_REF] Chang | A maternal store of macroH2A is removed from pronuclei prior to onset of somatic macroH2A expression in preimplantation embryos[END_REF].

During oocyte maturation, chromatin undergoes dramatic conformational changes in GV oocytes, from non-surrounded nucleolus (NSN) state to partially surrounded nucleolus (PSN) state, and eventually surrounded nucleolus (SN) state [START_REF] Mattson | Oogenesis: chromatin and microtubule dynamics during meiotic prophase[END_REF][START_REF] Zuccotti | Chromatin organization during mouse oocyte growth[END_REF][START_REF] Bogolyubov | Karyosphere (Karyosome): A Peculiar Structure of the Oocyte Nucleus[END_REF]. Those conformational changes correlate with, but do not determine oocyte transcriptional activity: NSN oocyte shows transcriptional activity while SN oocyte is transcriptionally silenced [START_REF] De La Fuente | Major chromatin remodeling in the germinal vesicle (GV) of mammalian oocytes is dispensable for global transcriptional silencing but required for centromeric heterochromatin function[END_REF].

Hi-C studies has shown that chromosome interactions, such as TADs and chromosome loops, exist in GV oocytes and start to decrease with the NSN to SN transition [START_REF] Flyamer | Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition[END_REF]. With resumption of meiosis, oocytes lose typical higher-order chromatin structures, including TADs and chromatin compartments. Instead, MII oocytes show a uniform interaction pattern along the entire chromosomes that appears to be locus-independent [START_REF] Ke | 3D Chromatin Structures of Mature Gametes and Structural Reprogramming during Mammalian Embryogenesis[END_REF][START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF].

Given the fact that H3K4me3 interacts with transcription regulators such as TAF3 [START_REF] Vermeulen | Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4[END_REF][START_REF] Van Ingen | Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3[END_REF][START_REF] Lauberth | H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation[END_REF], it was proposed that ncH3K4me3 at distal sites could function as 'sponges' that absorb and sequester transcription factors (TFs) and regulators, therefore diluting transcription resources away from promoters to modulate transcription [START_REF] Zhang | Allelic reprogramming of the histone modification H3K4me3 in early mammalian development[END_REF]. Moreover, the unusual epigenetic patterns in oocytes is not limited to H3K4me3.

In this scenario, the distinct epigenetic patterns in oocytes may confer oocytes different mechanisms of transcription regulation, even on the level of basal transcription machinery, together with specific transcription factors, facilitating the acquisition of competencies required for fertilization and embryogenesis.

Chromatin dynamics during early embryo development

Following fertilization, the paternal DNA methylation is rapidly lost mainly through a combination of TET3-mediated active demethylation and passive dilution, whereas maternal DNA methylation is mainly lost passively over cell divisions [START_REF] Wu | TET-mediated active DNA demethylation: mechanism, function and beyond[END_REF]. However, the main decrease of 5mC in paternal genome occurs before early pronuclear stage (PN) 3, while 5hmC starts to accumulate only after main drop of 5mC has occurred [START_REF] Santos | Active demethylation in mouse zygotes involves cytosine deamination and base excision repair[END_REF][START_REF] Amouroux | De novo DNA methylation drives 5hmC accumulation in mouse zygotes[END_REF], and ablation of maternal TET3 prevents accumulation of 5hmC but does not influence early loss of paternal 5mC in zygote [START_REF] Amouroux | De novo DNA methylation drives 5hmC accumulation in mouse zygotes[END_REF]. Together, these findings suggest the existences of de novo methylation and of an alternative unknown mechanism for DNA demethylation in early mouse zygote [START_REF] Amouroux | De novo DNA methylation drives 5hmC accumulation in mouse zygotes[END_REF]. As a result, global DNA methylome is largely erased by blastocyst stage except in selected regions, including but not limited to, imprinted regions and some classes of repetitive elements [START_REF] Smallwood | Dynamic CpG island methylation landscape in oocytes and preimplantation embryos[END_REF][START_REF] Lee | Reprogramming the methylome: erasing memory and creating diversity[END_REF]. Subsequently, DNA methylation is re-established in canonical patterns in the post-implantation embryo during lineage specification (Hanna et al., 2018a).

Paternal protamines are replaced by maternal histones immediately after fertilization, and the paternal genome acquires weak ncH3K4me3 and ncH3K27me3 domains [START_REF] Zhang | Allelic reprogramming of the histone modification H3K4me3 in early mammalian development[END_REF][START_REF] Zheng | Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals[END_REF]. Both paternal and maternal ncH3K4me3 are maintained until early 2-cell stage and removed after zygotic genome activation (ZGA) at the late 2-cell stage.

Meanwhile, canonical H3K4me3 forms at promoters after ZGA [START_REF] Zhang | Allelic reprogramming of the histone modification H3K4me3 in early mammalian development[END_REF][START_REF] Dahl | Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition[END_REF][START_REF] Liu | Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos[END_REF]. Concomitant with the erasure of ncH3K4me3 after ZGA, H3K27ac appears at promoters and putative enhancers near ZGA genes [START_REF] Dahl | Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition[END_REF][START_REF] Wu | The landscape of accessible chromatin in mammalian preimplantation embryos[END_REF].

Maternal ncH3K27me3 is erased specifically from the promoters of developmental genes after fertilization, while those at distal regions are retained. Weak canonical H3K27me3 starts to form at promoters of polycomb target genes in the blastocyst, and both paternal and maternal distal ncH3K27me3 persist to blastocyst stage before being converted to canonical pattern in post-implantation embryo [START_REF] Zheng | Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals[END_REF]. Notably, maternal H3K27me3 could control DNA methylation-independent imprinting (Inoue et al., 2017a;[START_REF] Inoue | Genomic imprinting of Xist by maternal H3K27me3[END_REF], and in Drosophila, maternally inherited H3K27me3 regulates the activation of enhancers in the early embryos [START_REF] Zenk | Germ line-inherited H3K27me3 restricts enhancer function during maternal-to-zygotic transition[END_REF].

The zygote genome also undergoes large-scale H3K9me3 re-establishment. H3K9me3 is highly enriched on LTRs in early embryos and is involved in transcriptional repression of LTRs, activation of which is triggered by DNA demethylation [START_REF] Wang | Reprogramming of H3K9me3-dependent heterochromatin during mammalian embryo development[END_REF].

Moreover, TADs and compartments exist in a priming state after fertilization, and become more mature from the 8-cell stage. Consolidation of TADs is independent of ZGA and proceeds during cell cycles. Overall, chromatin of zygote exists in a relatively relaxed state with weak TADs and depleted distal chromatin interactions, and is gradually resolved to the canonical state through preimplantation development [START_REF] Du | Allelic reprogramming of 3D chromatin architecture during early mammalian development[END_REF][START_REF] Ke | 3D Chromatin Structures of Mature Gametes and Structural Reprogramming during Mammalian Embryogenesis[END_REF][START_REF] Xu | Epigenome in Early Mammalian Development: Inheritance, Reprogramming and Establishment[END_REF].

Transcription by RNA polymerase II

The basal transcription machinery

The finding that crude HeLa cell extract directs selective and accurate transcription initiation by purified Pol II at the adenovirus major late promoter [START_REF] Weil | Selective and accurate initiation of transcription at the Ad2 major late promotor in a soluble system dependent on purified RNA polymerase II and DNA[END_REF] provided direct biochemical evidences that accessory factors are necessary for site-specific initiation by

Pol II.

Further fractionation of this HeLa extract yielded four enzymatically active fractions (A, B, C and D) (Figure 2-1 A), and the components within fractions A, C, and D were necessary for accurate transcription initiation by Pol II [START_REF] Matsui | Multiple factors required for accurate initiation of transcription by purified RNA polymerase II[END_REF]. The complex in fraction A and D

were named TFIIA and TFIID, respectively, while fraction C was subsequently fractionated into different complexes named TFIIB, TFIIE, TFIIF and TFIIH [START_REF] Matsui | Multiple factors required for accurate initiation of transcription by purified RNA polymerase II[END_REF][START_REF] Sawadogo | Factors involved in specific transcription by human RNA polymerase II: analysis by a rapid and quantitative in vitro assay[END_REF][START_REF] Reinberg | Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site[END_REF][START_REF] Flores | Factors involved in specific transcription by mammalian RNA polymerase II. Factors IIE and IIF independently interact with RNA polymerase II[END_REF][START_REF] Gerard | Purification and interaction properties of the human RNA polymerase B(II) general transcription factor BTF2[END_REF][START_REF] Flores | Factors involved in specific transcription by mammalian RNA polymerase II. Identification and characterization of factor IIH[END_REF].

Accessory factors necessary for site-specific transcription initiation by Pol II were similarly purified from different species like, rat liver [START_REF] Conaway | Transcription initiated by RNA polymerase II and purified transcription factors from liver. Cooperative action of transcription factors tau and epsilon in initial complex formation[END_REF], Drosophila [START_REF] Parker | A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity[END_REF][START_REF] Heberlein | Characterization of Drosophila transcription factors that activate the tandem promoters of the alcohol dehydrogenase gene[END_REF][START_REF] Price | Fractionation of transcription factors for RNA polymerase II from Drosophila Kc cell nuclear extracts[END_REF] and S. cerevisiae [START_REF] Lue | Accurate initiation at RNA polymerase II promoters in extracts from Saccharomyces cerevisiae[END_REF][START_REF] Sayre | Reconstitution of transcription with five purified initiation factors and RNA polymerase II from Saccharomyces cerevisiae[END_REF], and amazingly, organisms as diverse as human, rat, Drosophila and yeast use the same set of conserved complexes to initiate Pol II transcription.

These essential accessory factors, TFIIA, TFIIB, TFIID, TFIIE, TFIIF and TFIIH were collectively defined as general transcription factors (GTFs) [START_REF] Orphanides | The general transcription factors of RNA polymerase II[END_REF], which together with Pol II are known as the basal / general transcription machinery (Figure 2-1 B) [START_REF] Smale | The RNA polymerase II core promoter[END_REF][START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. HeLa nuclear extract, from [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. (B) Basal transcription machinery on the promoter.

RNA Polymerase II

Pol II is a multi-subunit complex responsible for the transcription of all protein-coding genes, long noncoding RNA, microRNA genes and most snRNA genes, which is composed of 12 highly conserved subunits (RPB1 to RPB12) (Table 2-1) [START_REF] Young | RNA polymerase II[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF].

However, not all the subunits are exclusive to Pol II: [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. polymerase core represents the Pol II during elongation, and is not able to initiated transcription [START_REF] Cramer | Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution[END_REF]. The RPB4/RPB7 heterodimeric subcomplex binds to a pocket formed by RPB1, RPB2 and RPB6 at the base of clamp module [START_REF] Bushnell | Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription[END_REF], forming the polymerase stalk (Figure 2-1 B). This binding induces a conformational change and locks the clamp in the closed conformation, which suggest that single-stranded DNA enters into the cleft of the polymerase core before binding of RPB4/RPB7 [START_REF] Bushnell | Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription[END_REF]. The RPB4/RPB7 heterodimer is located in the vicinity of the CTD, however, unstructured CTD forms a flexible, tail-like extension from the catalytic core of Pol II, and is not visible due to high mobility in the crystal structures [START_REF] Cramer | Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution[END_REF][START_REF] Meinhart | A structural perspective of CTD function[END_REF]. The CTD of RPB1 consists of tandem heptapeptide repeats with the consensus sequence Y1S2P3T4S5P6S7 [START_REF] Schuller | Heptad-Specific Phosphorylation of RNA Polymerase II CTD[END_REF][START_REF] Harlen | The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain[END_REF] (Figure 23). The repeats number varies between species from 26 in S. cerevisiae to 52 in vertebrates [START_REF] Young | RNA polymerase II[END_REF][START_REF] Hsin | The RNA polymerase II CTD coordinates transcription and RNA processing[END_REF]. However, in vertebrate, out of the 52, only 21 repeats match the consensus perfectly, and are mainly located in the N-terminal half of the CTD, while the remaining 31 heptads have one or more substitutions [START_REF] Hsin | The RNA polymerase II CTD coordinates transcription and RNA processing[END_REF]. A 10-residue sequence is present at the C-terminal end of CTD and is important for its stability [START_REF] Chapman | The last CTD repeat of the mammalian RNA polymerase II large subunit is important for its stability[END_REF].

Importantly, the CTD interacts with a wide range of regulatory factors via its dynamic binding surfaces generated by post-translational modifications, notably phosphorylation [START_REF] Buratowski | Progression through the RNA polymerase II CTD cycle[END_REF] [START_REF] Kwon | Phosphorylation-regulated binding of RNA polymerase II to fibrous polymers of low-complexity domains[END_REF][START_REF] Burke | Residue-by-Residue View of In Vitro FUS Granules that Bind the C-Terminal Domain of RNA Polymerase II[END_REF][START_REF] Harlen | The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain[END_REF][START_REF] Boehning | RNA polymerase II clustering through carboxy-terminal domain phase separation[END_REF]. In the following part, GTFs are described in the order that they join the PIC, with the exception of TFIIA , which can join the PIC at any step after TFIID binding [START_REF] Orphanides | The general transcription factors of RNA polymerase II[END_REF].

TFIID

TFIID is the first GTF that recognizes and binds to the core promoter, nucleating the assembly of the pre-initiation complex (PIC) (for PIC assembly, see section 2.2.2) [START_REF] Buratowski | Five intermediate complexes in transcription initiation by RNA polymerase II[END_REF]. It is a multi-subunit complex comprising of the TATA box binding protein (TBP) and 13 TBP-associated factors (TAFs, TAF1 to TAF13 in metazoan or 14 TAFs in yeast) with total molecular weight of 1.2 MDa. The subunits of TFIID are generally conserved from yeast to human, but also with the existence of TAF paralogs, TAF-like proteins and TBP-related proteins in different metazoans (Figure 2-5 a) (Tora, 2002;[START_REF] Muller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF].

TBP

TBP plays a crucial role in transcription initiation of all three RNA polymerases in eukaryotes (Hernandez, 1993). Tbp knockout in mice leads to growth arrest and apoptosis at the blastocyst stage (Martianov et al., 2002b), however, while Pol I and Pol III transcription is blocked, Pol II transcription is still active.

In Pol II transcription, TBP is the central DNA-binding subunit of TFIID [START_REF] Tora | The TATA box regulates TATA-binding protein (TBP) dynamics in vivo[END_REF], and it binds upstream of the transcription start site (TSS) of all promoters [START_REF] Rhee | Genome-wide structure and organization of eukaryotic pre-initiation complexes[END_REF].

Interestingly, most metazoans have multiple TBP paralogs, the TBP-related factors (TRFs), including insect specific TRF1, metazoan-specific TRF2/TBPL1/TLF/TLP/TRP and vertebrate-specific TBP2/TRF3/TBPL2. [START_REF] Torres-Padilla | TBP homologues in embryo transcription: who does what?[END_REF][START_REF] Muller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF]Goodrich et al., 2010). Function of these TBP-related factors will be discussed in section 3.2.

TBP has a highly conserved C-terminal half, which consists of two symmetric pseudo-repeats folding into a bipartite saddle-like structure (Figure 234). TBP initially binds the TATA box to form an unstable complex containing unbent DNA, subsequently forming a stable complex and inducing a 90° bend of the DNA, thus, providing an asymmetric platform for PIC assembly (Kim et al., 1993a;Kim et al., 1993b;[START_REF] Zhao | A regulated two-step mechanism of TBP binding to DNA: a solvent-exposed surface of TBP inhibits TATA box recognition[END_REF] (Figure 234).

However, it is unclear whether TATA-less promoters display any bending upon TBP or TFIID binding as the structure of a TFIID-containing PIC on TATA-less DNA has not been modeled [START_REF] Tora | The TATA box regulates TATA-binding protein (TBP) dynamics in vivo[END_REF][START_REF] Kamenova | Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription[END_REF]. Although it has been shown that single-site variants in the TATA box reduce TBP-induced bending in solution [START_REF] Wu | DNA bends in TATA-binding protein-TATA complexes in solution are DNA sequence-dependent[END_REF], however, systematic X-ray crystallographic study shows that co-crystal structures of A. thaliana TBP with 10 different TATA box variants are all very similar, indicating that structure of the TBP-DNA complex is independent of TATA element sequence [START_REF] Patikoglou | TATA element recognition by the TATA box-binding protein has been conserved throughout evolution[END_REF]. It is possible that TATA-less promoters are also bent during PIC formation, by TAFs, TFIIB and / or some other factors [START_REF] Kamenova | Mutations on the DNA binding surface of TBP discriminate between yeast TATA and TATA-less gene transcription[END_REF]. from Protein Data Bank in Europe (PDB EMDB id: 1cdw), [START_REF] Nikolov | Crystal structure of a human TATA box-binding protein/TATA element complex[END_REF] Although the affinity is ∼1000-fold lower compared with TATA sequences, TBP still exhibits binding ability to non-specific DNA, which may lead to the formation of non-productive PICs [START_REF] Coleman | Evidence for functional binding and stable sliding of the TATA binding protein on nonspecific DNA[END_REF]. Moreover, once TBP binds to DNA, no matter at TATA box or non-specific site, the half-life for TBP dissociation is very long in vitro (20-60 mins or more) [START_REF] Hoopes | Kinetic analysis of yeast TFIID-TATA box complex formation suggests a multi-step pathway[END_REF][START_REF] Coleman | Evidence for functional binding and stable sliding of the TATA binding protein on nonspecific DNA[END_REF]. Given the central role of TBP in eukaryotic transcription, its activity need to be tightly regulated, including the negative regulation of liberating TBP from stable TBP-promoter binding (especially TBP-TATA complex) and the removal of TBP from non-specific binding sites, which can be achieved by regulators such as ATPase BTAF1 (Mot1 in yeast) and Negative Cofactor 2 (NC2).

BTAF1 was initially identified as part of the B-TFIID complex, which consists of BTAF1 and TBP [START_REF] Timmers | Composition of transcription factor B-TFIID[END_REF]. BTAF1 can bind to the concave surface of TBP to block TBP-promoter binding [START_REF] Pereira | TAF(II)170 interacts with the concave surface of TATA-binding protein to inhibit its DNA binding activity[END_REF]. Moreover, as BTAF1/Mot1 belongs to the SWI2/SNF2-family ATPase, it can bind to the TBP-DNA complex and change TBP DNA binding properties upon ATP-binding and hydrolysis, ultimately leading to the displace and release of TBP from DNA [START_REF] Pereira | Roles for BTAF1 and Mot1p in dynamics of TATA-binding protein and regulation of RNA polymerase II transcription[END_REF][START_REF] Gumbs | High-affinity DNA binding by a Mot1p-TBP complex: implications for TAF-independent transcription[END_REF], thus facilitating the redistribution of TBP [START_REF] Klejman | Mutational analysis of BTAF1-TBP interaction: BTAF1 can rescue DNA-binding defective TBP mutants[END_REF].

NC2 is a conserved heterodimeric complex consisting of NC2α and NC2β (Goppelt et al., 1996a). Binding of NC2 to the TBP-promoter complex inhibits the association of TFIIA and TFIIB with TBP, therefore blocking PIC formation [START_REF] Inostroza | Dr1, a TATA-binding protein-associated phosphoprotein and inhibitor of class II gene transcription[END_REF][START_REF] Mermelstein | Requirement of a corepressor for Dr1-mediated repression of transcription[END_REF]Goppelt et al., 1996b). In addition, NC2 binding can induce significant conformational changes in the TBP-DNA complex, leading to TBP sliding on DNA [START_REF] Schluesche | NC2 mobilizes TBP on core promoter TATA boxes[END_REF].

BTAF1/Mot1 function is intimately linked with NC2. Consistent with that, the binding profiles of BTAF1 and NC2 strongly overlap, furthermore, a stable TBP-NC2-Mot1-DNA complex also exists [START_REF] Van Werven | Cooperative action of NC2 and Mot1p to regulate TATA-binding protein function across the genome[END_REF]. It has been proposed that bent DNA conformation induced by TBP binding could act as a ''spring'' for rapid BTAF1-NC2 mediated TBP release from TATA-containing promoters, allowing TBP redistribution to TATA-less promoters [START_REF] Tora | The TATA box regulates TATA-binding protein (TBP) dynamics in vivo[END_REF][START_REF] Zentner | Mot1 redistributes TBP from TATA-containing to TATA-less promoters[END_REF].

TBP recognizes and binds directly to TATA-containing promoters to start nucleation of the PIC. However, only about 24% of human promoters have a TATA-like element, among which only approximately 10% have the canonical TATA box (TATAWAWR) [START_REF] Yang | Prevalence of the initiator over the TATA box in human and yeast genes and identification of DNA motifs enriched in human TATA-less core promoters[END_REF].

Importantly, several TAFs are also able to recognize and bind different elements within the core promoter, or even acetylated or methylated histone tails around.

TAFs

As mentioned above, metazoan TFIID contains 13 TAFs (Figure 2345). TAFs mediate a broad range of interactions, which allow the regulation of TFIID recruitment and stabilization at both TATA-containing and TATA-less promoter (elements of core promoter, see section 2.3.1).

TAFs recognize and bind to promoters [START_REF] Muller | New problems in RNA polymerase II transcription initiation: matching the diversity of core promoters with a variety of promoter recognition factors[END_REF] (Figure 23456), which is of particularly importance since the majority of vertebrate promoters are lacking of TATA-box [START_REF] Yang | Prevalence of the initiator over the TATA box in human and yeast genes and identification of DNA motifs enriched in human TATA-less core promoters[END_REF]. TAF1 and TAF2 can bind to the initiator element (Inr) which overlaps with the transcription start site (TSS) [START_REF] Chalkley | DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator[END_REF], and possibly bind motif ten element (MTE) [START_REF] Louder | Structure of promoter-bound TFIID and model of human pre-initiation complex assembly[END_REF]. TAF1 was also shown to bind downstream core element (DCE) [START_REF] Lee | Functional characterization of core promoter elements: the downstream core element is recognized by TAF1[END_REF]. TAF6/TAF9 heterodimers bind to the downstream promoter element (DPE) [START_REF] Burke | The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila[END_REF][START_REF] Shao | Core promoter binding by histone-like TAF complexes[END_REF], which could be the target of TAF1 as well [START_REF] Louder | Structure of promoter-bound TFIID and model of human pre-initiation complex assembly[END_REF]. TAFs mediated promoter recognition and binding is essential for transcription of the genes with TATA-less promoters [START_REF] Huisinga | A genome-wide housekeeping role for TFIID and a highly regulated stress-related role for SAGA in Saccharomyces cerevisiae[END_REF]. 3) are labelled with a star. From [START_REF] Muller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF].

Moreover, TAFs can direct the recruitment of TFIID to promoters through interaction with histone post-translational modifications. Particularly, TAF3 can anchor TFIID to nucleosomes through its plant homeodomain (PHD) finger that selectively binds to the hallmark of active promoters H3K4me3 [START_REF] Vermeulen | Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4[END_REF][START_REF] Van Ingen | Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3[END_REF]. Interestingly, other active promoter marks, H3K9ac and H3K14ac increase TFIID binding to H3K4me3, while H3R2me2a (asymmetric di-methylation) inhibits this interaction [START_REF] Vermeulen | Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4[END_REF].

Further studies show that H3K4me3, through interaction with TAF3, can direct PIC formation either independently or cooperatively with the TATA box [START_REF] Lauberth | H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation[END_REF]. Besides, TAF1

contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides [START_REF] Jacobson | Structure and function of a human TAFII250 double bromodomain module[END_REF] [and it is important to mention that TAF1 has histone acetyltransferase (HAT) activity in vitro [START_REF] Mizzen | The TAF(II)250 subunit of TFIID has histone acetyltransferase activity[END_REF]].

Interactions between TAFs and histone PTMs provide novel insights for promoter recognition and TFIID recruitment, complementary to the mechanisms involving interactions of TBP and/or TAFs with core promoter elements. These new mechanisms are important specifically for TATA-less promoters [START_REF] Vermeulen | Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4[END_REF]. and downstream promoter element (DPE, light green), from [START_REF] Cianfrocco | Human TFIID binds to core promoter DNA in a reorganized structural state[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] In addition, TAFs can also modulate TBP/TFIID activity, for instance, TAF1 inhibits TBP/TFIID binding to TATA-containing core promoter DNA with its N-terminal domains TAND1 and TAND2, which bind respectively to the DNA-binding surface of TBP though mimicking TATA-box and the convex surface of TBP. [START_REF] Kokubo | Interaction between the N-terminal domain of the 230-kDa subunit and the TATA box-binding subunit of TFIID negatively regulates TATA-box binding[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF].

Binding of TFIIA offsets the inhibition by TAF1 and stabilize the TBP-promoter interaction [START_REF] Ozer | Transcription factor IIA derepresses TATA-binding protein (TBP)-associated factor inhibition of TBP-DNA binding[END_REF]. Alternatively, TAF11/TAF13 also interacts with the DNA binding surface of TBP, thereby blocking TBP from binding TATA-containing promoters, and this inhibition is required for normal TFIID function [START_REF] Gupta | Architecture of TAF11/TAF13/TBP complex suggests novel regulation properties of general transcription factor TFIID[END_REF]. TAF7 binds to TAF1 and inhibits its HAT activity [START_REF] Gegonne | TAFII55 binding to TAFII250 inhibits its acetyltransferase activity[END_REF]. Besides, TAFs can also interact with transcription activators [START_REF] Liu | Structures of three distinct activator-TFIID complexes[END_REF] and GTFs, such as TFIIA [START_REF] Yokomori | Drosophila TFIIA-L is processed into two subunits that are associated with the TBP/TAF complex[END_REF], TFIIB [START_REF] Goodrich | Drosophila TAFII40 interacts with both a VP16 activation domain and the basal transcription factor TFIIB[END_REF] and TFIIH [START_REF] Ruppert | Human TAFII250 interacts with RAP74: implications for RNA polymerase II initiation[END_REF]. Notably, TAF7 interacts with TFIIH and p-TEFb, and regulates their kinase activities [START_REF] Gegonne | TFIID component TAF7 functionally interacts with both TFIIH and P-TEFb[END_REF], thus TAF7 may function as a checkpoint regulator for transcription initiation [START_REF] Gegonne | TAF7: a possible transcription initiation check-point regulator[END_REF].

Overall, TAFs modulate TFIID recruitment to promoters containing different combinations of binding elements (including histone PTMs), and also finetune TFIID stability and activity. ), TAF4-12 [START_REF] Werten | Crystal structure of a subcomplex of human transcription factor TFIID formed by TATA binding protein-associated factors hTAF4 (hTAF(II)135) and hTAF12 (hTAF(II)20)[END_REF], TAF6-9 [START_REF] Xie | Structural similarity between TAFs and the heterotetrameric core of the histone octamer[END_REF] and TAF11-13 [START_REF] Birck | Human TAF(II)28 and TAF(II)18 interact through a histone fold encoded by atypical evolutionary conserved motifs also found in the SPT3 family[END_REF]. Stoichiometry analysis of TFIID in yeast revealed that six TAFs (TAF4, TAF5, TAF6, TAF9, TAF10 and TAF12) are present in two copies in TFIID, while TBP and other TAFs are present in a single copy [START_REF] Sanders | Molecular characterization of Saccharomyces cerevisiae TFIID[END_REF].

Among the six two-copies TAFs, five (TAF4, TAF5, TAF6, TAF9 and TAF12) form a core-TFIID subcomplex [START_REF] Wright | TAF4 nucleates a core subcomplex of TFIID and mediates activated transcription from a TATA-less promoter[END_REF], which has a symmetric structure, comprising two copies of HFD-containing heterodimer TAF4-12, TAF6-9 and WD40 repeat domain-containing protein TAF5 [START_REF] Bieniossek | The architecture of human general transcription factor TFIID core complex[END_REF] (Figure 234567). Interestingly, the symmetry of the core-TFIID is broken upon addition of a TAF8-TAF10 building block, resulting in a 7TAF-TFIID complex with an asymmetric structure, which was proposed to serve as a functional scaffold, Adapted from [START_REF] Bieniossek | The architecture of human general transcription factor TFIID core complex[END_REF].

A recent study has shown that, in the cytoplasm, TAF2, TAF8 and TAF10 form a ternary subcomplex, which subsequently translocates into nucleus and incorporates into the core-TFIID complex, forming a 8TAF-TFIID intermediate [START_REF] Trowitzsch | Cytoplasmic TAF2-TAF8-TAF10 complex provides evidence for nuclear holo-TFIID assembly from preformed submodules[END_REF]. Moreover, it has been shown that TFIID is assembled co-translationally (termed co-translational assembly) [START_REF] Kamenova | Co-translation drives the assembly of mammalian nuclear multisubunit complexes[END_REF].

However, high resolution structure of holo-TFIID is still not solved [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF], and also further studies are needed to fully understand the stepwise assembly mechanism of holo-TFIID.

TFIIA, a controversial GTF

Unlike the yeast TFIIA that contains 2 subunits, TFIIA in metazoan is composed of three subunits (TFIIAα, TFIIAβ and TFIIAγ), which are encoded by two genes, GTF2A1 and GTF2A2 [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. GTF2A1 encodes the TFIIAαβ precursor, which is cleaved into TFIIAα and TFIIAβ post-translationally by Taspase1 at the conserved cleavage site "QVD ↓ G", while GTF2A2 encodes the smallest subunit TFIIAγ (Zhou et al., 2006;[START_REF] Hoiby | A facelift for the general transcription factor TFIIA[END_REF]. In addition, TFIIAαβ has a cell type-specific paralogue, called TFIIA-like factor (ALF) (Upadhyaya et al., 1999;[START_REF] Ozer | A testis-specific transcription factor IIA (TFIIAtau) stimulates TATA-binding protein-DNA binding and transcription activation[END_REF] (see section 3.3.2).

TFIIA was initially classified as a general transcription factor as early studies showed TFIIA was necessary to reconstitute basal transcription in vitro [START_REF] Reinberg | Factors involved in specific transcription in mammalian RNA polymerase II. Functional analysis of initiation factors IIA and IID and identification of a new factor operating at sequences downstream of the initiation site[END_REF].

However, later studies showed the requirement for TFIIA in reconstituted transcription varies depending on different reconstituted transcription systems [START_REF] Orphanides | The general transcription factors of RNA polymerase II[END_REF][START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. Indeed, TFIIA stimulates both basal and activated transcription in vitro when TFIID is used for promoter binding [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF][START_REF] Hoiby | A facelift for the general transcription factor TFIIA[END_REF].

TFIIA contributes to transcription initiation through different mechanisms. First, TFIIA can bind and stabilize the TBP-promoter complex through direct interaction with TBP and contact with DNA upstream of the TATA box, thus enhancing PIC assembly [START_REF] Orphanides | The general transcription factors of RNA polymerase II[END_REF].

Second, TFIIA can counteract the inhibitory effects on TBP binding to DNA that are caused by NC2, BTAF1 or TAF1 [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. In addition, TFIIA can also function as a coactivator to facilitate PIC assembly by direct contacts with several activators and GTFs [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF][START_REF] Hoiby | A facelift for the general transcription factor TFIIA[END_REF].

TFIIB

TFIIB is the only GTF that consist of a single polypeptide. Human TFIIB contains 316 amino acids (345 aa in yeast), which are organized into 5 functional domains [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] (Figure 2-8 A&B).

TFIIB is essential for Pol II transcription initiation. First of all, TFIIB can bind to the TBP/TFIID-DNA complex, resulting in the formation of a more stable TFIIB-TFIID-promoter or TFIIB-TFIIA-TFIID-promoter complex, and it can recognize and bind to promoters that contain TFIIB recognition element (BRE) as well [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF][START_REF] Deng | TFIIB and the regulation of transcription by RNA polymerase II[END_REF]. [START_REF] Kostrewa | RNA polymerase II-TFIIB structure and mechanism of transcription initiation[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] Secondly, its N-terminal B-ribbon that contacts the dock domain of Pol II (RPB1 subunit) is involved in Pol II recruitment [START_REF] Buratowski | Functional domains of transcription factor TFIIB[END_REF][START_REF] Bushnell | Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms[END_REF]) (Figure 2345678A&C). Thirdly, its C-terminal domain (B-core cyclin folds) interacts with both Pol II and the TBP-promoter complex to orients the DNA. More specifically, the B-core N-terminal cyclin fold binds to the wall of Pol II to position DNA over the Pol II active center cleft [START_REF] Bushnell | Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms[END_REF][START_REF] Kostrewa | RNA polymerase II-TFIIB structure and mechanism of transcription initiation[END_REF][START_REF] Liu | Structure of an RNA polymerase II-TFIIB complex and the transcription initiation mechanism[END_REF] [START_REF] Kostrewa | RNA polymerase II-TFIIB structure and mechanism of transcription initiation[END_REF][START_REF] Sainsbury | Structure and function of the initially transcribing RNA polymerase II-TFIIB complex[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] (Figure 2

-8 A&D).

Furthermore, TFIIB stimulates initial RNA synthesis [START_REF] Sainsbury | Structure and function of the initially transcribing RNA polymerase II-TFIIB complex[END_REF], and stabilizes the early initiation complex containing a short transcript [START_REF] Bushnell | Structural basis of transcription: an RNA polymerase II-TFIIB cocrystal at 4.5 Angstroms[END_REF]. B-reader loop of TFIIB blocks the path of the transcript longer than 6 nucleotides and directs it to its exit tunnel, and this blocking by the B-reader loop may play a role in DNA-RNA strand separation [START_REF] Sainsbury | Structure and function of the initially transcribing RNA polymerase II-TFIIB complex[END_REF] (Figure 2345678). Lastly, TFIIB is released from Pol II when transcript grows to 12-13 nucleotides, as the RNA starts to clash with B-ribbon, triggering TFIIB displacement [START_REF] Cabart | Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes[END_REF][START_REF] Sainsbury | Structure and function of the initially transcribing RNA polymerase II-TFIIB complex[END_REF].

TFIIF

In mammals, TFIIF is a heterodimer comprising of the subunits TFIIFα and TFIIFβ (also known as RAP74 and RAP30, respectively) [START_REF] Burton | RAP30/74: a general initiation factor that binds to RNA polymerase II[END_REF][START_REF] Flores | Factors involved in specific transcription by mammalian RNA polymerase II. RNA polymerase II-associating protein 30 is an essential component of transcription factor IIF[END_REF][START_REF] Flores | Factors involved in specific transcription by mammalian RNA polymerase II. Purification and subunit composition of transcription factor IIF[END_REF]. However, yeast TFIIF has 3 subunits [START_REF] Henry | Purification and characterization of yeast RNA polymerase II general initiation factor g[END_REF][START_REF] Henry | TFIIF-TAF-RNA polymerase II connection[END_REF]: Tfg1

and Tfg2 are essential and correspond to human TFIIFα and TFIIFβ, respectively, while the third subunit Tfg3 is non-essential for transcription, and it is also present in yeast TFIID (as TAF14) and SWI/SNF chromatin remodeling complexes (as ANC1) [START_REF] Cairns | TFG/TAF30/ANC1, a component of the yeast SWI/SNF complex that is similar to the leukemogenic proteins ENL and AF-9[END_REF].

Interesting, transcription can be initiated to some extent in vitro without TFIIE and TFIIH, but TFIIF is absolutely needed, indicating the critical role of TFIIF for transcription initiation [START_REF] Pan | Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site[END_REF]. Indeed, TFIIF plays multiple roles during PIC formation. First, TFIIF tightly associates with Pol II and enhances the affinity of Pol II for TFIIB-TFIID-promoter complex [START_REF] Robert | Wrapping of promoter DNA around the RNA polymerase II initiation complex induced by TFIIF[END_REF], preventing non-specific interaction of Pol II with DNA [START_REF] Conaway | Mechanism of promoter selection by RNA polymerase II: mammalian transcription factors alpha and beta gamma promote entry of polymerase into the preinitiation complex[END_REF], facilitating Pol II recruitment [START_REF] Flores | The small subunit of transcription factor IIF recruits RNA polymerase II into the preinitiation complex[END_REF] and stabilizing the PIC [START_REF] Tan | Roles for both the RAP30 and RAP74 subunits of transcription factor IIF in transcription initiation and elongation by RNA polymerase II[END_REF], in particular stabilizing TFIIB within the PIC [START_REF] Cabart | Transcription factor TFIIF is not required for initiation by RNA polymerase II, but it is essential to stabilize transcription factor TFIIB in early elongation complexes[END_REF][START_REF] Fishburn | Architecture of the yeast RNA polymerase II open complex and regulation of activity by TFIIF[END_REF].

Second, TFIIF is required for subsequent recruitment of TFIIE and TFIIH through direct interactions with TFIIE [START_REF] Maxon | Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance[END_REF][START_REF] Orphanides | The general transcription factors of RNA polymerase II[END_REF]. Third, TFIIF influences TSS selection [START_REF] Ghazy | Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II[END_REF].

After PIC assembly, TFIIF stimulates early RNA synthesis and is required for efficient Pol II promoter escape [START_REF] Yan | Dual roles for transcription factor IIF in promoter escape by RNA polymerase II[END_REF]. TFIIF also enhances the efficiency of Pol II elongation and suppresses transient Pol II pausing [START_REF] Zhang | Transcription factors IIF and IIS and nucleoside triphosphate substrates as dynamic probes of the human RNA polymerase II mechanism[END_REF][START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. Moreover, TFIIF assists in the stabilization of the transcription bubble [START_REF] Pan | Initiation of transcription by RNA polymerase II is limited by melting of the promoter DNA in the region immediately upstream of the initiation site[END_REF].

TFIIE

Like TFIIF, human TFIIE is also a heterodimer consisting of the two subunits, TFIIEα and TFIIEβ (Tfa1 and Tfa2 in yeast) [START_REF] Peterson | Structure and functional properties of human general transcription factor IIE[END_REF][START_REF] Ohkuma | Structural motifs and potential sigma homologies in the large subunit of human general transcription factor TFIIE[END_REF][START_REF] Itoh | Investigation of molecular size of transcription factor TFIIE in solution[END_REF][START_REF] Jawhari | Structure and oligomeric state of human transcription factor TFIIE[END_REF]. TFIIEα contains an N-terminal extended winged WH domain (eWH), a central zinc ribbon domain (E-ribbon) connected by E-linker and a C-terminal acidic domain.

TFIIEβ has two WH domains and an E-tether [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF][START_REF] Plaschka | Transcription initiation complex structures elucidate DNA opening[END_REF] (Figure 23456789). E-tether binds the E-linker and is essential for TFIIE subunit dimerization Adapted from [START_REF] Plaschka | Transcription initiation complex structures elucidate DNA opening[END_REF].

Once recruited, it interacts directly with TFIIF, TFIIB, Pol II and promoter DNA [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. TFIIE is located between the clamp and the RPB4-RPB7 stalk of Pol II. Furthermore, TFIIE assists in the recruitment of TFIIH [START_REF] Maxon | Transcription factor IIE binds preferentially to RNA polymerase IIa and recruits TFIIH: a model for promoter clearance[END_REF][START_REF] Holstege | Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH[END_REF], and it also stimulates ATPase and kinase activity of TFIIH [START_REF] Ohkuma | Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation[END_REF], thus

facilitating the formation of an initiation-competent Pol II complex. In addition, TFIIE, together with TFIIH, is essential for promoter melting and the transition from initiation to elongation [START_REF] Holstege | Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH[END_REF].

TFIIH

TFIIH is a complex with 10 subunits, organized into a seven-subunit core and a three-subunit kinase module [START_REF] Compe | TFIIH: when transcription met DNA repair[END_REF][START_REF] Schilbach | Structures of transcription pre-initiation complex with TFIIH and Mediator[END_REF][START_REF] Greber | The cryo-electron microscopy structure of human transcription factor IIH[END_REF].

Human TFIIH core is composed of two ATPases (XPB and XPD) and 5 subunits (p62, p52, p44, p34 and p8), which are respectively known as Ssl2, Rad3, Tfb1, Tfb2, Tfb4, Tfb5 and Ssl1 in yeast [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF]. The TFIIH kinase module comprises of CDK7, cyclin H and MAT1 in human, which correspond respectively to Kin28, Ccl1 and Tfb3 in yeast [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] (Table 2-2). Over all, TFIIH accommodates three different enzymatic activities: ATPase activity and helicase activity from XPB and XPD, and kinase activity from CDK7 [START_REF] Compe | TFIIH: when transcription met DNA repair[END_REF]. After its recruitment by TFIIE, TFIIH binds to Pol II and functions in promoter opening and escape [START_REF] Goodrich | Transcription factors IIE and IIH and ATP hydrolysis direct promoter clearance by RNA polymerase II[END_REF][START_REF] Holstege | Opening of an RNA polymerase II promoter occurs in two distinct steps and requires the basal transcription factors IIE and IIH[END_REF][START_REF] Moreland | A role for the TFIIH XPB DNA helicase in promoter escape by RNA polymerase II[END_REF]. In general, without TFIIH, Pol II tends to stall on the promoter-proximal region, resulting in abortive transcription [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF]. It has been shown that promoter opening by TFIIH is dependent on the ATPase activity of XPB but not XPD [START_REF] Tirode | Reconstitution of the transcription factor TFIIH: assignment of functions for the three enzymatic subunits, XPB, XPD, and cdk7[END_REF][START_REF] Coin | Mutations in XPB and XPD helicases found in xeroderma pigmentosum patients impair the transcription function of TFIIH[END_REF]. As XPB engages with promoter DNA around 25-30 bp downstream of TSS [START_REF] Schilbach | Structures of transcription pre-initiation complex with TFIIH and Mediator[END_REF] (Figure 2-10 A) and does not bind the transcription bubble, it apparently does not function as a classical helicase, which would bind to the unwound region. Consistently, mutational analysis of XPB revealed that DNA opening does not rely on its helicase activity [START_REF] Lin | TFIIH XPB mutants suggest a unified bacterial-like mechanism for promoter opening but not escape[END_REF]. Current The TFIIH kinase module, specifically CDK7, is responsible for the phosphorylation of Pol II CTD at serine 5 residue [START_REF] Serizawa | Association of Cdk-activating kinase subunits with transcription factor TFIIH[END_REF], which aids in promoter escape by Pol II [START_REF] Harlen | The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain[END_REF] and enhances the association of the Pol II CTD with the 7-methylguanosine (m7G) RNA capping machinery [START_REF] Cho | mRNA capping enzyme is recruited to the transcription complex by phosphorylation of the RNA polymerase II carboxy-terminal domain[END_REF][START_REF] Komarnitsky | Different phosphorylated forms of RNA polymerase II and associated mRNA processing factors during transcription[END_REF].

In addition, CDK7 also phosphorylates Ser7 of CTD, which seems to be important for snRNA gene expression [START_REF] Egloff | Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression[END_REF][START_REF] Compe | TFIIH: when transcription met DNA repair[END_REF].

Besides its roles in Pol II transcription, TFIIH also participates in Pol I transcription, probably Pol III transcription as well, and it is also known be to essential for DNA repair [START_REF] Compe | TFIIH: when transcription met DNA repair[END_REF].

The cycle of Pol II transcription

The cycle of Pol II-mediated transcription contains at least eight major steps (Figure 2-11) [START_REF] Fuda | Defining mechanisms that regulate RNA polymerase II transcription in vivo[END_REF]. It begins with chromatin opening (Step 1), which facilitates GTFs and Pol II gaining access to the promoter. GTFs and Pol II bind the core promoter and form a preinitiation abstract book, which is adapted from [START_REF] Fuda | Defining mechanisms that regulate RNA polymerase II transcription in vivo[END_REF].

Chromatin opening

Binding of activators

Gene-specificity of transcription is achieved and greatly stimulated by activators or conversely repressors (also referred as gene-specific transcription factors), which mark or pre-mark promoter-proximal regions and/or distant enhancers for activity through DNA sequence-specific binding [START_REF] Koster | Genesis of Chromatin and Transcription Dynamics in the Origin of Species[END_REF] (Figure 234567891011121314).

Among the activators, some can be referred as pioneer factors (Table 2-2), and have the unique ability to engage their nucleosomal target sites in condensed or inaccessible chromatin [START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF][START_REF] Iwafuchi-Doi | The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation[END_REF]. AP-2γ (TFAP2C) Basic helix-span-helix [START_REF] Tan | AP-2gamma regulates oestrogen receptor-mediated long-range chromatin interaction and gene transcription[END_REF] FOXA1 (HNF-3α) Forkhead [START_REF] Cirillo | Binding of the winged-helix transcription factor HNF3 to a linker histone site on the nucleosome[END_REF][START_REF] Serandour | Epigenetic switch involved in activation of pioneer factor FOXA1-dependent enhancers[END_REF] FOXA2 (HNF-3β) Forkhead [START_REF] Cirillo | Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4[END_REF][START_REF] Donaghey | Genetic determinants and epigenetic effects of pioneer-factor occupancy[END_REF] FOXE1 Forkhead [START_REF] Cuesta | The forkhead factor FoxE1 binds to the thyroperoxidase promoter during thyroid cell differentiation and modifies compacted chromatin structure[END_REF] FOXD3 Forkhead [START_REF] Xu | Transcriptional competence and the active marking of tissue-specific enhancers by defined transcription factors in embryonic and induced pluripotent stem cells[END_REF] GATA2 2X GATA-type zinc fingers [START_REF] Wu | Three-tiered role of the pioneer factor GATA2 in promoting androgen-dependent gene expression in prostate cancer[END_REF] GATA3 2X GATA-type zinc fingers [START_REF] Shoemaker | GATA-3 directly remodels the IL-10 locus independently of IL-4 in CD4+ T cells[END_REF] GATA4 2X GATA-type zinc fingers [START_REF] Cirillo | Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4[END_REF][START_REF] Donaghey | Genetic determinants and epigenetic effects of pioneer-factor occupancy[END_REF] KLF4 3X C2H2-type zinc fingers [START_REF] Soufi | Facilitators and impediments of the pluripotency reprogramming factors' initial engagement with the genome[END_REF][START_REF] Soufi | Pioneer transcription factors target partial DNA motifs on nucleosomes to initiate reprogramming[END_REF] NF-Y (CBF) NF-YA/HAP2 [START_REF] Oldfield | Histone-fold domain protein NF-Y promotes chromatin accessibility for cell type-specific master transcription factors[END_REF] OCT4 POU-specific + POU-Homeodomain (Buecker et al., 2014; Donaghey et al., 2018) OTX2 Homeodomain [START_REF] Buecker | Reorganization of enhancer patterns in transition from naive to primed pluripotency[END_REF] PAX7 Paired + Homeodomain [START_REF] Budry | The selector gene Pax7 dictates alternate pituitary cell fates through its pioneer action on chromatin remodeling[END_REF][START_REF] Mayran | Pioneer factor Pax7 deploys a stable enhancer repertoire for specification of cell fate[END_REF] PBX1 Homeodomain [START_REF] Berkes | Pbx marks genes for activation by MyoD indicating a role for a homeodomain protein in establishing myogenic potential[END_REF] PU. In the following sections, I will mainly focus on the Mediator complex, the SAGA complex and NuA4/TIP60 complex.

The Mediator complex

The Mediator is an evolutionarily conserved multi-subunit complex that is generally required for Pol II transcription [START_REF] Allen | The Mediator complex: a central integrator of transcription[END_REF]. As mentioned above, the Mediator is generally required for transcription. After its recruitment at enhancers (or UASs in yeast) by activator, the Mediator serves as a 'molecular bridge' between activators and the basal transcriptional machinery, integrating and transmitting regulatory signals from activators directly to Pol II and GTFs, and promoting PIC assembly at core promoters [START_REF] Kornberg | Mediator and the mechanism of transcriptional activation[END_REF][START_REF] Soutourina | Direct interaction of RNA polymerase II and mediator required for transcription in vivo[END_REF][START_REF] Soutourina | Transcription regulation by the Mediator complex[END_REF].

It
After PIC formation, the Mediator stimulates phosphorylation of Pol II CTD through regulating the enzymatic activity of TFIIH CDK7 [START_REF] Kim | A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II[END_REF][START_REF] Nair | Mediator and TFIIH govern carboxyl-terminal domain-dependent transcription in yeast extracts[END_REF][START_REF] Boeing | RNA polymerase II C-terminal heptarepeat domain Ser-7 phosphorylation is established in a mediator-dependent fashion[END_REF], thus triggering Pol II release from promoters. Moreover, the Mediator also appears to play a role in promoter-proximal pausing and / or pause release (Wang et al., 2005a;[START_REF] Kremer | Role of Mediator in regulating Pol II elongation and nucleosome displacement in Saccharomyces cerevisiae[END_REF][START_REF] Allen | The Mediator complex: a central integrator of transcription[END_REF], and even transcription re-initiation [START_REF] Yudkovsky | A transcription reinitiation intermediate that is stabilized by activator[END_REF]. In addition to its canonical roles in transcription, Mediator also interacts with the TREX2 complex to couple transcription with mRNA export [START_REF] Schneider | The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression[END_REF].

The SAGA general coactivator

The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex is an evolutionarily conserved, multi-functional and multi-subunit coactivator (Spedale et al., 2012). It contains 18-20 subunits, which are organized into 4 or 5 separate modules with distinct activities: an activator-binding module, a histone acetyltransferase (HAT) module, a histone deubiquitinase (DUB) module, a core structural module and a metazoan-specific splicing module [START_REF] Helmlinger | Sharing the SAGA[END_REF].

Interestingly, most subunits within different modules are not exclusive to SAGA, instead, they are shared with other regulatory complexes [START_REF] Helmlinger | Sharing the SAGA[END_REF] (Figure 2

-13).

As indicated in the modular names, SAGA plays multiple roles in regulating transcription, including but not limited to activator interaction, histone acetylation, histone deubiquitination and regulation of the basal transcription machinery (Spedale et al., 2012).

The activator-binding module contains TRRAP (Tra1 in yeast) is the largest component of SAGA (∼420 kDa), and is also present in the NuA4/TIP60 complex (see 2.2.1.5). TRRAP interacts with different transcription factors and may serve as a major target of promoter-bound activators, thus plays a crucial role in SAGA recruitment at gene-specific promoters [START_REF] Mcmahon | The novel ATM-related protein TRRAP is an essential cofactor for the c-Myc and E2F oncoproteins[END_REF][START_REF] Brown | Recruitment of HAT complexes by direct activator interactions with the ATM-related Tra1 subunit[END_REF][START_REF] Bhaumik | In vivo target of a transcriptional activator revealed by fluorescence resonance energy transfer[END_REF][START_REF] Helmlinger | Tra1 has specific regulatory roles, rather than global functions, within the SAGA co-activator complex[END_REF][START_REF] Weake | SAGA function in tissue-specific gene expression[END_REF]. It is important to mention that, in addition to TRRAP, SAGA can also be recruited potentially by interactions with chromatin marks, TAF12 interactions with activators and/or binding of Spt3 and Spt8 to TBP in yeast [START_REF] Weake | SAGA function in tissue-specific gene expression[END_REF].

After SAGA recruitment, the HAT module preferentially acetylates histone H3 [START_REF] Helmlinger | Sharing the SAGA[END_REF], and the DUB module deubiquitinates both histone (H2Bub and H2Aub) and non-histone substrates [START_REF] Weake | SAGA function in tissue-specific gene expression[END_REF]. From [START_REF] Helmlinger | Sharing the SAGA[END_REF].

Moreover, recent work showed that SAGA is required for essentially all RNA polymerase II transcription, acting as a general cofactor [START_REF] Baptista | SAGA Is a General Cofactor for RNA Polymerase II Transcription[END_REF].

NuA4/TIP60 complex

The mammalian NuA4/TIP60 complex is a conserved transcriptional coactivator with multiple subunits [START_REF] Jha | RVB1/RVB2: running rings around molecular biology[END_REF], among which, TIP60 is a histone acetyltransferase (HAT)

belonging to the MYST family of HATs (Sapountzi et al., 2011), and p400 is a ATPase belonging to the SWI2/SNF2 class of chromatin remodelers (Fuchs et al., 2001) and a activator-binding subunit TRRAP shared with SAGA (Figure 2345678910111213). Thus the NuA4/TIP60 complex accommodates three distinct enzymatic activities: a histone H2A/H4 acetyltransferase activity, an ATP-dependent histone (H2A/H2A.Z) exchange activity and a helicase activity (Ikura et al., 2000;[START_REF] Gevry | p21 transcription is regulated by differential localization of histone H2A[END_REF]Auger et al., 2008). After its recruitment by activators, NuA4/TIP60 complex acetylates histone H4, H2A and H2A.Z [START_REF] Allard | NuA4, an essential transcription adaptor/histone H4 acetyltransferase complex containing Esa1p and the ATM-related cofactor Tra1p[END_REF][START_REF] Keogh | The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4[END_REF][START_REF] Babiarz | Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomyces cerevisiae[END_REF] and facilitates histone (H2A/H2A.Z) exchange, therefore allowing chromatin opening and playing important roles in transcription regulation.

In addition, TIP60 not only acetylates histones, but also acetylates other cellular proteins involved in transcription, for instance, the Androgen Receptor [START_REF] Gaughan | Tip60 and histone deacetylase 1 regulate androgen receptor activity through changes to the acetylation status of the receptor[END_REF] and p53 [START_REF] Sykes | Acetylation of the p53 DNA-binding domain regulates apoptosis induction[END_REF][START_REF] Tang | Tip60-dependent acetylation of p53 modulates the decision between cell-cycle arrest and apoptosis[END_REF]. Besides, the NuA4/TIP60 complex also plays a key role in DNA repair [START_REF] Rossetto | Epigenetic modifications in double-strand break DNA damage signaling and repair[END_REF][START_REF] Jacquet | The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation[END_REF].

Pre-initiation complex assembly

After chromatin opening, GTFs and Pol II start to form PIC. According to the canonical sequential assembly model, PIC assembly starts with core promoter binding by TFIID, followed by the recruitment of TFIIA and TFIIB, which stabilize the TFIID-promoter complex. As mentioned above (2.1.3.1), TFIIA is not absolutely required for basal transcription, but it can stabilize the TFIID-DNA complex, and it can join the PIC at any step after TFIID binding. TFIIB subsequently anchors Pol II to the promoter with the associated TFIIF. TFIIF stabilizes the PIC, in particular TFIIB within the PIC, forming a stable TFIID-TFIIA-TFIIB-Pol II-TFIIF-promoter complex (core PIC). Recruitment of TFIIE and entry of TFIIH complete the PIC assembly, leading to the formation of a closed PIC [START_REF] Buratowski | Five intermediate complexes in transcription initiation by RNA polymerase II[END_REF][START_REF] Orphanides | The general transcription factors of RNA polymerase II[END_REF][START_REF] Cheung | A movie of RNA polymerase II transcription[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF][START_REF] Plaschka | Transcription initiation complex structures elucidate DNA opening[END_REF][START_REF] Schilbach | Structures of transcription pre-initiation complex with TFIIH and Mediator[END_REF] (Figure 2 From [START_REF] Koster | Genesis of Chromatin and Transcription Dynamics in the Origin of Species[END_REF].

It is important to mention that, besides the sequential assembly model, another model, the RNA Pol II holoenzyme pathway also has been proposed. This model came up after the observation that Pol II could be purified as a preassembled holoenzyme containing several GTFs (but no TFIID), Mediator and chromatin remodelers/modifiers. According to this model, TFIID binds to the core promoter and is stabilized by TFIIA, facilitating the recruitment of preassembled Pol II holoenzyme [START_REF] Koleske | An RNA polymerase II holoenzyme responsive to activators[END_REF][START_REF] Kim | A multiprotein mediator of transcriptional activation and its interaction with the C-terminal repeat domain of RNA polymerase II[END_REF][START_REF] Ossipow | A mammalian RNA polymerase II holoenzyme containing all components required for promoter-specific transcription initiation[END_REF][START_REF] Thomas | The general transcription machinery and general cofactors[END_REF].

Both models are supported by in vitro studies [START_REF] Thomas | The general transcription machinery and general cofactors[END_REF], however, there is no conclusive evidence which one is used in vivo. Given the fact that both pathways are not mutually exclusive, they could co-exist or occur in an integrated middle way. Adapted from [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF].

Transcription initiation and Pol II promoter escape

In the presence of ATP, the promoter DNA (around TSS) inside the closed PIC is unwound by the PIC, especially TFIIH with the help of TFIIB and TFIIE (model for DNA opening see is regulated by TFIIE [START_REF] Ohkuma | Regulation of TFIIH ATPase and kinase activities by TFIIE during active initiation complex formation[END_REF] and Mediator [START_REF] Allen | The Mediator complex: a central integrator of transcription[END_REF]. Transcription initiation, Pol II promoter escape and promoter-proximal pausing are coupled continuous processes.

Promoter-proximal pausing

In metazoans, at many genes, after promoter escape, Pol II transcribes only a short stretch of nascent RNA (~30-50 nucleotides) and then pauses at downstream of the TSS, undergoing promoter-proximal pausing [START_REF] Zeitlinger | RNA polymerase stalling at developmental control genes in the Drosophila melanogaster embryo[END_REF][START_REF] Muse | RNA polymerase is poised for activation across the genome[END_REF][START_REF] Guenther | A chromatin landmark and transcription initiation at most promoters in human cells[END_REF][START_REF] Core | Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters[END_REF]Adelman et al., 2012;[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] Pol II promoter-proximal pausing is mediated by the binding of the negative elongation factor (NELF) and the DRB sensitivity-inducing factor (DSIF, a heterodimer of STP4 and SPT5) (Adelman et al., 2012). It has been suggested that the core promoter features and barrier effect of +1 nucleosome play important roles in triggering Pol II pausing (Kwak et al., 2013a;Li et al., 2013a;[START_REF] Weber | Nucleosomes are context-specific, H2A.Z-modulated barriers to RNA polymerase[END_REF][START_REF] Jonkers | Getting up to speed with transcription elongation by RNA polymerase II[END_REF].

However, interestingly, NELF and DSIF require a nascent transcript longer than 18 nucleotides to stably associate with the Pol II elongation complex [START_REF] Missra | Interactions between DSIF (DRB sensitivity inducing factor), NELF (negative elongation factor), and the Drosophila RNA polymerase II transcription elongation complex[END_REF][START_REF] Yamaguchi | Transcription elongation factors DSIF and NELF: promoter-proximal pausing and beyond[END_REF], and both NELF and DSIF can contact nascent RNA exiting from Pol II [START_REF] Bernecky | Structure of a transcribing RNA polymerase II-DSIF complex reveals a multidentate DNA-RNA clamp[END_REF][START_REF] Qiu | Identification of Regions in the Spt5 Subunit of DRB Sensitivity-inducing Factor (DSIF) That Are Involved in Promoter-proximal Pausing[END_REF][START_REF] Ehara | Structure of the complete elongation complex of RNA polymerase II with basal factors[END_REF]Vos et al., 2018a;[START_REF] Vos | Structure of paused transcription complex Pol II-DSIF-NELF[END_REF], together suggesting that NELF and DSIF interaction with nascent RNA plays a role in establishing Pol II pausing, and that Pol II pausing might be triggered independently of promoter sequence and chromatin properties [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF].

Different hypotheses for the functions of Pol II pausing have been proposed, including establishing permissive chromatin, rapid / synchronous gene activation, integrating multiple regulatory signals and acting as a checkpoint for coupling elongation and RNA processing (Adelman et al., 2012).

Escape from the pausing (pause-release)

Release of paused Pol II is mediated by the positive transcription elongation factor b (P-TEFb), which is composed cyclin-dependent kinase 9 (CDK9) and cyclin T [START_REF] Peterlin | Controlling the elongation phase of transcription with P-TEFb[END_REF][START_REF] Zhou | RNA polymerase II elongation control[END_REF]. P-TEFb can be recruited directly by activators like NF-κB and MYC, or indirectly via coactivator such as BRD4 [START_REF] Jonkers | Getting up to speed with transcription elongation by RNA polymerase II[END_REF]. After its recruitment, P-TEFb phosphorylates NELF, DSIF and Ser2 of the Pol II CTD (Kwak et al., 2013b)

Productive elongation

After escaping from the promoter-proximal pausing, Pol II enters into productive elongation. During this process, the dynamic nucleosome turnover that allows Pol II to move forward while prevents cryptic intragenic transcription, is mainly driven by chromatin remodelers and histone chaperones [START_REF] Venkatesh | Histone exchange, chromatin structure and the regulation of transcription[END_REF][START_REF] Talbert | Histone variants on the move: substrates for chromatin dynamics[END_REF][START_REF] Lai | Understanding nucleosome dynamics and their links to gene expression and DNA replication[END_REF]. For instance, FACT travels with Pol II during elongation and mediates eviction of Moreover, transition from Pol II pausing to productive elongation is not an ON-OFF switch.

The Pol II elongation rate (kb/min) varies between genes, and also varies between different parts of the gene. In mammals, productive elongation is not very efficient within the first kilobase, and it increases from ∼0.5 kb/min within the first few kilobases to 2-5 kb/min after ∼15 kb [START_REF] Jonkers | Getting up to speed with transcription elongation by RNA polymerase II[END_REF]. In addition, Pol II can be slowdown by mRNA cleavage and presence of exons, as well as polyadenylation sites [START_REF] Jonkers | Getting up to speed with transcription elongation by RNA polymerase II[END_REF].

Transcription termination

Transcription termination is necessary to partition the genome by defining the boundaries of transcription units [START_REF] Porrua | Transcription termination and the control of the transcriptome: why, where and how to stop[END_REF].

In metazoans, transition from productive elongation to termination is triggered by cleavage and polyadenylation specificity factor (CPSF), cleavage stimulatory factor (CSTF), cleavage factor I (CFI) and CFII, which are all bound to Pol II. Notably, CSTF and CFI-CFII bind to phosphorylated Ser2 of CTD [START_REF] Kuehner | Unravelling the means to an end: RNA polymerase II transcription termination[END_REF][START_REF] Porrua | Transcription termination and the control of the transcriptome: why, where and how to stop[END_REF]. CPSF and CSTF recognize the 3' UTR of the nascent RNA, cleavage of which is mediated by CPSF at 18-30 nucleotides downstream of the polyadenylation signal 'AAUAAA'. Released nascent RNA is polyadenylated at the 3'end and subsequently exported to the cytoplasm [START_REF] Porrua | Transcription termination and the control of the transcriptome: why, where and how to stop[END_REF] (Figure 2

-20 A&B).

Two alternative models have been proposed for Pol II expulsion after transcript cleavage, namely, the torpedo model [START_REF] West | Human 5' --> 3' exonuclease Xrn2 promotes transcription termination at co-transcriptional cleavage sites[END_REF]) and the allosteric model [START_REF] Zhang | Pcf11 is a termination factor in Drosophila that dismantles the elongation complex by bridging the CTD of RNA polymerase II to the nascent transcript[END_REF].

According [START_REF] Porrua | Transcription termination and the control of the transcriptome: why, where and how to stop[END_REF].

Transcription termination occurs when the when Pol II is released from chromatin. Free

Introduction

Transcription by RNA Polymerase II

51

Pol II can be recycled and reinitiate to start a new round of transcription.

Transcription re-initiation

It has been reported that after transcription initiation, a subset of the transcription machinery remains at the promoter, forming a platform for the assembly of a second PIC [START_REF] Roberts | A role for activator-mediated TFIIB recruitment in diverse aspects of transcriptional regulation[END_REF][START_REF] Zawel | Recycling of the general transcription factors during RNA polymerase II transcription[END_REF][START_REF] Sandaltzopoulos | Heat shock factor increases the reinitiation rate from potentiated chromatin templates[END_REF][START_REF] Hahn | Activation and the role of reinitiation in the control of transcription by RNA polymerase II[END_REF].

Notably, after Pol II promoter escape, some GTFs (including TFIID, TFIIA, TFIIH and TFIIE) and Mediator can remain associated with the promoter as a re-initiation intermediate, which can be stabilized by activator, act as a scaffold for re-initiation complex formation and allow Pol II to initiate efficiently in successive rounds of transcription [START_REF] Yudkovsky | A transcription reinitiation intermediate that is stabilized by activator[END_REF].

Moreover, a recent study showed that transcription induced downstream promoter binding of TAFs promotes subsequent activator-independent transcription re-initiation, suggesting that TAFs could function as re-initiation factors [START_REF] Joo | Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation[END_REF]. It was proposed that after the pioneer round of PIC assembly and transcription initiation, TAFs stably interact with downstream promoter DNA and the acetylated +1 nucleosome, acting as a re-initiation intermediate, which could further stimulate re-initiation, or facilitate the incorporation of H2A

and may act as a memory of recent transcription to allow rapid re-initiation, or return to the inactive state [START_REF] Joo | Downstream promoter interactions of TFIID TAFs facilitate transcription reinitiation[END_REF].

Core promoter

Core promoter, the gateway to transcription (Vo Ngoc et al., 2017b), is defined as a short sequence flanking the TSS that is sufficient to assemble the RNA polymerase II transcription machinery and to initiate transcription. It encompasses ∼50 bp upstream and ∼50 bp downstream of the TSS [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF].

Extensive works from many groups have characterized different features of core promoters, including the sequence motifs, TSS patterns and chromatin properties [reviewed in [START_REF] Muller | New problems in RNA polymerase II transcription initiation: matching the diversity of core promoters with a variety of promoter recognition factors[END_REF][START_REF] Juven-Gershon | Regulation of gene expression via the core promoter and the basal transcriptional machinery[END_REF][START_REF] Lenhard | Metazoan promoters: emerging characteristics and insights into transcriptional regulation[END_REF][START_REF] Muller | Chromatin and DNA sequences in defining promoters for transcription initiation[END_REF][START_REF] Roy | Core promoters in transcription: old problem, new insights[END_REF]Vo Ngoc et al., 2017b;[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF]]. The TATA box was the first identified core promoter motif in eukaryotes [START_REF] Goldberg | Sequence analysis of Drosophila histone genes[END_REF], and is located ∼30 bp upstream of TSS and serves as the conserved binding site for the TBP [START_REF] Patikoglou | TATA element recognition by the TATA box-binding protein has been conserved throughout evolution[END_REF]. However, only a minority of core promoters contain a TATA box, for instance, ∼5% of core promoters in flies and ∼10% in humans [START_REF] Ohler | Computational analysis of core promoters in the Drosophila genome[END_REF][START_REF] Fitzgerald | Comparative genomics of Drosophila and human core promoters[END_REF][START_REF] Yang | Prevalence of the initiator over the TATA box in human and yeast genes and identification of DNA motifs enriched in human TATA-less core promoters[END_REF]. Among the TATA-less promoters, many contain the initiator (Inr) motif, although it is also present often in TATA-containing promoters [START_REF] Roy | Core promoters in transcription: old problem, new insights[END_REF].

The Inr motif spans the TSS and can be recognized by TAF1 and TAF2 [START_REF] Chalkley | DNA binding site selection by RNA polymerase II TAFs: a TAF(II)250-TAF(II)150 complex recognizes the initiator[END_REF][START_REF] Louder | Structure of promoter-bound TFIID and model of human pre-initiation complex assembly[END_REF]. It is more abundant than the TATA box but still not universal. In TATA-less promoters, the Inr motif is often accompanied by the downstream promoter element (DPE).

DPE was initially discovered in Drosophila, it is located downstream of TSS and functions cooperatively with the Inr for TFIID binding and transcriptional activity [START_REF] Burke | Drosophila TFIID binds to a conserved downstream basal promoter element that is present in many TATA-box-deficient promoters[END_REF].

DPE commonly exists in flies (∼30% of core promoters) but appears to be rare in humans [START_REF] Burke | The downstream core promoter element, DPE, is conserved from Drosophila to humans and is recognized by TAFII60 of Drosophila[END_REF][START_REF] Kutach | The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters[END_REF]. Moreover, DPE rarely co-exists with TATA box in flies, therefore they were suggested to be associated with genes of different functional categories [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF].

The motif ten element (MTE) was identified as an overrepresented sequence in Drosophila core promoters [START_REF] Ohler | Computational analysis of core promoters in the Drosophila genome[END_REF], and it was found to be a TFIID binding motif [START_REF] Lim | The MTE, a new core promoter element for transcription by RNA polymerase II[END_REF][START_REF] Theisen | Three key subregions contribute to the function of the downstream RNA polymerase II core promoter[END_REF]. The downstream core element (DCE) comprising three sub-elements, is another core promoter motif that can be recognized by TFIID [START_REF] Lewis | A downstream element in the human beta-globin promoter: evidence of extended sequence-specific transcription factor IID contacts[END_REF][START_REF] Lee | Functional characterization of core promoter elements: the downstream core element is recognized by TAF1[END_REF].

The TFIIB recognition element (BRE) was initially identified as a TFIIB binding site that is located immediately upstream of TATA box [START_REF] Lagrange | New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB[END_REF]. Later on, a second BRE located downstream of TATA box was found [START_REF] Deng | A core promoter element downstream of the TATA box that is recognized by TFIIB[END_REF], thus they were renamed as BRE u and BRE d , respectively. During PIC assembly, TFIIB binds to TFIID-DNA complex forming a ternary complex, in which TFIIB interacts with TBP as well as DNA in both flanks of TATA box [START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF], namely, BRE u and BRE d .

The TCT motif is also known as the polypyrimidine initiator, which is a rare motif that was estimated can be found in only ∼1% of human core promoters (Vo Ngoc et al., 2017a).

However, interestingly, the TCT motif is present in the core promoters of nearly all ribosomal protein genes, as well as some translation initiation and elongation factor coding genes in both flies and humans [START_REF] Hariharan | Functional dissection of a mouse ribosomal protein promoter: significance of the polypyrimidine initiator and an element in the TATA-box region[END_REF][START_REF] Perry | The architecture of mammalian ribosomal protein promoters[END_REF][START_REF] Roepcke | Identification of highly specific localized sequence motifs in human ribosomal protein gene promoters[END_REF][START_REF] Parry | The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery[END_REF].

Moreover, in vitro foot-printing experiments revealed that TCT motif is not recognized by the canonical TFIID [START_REF] Parry | The TCT motif, a key component of an RNA polymerase II transcription system for the translational machinery[END_REF]. It seems that TCT motif-containing promoters preferentially employ a specialized PIC without canonical TFIID to transcribe translation related genes [START_REF] Muller | Chromatin and DNA sequences in defining promoters for transcription initiation[END_REF]. Specifically, in Drosophila, TCT-dependent transcription involves the use of TRF2/TBPL1 instead of the canonical TBP (Wang et al., 2014) (discussed in more detail in section 3.

2.2.4).

In addition to the core promoter motifs discussed above, some other motifs also have been discovered (see Table 2345). These motifs, together with other promoter features, can be recognized and bound by GTFs, thus potentially mediating PIC recruitment and assembly.

It is important to mention that, the sequence motifs described above are mostly studied in 'focused' promoters (see section 2.3.1.2) with single dominant TSS, and there are no universal core promoter motifs. Indeed, although various core promoter motifs have been identified, many core promoters do not contain any of these known motifs, probably these promoters possess other features or there are other core promoter motifs remain to be discovered. at the 5ʹ end of a gene. However, transcription initiation sites mapping at single nucleotide resolution revealed that core promoters have distinct TSS patterns [START_REF] Carninci | Genome-wide analysis of mammalian promoter architecture and evolution[END_REF], according to which core promoters have been classified into three types: 'focused', 'dispersed' and 'mixed' core promoters [START_REF] Carninci | Genome-wide analysis of mammalian promoter architecture and evolution[END_REF][START_REF] Juven-Gershon | Regulation of gene expression via the core promoter and the basal transcriptional machinery[END_REF]Vo Ngoc et al., 2017b). Specifically, 'focused' core promoters have a single well-defined TSS or a narrow H3K4me3 and facilitates PIC assembly [START_REF] Vermeulen | Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4[END_REF][START_REF] Van Ingen | Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3[END_REF][START_REF] Lauberth | H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation[END_REF]. Although it has been reported that, in yeast, H3K4me3 deposition is mediated by SET1 that recruited by Pol II and thus occurs downstream of transcription [START_REF] Ng | Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity[END_REF]. However, in mammals, it is known that zinc finger CXXC proteins such as CFP1, MLL and KDM proteins specifically recognize non-methylated CpG islands (CGIs) and regulate lysine methylation on histone tails [START_REF] Long | ZF-CxxC domain-containing proteins, CpG islands and the chromatin connection[END_REF]. CFP1 and MLL proteins can deposit H3K4me3, and as a result, CGIs at promoters tend to be marked by H3K4me3 independently of gene activity (Mikkelsen et al., 2007;[START_REF] Thomson | CpG islands influence chromatin structure via the CpG-binding protein Cfp1[END_REF][START_REF] Muller | Chromatin and DNA sequences in defining promoters for transcription initiation[END_REF]. Besides, TAF1 binds to multiply acetylated histone H4 peptides [START_REF] Jacobson | Structure and function of a human TAFII250 double bromodomain module[END_REF], thus H4ac on promoters may serve as a target for TFIID recruitment as well. The role of promoter associated Secondly, in vertebrates, many core promoters overlap with CGIs, but the mechanisms by which CGIs confer core promoter function are still unknown [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF]. Although non-methylated CGIs can nucleate the deposition of H3K4me3, there is no evidence that they can nucleate the recruitment of transcriptional machinery to a promoter, instead, 5mC is a stable repressive regulator of promoter activity. However in plants, it has been shown that Pol V (a variant of Pol II) can be recruited to promoters containing methylated DNA via factors that bind to both polymerase and methylated DNA [START_REF] Johnson | SRA-and SET-domain-containing proteins link RNA polymerase V occupancy to DNA methylation[END_REF][START_REF] Liu | The SET domain proteins SUVH2 and SUVH9 are required for Pol V occupancy at RNA-directed DNA methylation loci[END_REF]. It is possible that similar mechanisms also exist in vertebrates. Indeed, in vitro studies have uncovered that many TFs bind the methylated motifs [START_REF] Hu | DNA methylation presents distinct binding sites for human transcription factors[END_REF][START_REF] Zhu | Transcription factors as readers and effectors of DNA methylation[END_REF].

Moreover, many TFs, including some developmentally important proteins, even have higher affinity for CpG-methylated sequences [START_REF] Yin | Impact of cytosine methylation on DNA binding specificities of human transcription factors[END_REF]. Further studies in physiological contexts are needed. In addition, DNA hydroxy-methylation and 6mA are also the potential targets of the TFs (Vo Ngoc et al., 2017b).

Thirdly, another hallmark of active core promoters is the presence of nucleosome depleted regions (NDRs) [START_REF] Yuan | Genome-scale identification of nucleosome positions in S. cerevisiae[END_REF][START_REF] Mavrich | Nucleosome organization in the Drosophila genome[END_REF][START_REF] Jiang | Nucleosome positioning and gene regulation: advances through genomics[END_REF]. Interestingly, it has been shown that those NDRs can be occupied by prenucleosome (nucleosome isomer associated with ∼80bp of DNA) [START_REF] Fei | The prenucleosome, a stable conformational isomer of the nucleosome[END_REF][START_REF] Khuong | Prenucleosomes and Active Chromatin[END_REF], prenucleosome-like particles (histone-containing particles associated with ∼61 to 100bp of DNA) [START_REF] Rhee | Subnucleosomal structures and nucleosome asymmetry across a genome[END_REF][START_REF] Ishii | MPE-seq, a new method for the genome-wide analysis of chromatin structure[END_REF], or H3.3-and H2A.Z-containing nucleosomes [START_REF] Jin | H3.3/H2A.Z double variant-containing nucleosomes mark 'nucleosome-free regions' of active promoters and other regulatory regions[END_REF]. These features are proposed to ensure chromatin accessibility to facilitate transcription, however, it is not clear whether they participate directly in the recruitment of the transcriptional machinery, for instance, by being the TF targets. Given that H3.3 constitutes ∼10% of total histone H3 and H2A.Z constitutes ∼1%-3% of total H2A, it has been suggested that presence of H3.3 and H2A.Z at the promoters could provide some specificity to core promoter function [START_REF] Dang | Label-Free Relative Quantitation of Isobaric and Isomeric Human Histone H2A and H2B Variants by Fourier Transform Ion Cyclotron Resonance Top-Down MS/MS[END_REF]Vo Ngoc et al., 2017b). Pol II from their own reverse-directed core promoters [START_REF] Duttke | Human promoters are intrinsically directional[END_REF][START_REF] Scruggs | Bidirectional Transcription Arises from Two Distinct Hubs of Transcription Factor Binding and Active Chromatin[END_REF] .

Enhancer

Enhancers are distal regulatory DNA sequences that contain activator binding sites and can increase the transcription level of target genes independently of distance and orientation [START_REF] Benoist | In vivo sequence requirements of the SV40 early promotor region[END_REF][START_REF] Banerji | Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences[END_REF][START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] Based on these features, large number of putative enhancers (>400,000 to ∼1 million) have been annotated in the human genome. Active enhancer can turn to poised state, which is characterized by the presence of both H3K4me1 and H3K27me3. In addition, latent enhancers also exist: they are located in closed chromatin and are not labelled by any enhancer-associated marks, but they acquire H3K4me1 and H3K27ac marks upon stimulation [START_REF] Spitz | Transcription factors: from enhancer binding to developmental control[END_REF][START_REF] Calo | Modification of enhancer chromatin: what, how, and why?[END_REF][START_REF] Rivera | Mapping human epigenomes[END_REF][START_REF] Shlyueva | Transcriptional enhancers: from properties to genome-wide predictions[END_REF][START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF][START_REF] Li | Enhancers as non-coding RNA transcription units: recent insights and future perspectives[END_REF].

Enhancer-promoter communication

As mentioned above (section 2.3.2), enhancers increase the transcription level of target genes independently of distance. The distance between enhancers and promoters can range from a few kilobases to over 1000 kb in metazoans [START_REF] Noonan | Genomics of long-range regulatory elements[END_REF]. How can enhancer convey regulatory information across such distance? Several models for enhancer-promoter communication have been proposed based on studies of specific loci and genome-wide analysis [START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF][START_REF] Furlong | Developmental enhancers and chromosome topology[END_REF] (Figure 2-29).

The tracking model (Figure 2-29 A) proposes that enhancer-bound Pol II / transcription complexes move progressively along the DNA, dragging the enhancer towards the target promoter, thus resulting in the formation of a loop, and size of the loop increases progressively until Pol II reaches the promoter [START_REF] Kong | Transcription of the HS2 enhancer toward a cis-linked gene is independent of the orientation, position, and distance of the enhancer relative to the gene[END_REF][START_REF] Blackwood | Going the distance: a current view of enhancer action[END_REF][START_REF] Hatzis | Dynamics of enhancer-promoter communication during differentiation-induced gene activation[END_REF]Wang et al., 2005b;[START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF]. The linking model (Figure 2-29 B) suggests that an activator binds to enhancer first and facilitates the recruitment of other TFs, forming protein-protein oligomers to bridge the enhancer and the target promoter [START_REF] Bulger | Looping versus linking: toward a model for long-distance gene activation[END_REF][START_REF] Dorsett | Distant liaisons: long-range enhancer-promoter interactions in Drosophila[END_REF][START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF][START_REF] Furlong | Developmental enhancers and chromosome topology[END_REF]. The looping model (Figure 2

Diversity of basal transcription machinery

TBP and TAFs are important for embryogenesis. For instance, in mice, knockout of Tbp (Martianov et al., 2002b),Taf7 (Gegonne et al., 2012), Taf8 [START_REF] Voss | Taube nuss is a novel gene essential for the survival of pluripotent cells of early mouse embryos[END_REF] and Taf10 (Mohan et al., 2003) lead to embryonic lethality between E3.5 and E6.5. Taf4 knockout embryos survive until E9.5 because of the compensation of the paralog TAF4b, and from E9.5

TAF4b cannot fully compensate the loss of TAF4 anymore, especially for the transcription of critical differentiation genes [START_REF] Langer | Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation[END_REF]. No knockout of other prototypical TAFs has been reported. However, it has been shown that TAFs are differentially required during development [START_REF] Metzger | Mammalian TAF(II)30 is required for cell cycle progression and specific cellular differentiation programmes[END_REF]Indra et al., 2005;[START_REF] Fadloun | Retinoic acid induces TGFbeta-dependent autocrine fibroblast growth[END_REF][START_REF] Liu | Control of embryonic stem cell lineage commitment by core promoter factor[END_REF]Bardot et al., 2017;[START_REF] El-Saafin | Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription[END_REF], which provide evidences for the heterogeneity of TFIID. Moreover, emerging evidences show that, apart from epigenetic regulations and gene-specific transcription factors, variation in the composition of basal transcription machinery, especially the heterogeneity and specialized functions of TFIID with different TAFs and TAF paralogs, as well as specialized role of TBP-related factors, plays vital roles in driving cell-type-specific and gene-specific transcriptions [START_REF] D'alessio | Shifting players and paradigms in cell-specific transcription[END_REF][START_REF] Muller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF]Goodrich et al., 2010;[START_REF] Levine | Looping back to leap forward: transcription enters a new era[END_REF]. It is important to mention that, many evidences for diversity of the basal transcription machinery come from germ cells. In this chapter, I will mainly focus on the studies from mice, and if necessary, in comparation with studies in Xenopus, zebrafish, Drosophila and C. elegans. TAF10 is a ubiquitously expressed TFIID subunit, and knockout of Taf10 leads to embryonic lethality between E3.5 and E6.5 (Mohan et al., 2003). Many evidences show requirement of TAF10 varies in different contexts.

Heterogeneity and specialized functions of TFIID

First of all, TAF10 disruption leads to early embryonic lethality shortly after implantation, while inner cell mass cells die by apoptosis, trophoblastic cells survive. Although with reduced DNA replication and transcription, these trophoblastic cells can survive in culture for at least 12 days (Mohan et al., 2003). Secondly, TAF10 is essential for the survival and proliferation of F9 carcinoma cells, however, interestingly, it is dispensable for retinoic acid (RA)-induced primitive endodermal differentiation [START_REF] Metzger | Mammalian TAF(II)30 is required for cell cycle progression and specific cellular differentiation programmes[END_REF]. Thirdly, Taf10 conditional deletion in keratinocytes has shown that ablation of Taf10 in foetal keratinocytes impairs their terminal differentiation and alters skin barrier function. However, TAF10 is dispensable for epidermal keratinocyte proliferation and differentiation in adult mice (Indra et al., 2005). Fourthly, conditional knockout of Taf10 in foetal and adult hepatocytes also has shown different requirement of TAF10 for transcription in embryo and adult (Tatarakis et al., 2008). Moreover, recently, it has been shown that absence of TAF10 does not affect global steady-state mRNA and the dynamic transcription initiation of cyclic genes in presomitic mesoderm (PSM) at ∼E9.5 (Bardot et al., 2017).

In addition, in chicken DT40 cells, loss of TAF10 eventually leads to apoptotic cell death, however, activation of c-fos transcription can still occur efficiently in absence of TAF10 [START_REF] Chen | Robust mRNA transcription in chicken DT40 cells depleted of TAF(II)31 suggests both functional degeneracy and evolutionary divergence[END_REF]. In C. elegans, TAF10 is needed for a significant fraction of transcription, but apparently is not required for the expression of many metazoan-specific genes [START_REF] Walker | Distinct requirements for C.elegans TAF(II)s in early embryonic transcription[END_REF]. Altogether, these studies indicate that TAF10 is differentially required depending on the cellular and developmental context. frameshift mutation, global Pol II transcription is not impaired, although canonical TFIID assembly is altered as unstable TAF8 mutant protein is undetectable [START_REF] El-Saafin | Homozygous TAF8 mutation in a patient with intellectual disability results in undetectable TAF8 protein, but preserved RNA polymerase II transcription[END_REF]. In addition, it also has been reported that, TAF8 is not detected in preadipocytes, but is dramatically upregulated during adipogenesis [START_REF] Guermah | The TBN protein, which is essential for early embryonic mouse development, is an inducible TAFII implicated in adipogenesis[END_REF]. These observations suggest that partial TFIID without TAF8 or altered TFIID with mutated TAF8 are sufficient for

Pol II transcription, at least in certain context.

3.1.1.3 TAF7 is not essential for mature T cell survival or differentiation Homozygous Taf7 deletion results in early embryonic lethality between E3.5 and E5.5, and ablation of Taf7 in embryonic fibroblasts ceases global transcription. However, functional analysis of TAF7 in thymocytes shows that, while TAF7 is required during the early steps in

thymic development [double-negative (CD4 - CD8 -) to double-positive (CD4 + CD8 + ) transition]
for the differentiation and proliferation of immature thymocytes, it is not essential for the survival or differentiation of mature T cells (Gegonne et al., 2012).

TAF4 is differentially required in T-RA-induced gene activation

Characterization of the role of TAF4 in the activation of cellular genes by all-trans retinoic acid (T-RA) revealed that T-RA regulates ∼1000 genes in Taf4 lox/-MEFs and less than 300 genes in Taf4 -/-MEFs, indicating the existence of TAF4 dependent and independent mechanisms for T-RA induced transcription activation [START_REF] Fadloun | Retinoic acid induces TGFbeta-dependent autocrine fibroblast growth[END_REF]. The TAF4 independent activation could due to compensation of TAF4 by its paralog TAF4b or TAF4-independent transcription initiation. Moreover, it has been shown that Taf4 -/-ESCs are viable due to the compensation by TAF4b, and the observation that Taf4 is dispensable for primordial germ cell generation may also due to the same mechanism [START_REF] Langer | Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation[END_REF].

It also has been shown that TAF4 (also TBP and some other TAFs) protein level is dramatically decreased in adult hepatocytes compared with E13.5 hepatoblasts, suggesting the a change in core promoter recognition complex in hepatocytes [START_REF] D'alessio | Core promoter recognition complex changes accompany liver development[END_REF].

Later studies confirmed the reduced expression of TAF4 in adult hepatocytes, but not TBP, and showed that TAF4 is required for post-natal hepatocyte differentiation [START_REF] Alpern | TAF4, a subunit of transcription factor II D, directs promoter occupancy of nuclear receptor HNF4A during post-natal hepatocyte differentiation[END_REF].

TAF3 is differentially required during ESCs lineage commitment

In mice, TAF3 is highly expressed in embryonic stem cells (ESCs), interestingly, TAF3 expression (protein level) is reduced progressively during the formation of embryoid bodies (EBs) while expression of other TFIID subunits (TAF4 and TBP) remain mostly unchanged [START_REF] Liu | Control of embryonic stem cell lineage commitment by core promoter factor[END_REF]. Functional analysis showed that TAF3 is required for endoderm lineage differentiation, interacting with CTCF and mediating DNA looping between distal enhancer sites and core promoters of endoderm specification genes, while it prevents premature specification of neuroectoderm and mesoderm by repressing neuroectodermal genes [START_REF] Liu | Control of embryonic stem cell lineage commitment by core promoter factor[END_REF].

Altogether, these observations show that the composition of TFIID can be variable depending on the cellular and developmental context.

Role of TAF paralogs during development

Besides differential requirement of TAFs, the heterogeneity of TFIID also arises from the TAF paralogs. In vertebrates, apart from the 13 prototypical TAFs, several TAF paralogs have been identified, including TAF1L, TAF4b, TAF5L, TAF6L, TAF7L and TAF9b [reviewed in [START_REF] Kolthur-Seetharam | Specialization of the general transcriptional machinery in male germ cells[END_REF][START_REF] Muller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF]Goodrich et al., 2010)].

TAF1L, a retroposed copy of TAF1, has been identified as a human specific TAF1 paralog (also present in some but not all primates) and is specifically expressed in human male germ cells [START_REF] Wang | Functional substitution for TAF(II)250 by a retroposed homolog that is expressed in human spermatogenesis[END_REF]. TAF5L and TAF6L are SAGA specific TAF paralogs [START_REF] Helmlinger | Sharing the SAGA[END_REF]. Thus in the following, I mainly focus on TAF4b, TAF7L and TAF9b.

Specialized roles of TAF4b in germ cell differentiation

TAF4b was initially identified as a cell-type-specific TAF in human differentiated B-cells [START_REF] Dikstein | Human TAFII 105 is a cell type-specific TFIID subunit related to hTAFII130[END_REF]. However, it was later found to be expressed in many tissues in mice and highly enriched in the testis and the ovary (Figure 3-2 a & b) [START_REF] Freiman | Requirement of tissue-selective TBP-associated factor TAFII105 in ovarian development[END_REF].

In mouse testis, the TAF4b protein is present in post-natal gonocytes, spermatogonia and spermatids (Falender et al., 2005a). Taf4b-null males are initially fertile, but progressively become infertile (by around 3 months), as testes degenerate with age resulting in seminiferous tubules devoid of germ cells (Falender et al., 2005a). Furthermore, in Taf4b-deficient males, In embryonic and early neonatal mouse ovary [from 14.5 d.p.c (day post coitum) up to postnatal day (P) 2], TAF4b protein selectively expressed in the oocytes but not somatic cells (Falender et al., 2005b). In adult mouse ovary, Taf4b mRNA is restricted to the granulosa cells [START_REF] Freiman | Requirement of tissue-selective TBP-associated factor TAFII105 in ovarian development[END_REF], and required for granulosa cell survival and proliferation [START_REF] Voronina | Ovarian granulosa cell survival and proliferation requires the gonad-selective TFIID subunit TAF4b[END_REF]. Female mice lacking Taf4b are viable but infertile, developmental defects was observed as early as P3 when the number of oocytes is significantly reduced (Falender et al., 2005b).

Females mice are with smaller ovaries that undergo progressive follicle loss and lack mature follicles, and many ovarian-specific genes are downregulated [START_REF] Freiman | Requirement of tissue-selective TBP-associated factor TAFII105 in ovarian development[END_REF][START_REF] Lovasco | Accelerated ovarian aging in the absence of the transcription regulator TAF4B in mice[END_REF]. It was found later that Taf4b null females are able to ovulate, although many of the ovulated oocytes display defects in spindle formation and/or extrusion of polar body, some oocytes achieve metaphase II of meiosis (Falender et al., 2005b). However, few oocytes that are fertilized and embryos cannot proceed beyond the 1-cell stage (Falender et al., 2005b).

Moreover, TAF4b controls granulosa-cell-specific expression of c-Jun [START_REF] Geles | Cell-type-selective induction of c-jun by TAF4b directs ovarian-specific transcription networks[END_REF], and it has been suggested that TAF4b works with c-Jun to regulate the transcription of genes involved in follicle growth (Goodrich et al., 2010).

The fact that Taf4b -/-mice are viable and only show defects in germ cells, suggests that TAF4b can be fully replace by TAF4 during embryogenesis, while TAF4b can only partially compensate for TAF4 functions as TAF4-deficient embryo die at ∼E9.5 [START_REF] Langer | Essential role of the TFIID subunit TAF4 in murine embryogenesis and embryonic stem cell differentiation[END_REF]. TAF7L, the paralogue of TAF7, was initially discovered as a spermatogonial expressed gene located on the X chromosome (Wang et al., 2001b;[START_REF] Pointud | The intracellular localisation of TAF7L, a paralogue of transcription factor TFIID subunit TAF7, is developmentally regulated during male germ-cell differentiation[END_REF][START_REF] Cheng | Abnormal sperm in mice lacking the Taf7l gene[END_REF], but later also was found in different mouse cells and tissues, and is highly enriched in testis and adipocytes [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF][START_REF] Zhou | TAF7L modulates brown adipose tissue formation[END_REF].

In mouse testis, TAF7L protein is localized in the cytoplasm of spermatogonia and early primary spermatocytes, and is imported into the nucleus from mid-pachytene stage onwards, eventually accumulates strongly in the nucleus of post-meiotic round spermatids [START_REF] Pointud | The intracellular localisation of TAF7L, a paralogue of transcription factor TFIID subunit TAF7, is developmentally regulated during male germ-cell differentiation[END_REF] (Figure 3-2 a). Interestingly, import of TAF7L into the nucleus correlates with increased TBP expression and decreased TAF7 expression, and TAF7L is associated with TBP in pachytene spermatocytes and round spermatids, indicating it can replace TAF7 as a TFIID subunit in these stages [START_REF] Pointud | The intracellular localisation of TAF7L, a paralogue of transcription factor TFIID subunit TAF7, is developmentally regulated during male germ-cell differentiation[END_REF]. Knockout of the Taf7l gene leads to the development of deformed sperm [START_REF] Cheng | Abnormal sperm in mice lacking the Taf7l gene[END_REF]. The male mice are initially fertile with reduced litter size, however, after two to four additional rounds of back-crossings, the TAF7-deficient male mice become essentially sterile [START_REF] Cheng | Abnormal sperm in mice lacking the Taf7l gene[END_REF]Zhou et al., 2013a).

Importantly, TAF7 binds to the promoters of target genes in the testis and Taf7l ablation impairs the expression of many post-meiotic spermiogenic-specific as well as metabolic genes (Zhou et al., 2013a). Moreover, TAF7L forms a complex with TRF2 (for TRF2, see section 3.2.2), and they coregulate the expression of post-meiotic genes (Zhou et al., 2013a) (Figure 3-3 A). In adipocytes, TAF7L is required for adipocyte-specific gene expression [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF]. Depletion of TAF7L compromises adipocyte differentiation and white adipose tissue (WAT) development, while ectopic expression of TAF7L transdifferentiate C2C12 myoblast into adipocytes upon adipogenic induction [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF]. During adipogenesis, TAF7L binds to the promoters and/or promoter proximal regions of most adipocyte-specific genes, and interestingly, it co-localizes with PPARγ (a key adipogenic transcriptional activator) genome-widely [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF]. Moreover, TAF7L physically associates with PPARγ and TBP/TFIID, respectively [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF]. Altogether, these data suggest that TAF7L plays important roles during adipogenesis by serving as a cofactor for PPARγ and also a component of the core transcriptional machinery [START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF]) (Figure 3-3 B). Furthermore, TAF7L also modulates brown adipose tissue formation [START_REF] Zhou | TAF7L modulates brown adipose tissue formation[END_REF].

TAF9b

TAF9b, the paralog of TAF9, is present widely in many tested cell lines [START_REF] Frontini | TAF9b (formerly TAF9L) is a bona fide TAF that has unique and overlapping roles with TAF9[END_REF]. Human TAF9b can fully restore the function of chicken TAF9 in TAF9-deficient DT40 cells, suggesting partially redundant functions for these two proteins (Chen et al., 2003b). Like TAF9, TAF9b is also present in both TFIID and SAGA [START_REF] Frontini | TAF9b (formerly TAF9L) is a bona fide TAF that has unique and overlapping roles with TAF9[END_REF]. TAF9 and TAF9b are both essential for HeLa cell viability, however, transcriptome analysis revealed that they have minimal overlap of regulated genes, indicating they have distinct roles in gene transcription regulation [START_REF] Frontini | TAF9b (formerly TAF9L) is a bona fide TAF that has unique and overlapping roles with TAF9[END_REF]. Moreover, recent studies have shown that TAF9b is dispensable for global gene expression in mouse ES cells, and that TAF9b is up-regulated upon neuronal differentiation [START_REF] Herrera | Core promoter factor TAF9B regulates neuronal gene expression[END_REF]. TAF9b controls neuronal gene expression, thus it is required for efficient differentiation of ES cells to motor neurons, where it is preferentially associated with SAGA rather than the canonical TFIID complex [START_REF] Herrera | Core promoter factor TAF9B regulates neuronal gene expression[END_REF].

In addition, Drosophila homologues of TAF4 (No hitter), TAF5 (Cannonball), TAF6

(Meiosis I arrest), TAF8 (Spermatocyte arrest) and TAF12 (Ryan) are selectively expressed in primary spermatocytes and required for the regulation of testis-specific gene expression program during spermatogenesis [START_REF] Hiller | Developmental regulation of transcription by a tissue-specific TAF homolog[END_REF][START_REF] Hiller | Testis-specific TAF homologs collaborate to control a tissue-specific transcription program[END_REF].

Collectively, the differential requirement for TAFs and specialized functions of TAF paralogs suggest that, heterogeneous 'TFIIDs' with different combinations of subunits can participate in the gene-specific and tissue-specific transcriptional regulatory functions.

Specialized functions of TBP-related factors

Initially, TBP was thought to be a universal transcription factor, but later on, genomic and cDNA sequencing revealed that three TBP paralogs exist: the insect-specific TRF1 (also known as TRF), the metazoan-specific TRF2 (also known as TBPL1, TLP, TRP and TLF) and the vertebrate-specific TBP2 (also known as TBPL2 and TRF3) (Crowley et al., 1993;[START_REF] Berk | TBP-like factors come into focus[END_REF][START_REF] Davidson | The genetics of TBP and TBP-related factors[END_REF]Persengiev et al., 2003;[START_REF] Muller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF][START_REF] Akhtar | TBP-related factors: a paradigm of diversity in transcription initiation[END_REF]Vo Ngoc et al., 2017b).

TRF1, an insect-specific TBP-related factor

TRF1 was the first identified TBP-related factor (Crowley et al., 1993), and has been found only in insects. In Drosophila, TRF1 associates with BRF1 (an RNA Pol III transcription factor) to form the TFIIIB complex and mediates Pol III-dependent tRNA gene transcription instead of TBP [START_REF] Takada | A TRF1:BRF complex directs Drosophila RNA polymerase III transcription[END_REF]Isogai et al., 2007b;[START_REF] Verma | Differential utilization of TATA box-binding protein (TBP) and TBP-related factor 1 (TRF1) at different classes of RNA polymerase III promoters[END_REF]. Although TRF1 predominantly regulates Pol III transcription, it also has been reported that TRF1 can also bind to the TATA box and form a stable TRF1/TFIIA/TFIIB complex, substituting for TBP in Pol II transcription in vitro (Hansen et al., 1997;[START_REF] Holmes | Promoter-selective properties of the TBP-related factor TRF1[END_REF].

TRF2, a metazoan-specific TBP-related factor

TRF2 is the second identified TBP-related factor, and it has been given various names, such as TBP-like protein (TLP) (Ohbayashi et al., 1999a), TBP related factor 2 (TRF2) (Rabenstein et al., 1999;Teichmann et al., 1999), TBP-like factor (TLF) [START_REF] Dantonel | The TBP-like factor: an alternative transcription factor in metazoa?[END_REF], TBP-related protein (TRP) [START_REF] Moore | A human TATA binding protein-related protein with altered DNA binding specificity inhibits transcription from multiple promoters and activators[END_REF] and also TBPL1. As TRF2 is more commonly used in the literature, I will also use the term TRF2 (but not to be confused with telomere-repeat binding factors, which are also termed TRF1 and TRF2).

TRF2 is a metazoan-specific TBP-related factor that shares ∼40% identity with the TBP core domain and that interacts with TFIIA and TFIIB (Rabenstein et al., 1999;Teichmann et al., 1999). Unlike TBP and the other TRFs, TRF2 alone does not bind to the TATA box and does not appear to have any sequence-specific DNA-binding activity [START_REF] Dantonel | The TBP-like factor: an alternative transcription factor in metazoa?[END_REF]Rabenstein et al., 1999;Wang et al., 2014;Vo Ngoc et al., 2017b). However, TRF2 has many functions in different organisms.

TRF2 regulates male germ cell differentiation in mouse

In mammals, TRF2 is broadly expressed at low levels in different tissues but is highly enriched in testes (Ohbayashi et al., 1999b;Rabenstein et al., 1999;Teichmann et al., 1999;[START_REF] Moore | A human TATA binding protein-related protein with altered DNA binding specificity inhibits transcription from multiple promoters and activators[END_REF]Martianov et al., 2001).

In mice testis, dynamic expression of TRF2 is tightly controlled during spermatogenesis, TRF2 is highly expressed only in late-pachytene spermatocytes, late round spermatids and elongating spermatids, but not in other stages (Martianov et al., 2001;Zhang et al., 2001a;Martianov et al., 2002a) (Figure 34).

Trf2 -/-mice are viable, but mutant male mice are sterile due to a severe defect in spermiogenesis (Martianov et al., 2001;Zhang et al., 2001b). In the TRF2-deficient males, spermatogonia and spermatocytes develop normally, however, acrosome formation is impaired in early stage round spermatids, and spermiogenesis is arrested at the transition of round spermatids to elongating spermatids, and most round spermatids undergo apoptosis during the arrest (Martianov et al., 2001;Zhang et al., 2001b;Martianov et al., 2002a).

Importantly, transcription of multiple post-meiotic testes-specific genes is severely decreased, including the transition protein and protamine genes (Martianov et al., 2001;Zhang et al., 2001b). Further investigation showed that Trf2 -/-mutant round spermatids display a fragmentation of the chromocenter (Martianov et al., 2002a), a nuclear structure containing centromeric heterochromatin from each chromosome. However, it is not yet clear whether the defect of spermatogenesis in TRF2-deficient mice is mainly due to the impaired gene expression or due to the defect of chromocenter formation. ES, elongating spermatids; MS, mature spermatozoa. Adapted from (Martianov et al., 2002a).

Later on, it has been reported that TRF2 can activate a number of genes, and together with TFIIA, it even binds to the promoter of neurofibromatosis type 1 gene [START_REF] Chong | TATA-binding protein (TBP)-like factor (TLF) is a functional regulator of transcription: reciprocal regulation of the neurofibromatosis type 1 and c-fos genes by TLF/TRF2 and TBP[END_REF].

Moreover, TRF2 interacts with TAF7L in testis and they coregulated a subset of post-meiotic genes required for spermiogenesis (Zhou et al., 2013a). Furthermore, TRF2 associates with TFIIA and/or ALF, forming a stable complex chaperoned by heat shock proteins in the cytoplasm [START_REF] Catena | Proteolytic cleavage of ALF into alpha-and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells[END_REF][START_REF] Martianov | TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression[END_REF], and it is recruited to active spermatid gene promoters together with TBP, TAF7L and Pol II [START_REF] Martianov | TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression[END_REF]. Based on these observations, it has been proposed that TRF2 is recruited to the preinitiation complex as a testis-specific subunit of TFIIA/ALF that cooperates with TBP and TAF7L to regulate spermatid gene expression [START_REF] Martianov | TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression[END_REF]) (Figure 3

TRF2 functions in Xenopus

In Xenopus, TRF2 is highly expressed in ovary, testis and embryos, and knock down of Trf2 blocks embryos development past the mid-blastula stage, showing that TRF2 is essential for early embryogenesis and contributes to transcription in vivo [START_REF] Veenstra | Distinct roles for TBP and TBP-like factor in early embryonic gene transcription in Xenopus[END_REF]Xiao et al., 2006). Trf2 knockdown combined with transcriptome profiling in blastula stage embryos showed that a large number of transcripts require TRF2, and among these genes, a significant proportion is preferentially expressed in embryos (Jacobi et al., 2007). Moreover, TRF2 shows a functional specialization in catabolism (Jacobi et al., 2007).

TRF2 functions in zebrafish

In zebrafish, function of TRF2 has been investigated by inhibiting TRF2 through the expression of a dominant negative Trf2 mutant [START_REF] Muller | TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish[END_REF]. TRF2-blocked embryos develop normally until the mid-blastula stage, however, they fail to initiate epiboly or arrested before dome stage and eventually fail to gastrulate [START_REF] Muller | TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish[END_REF]. Interestingly, this phenotype is similar to inhibition of Pol II transcription by injecting α-amanitin, indeed, block of TRF2 abolishes the expression of many zygotic regulatory genes [START_REF] Muller | TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish[END_REF].

Versatile TRF2 in Drosophila

In Drosophila, two TRF2 protein isoforms of 632aa and 1715aa exist, and both isoforms are expressed in embryos and different tissues of adult flies [START_REF] Kopytova | Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes[END_REF]. Both isoforms can interact with ISWI [START_REF] Hochheimer | TRF2 associates with DREF and directs promoter-selective gene expression in Drosophila[END_REF][START_REF] Kopytova | Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes[END_REF]. Study of Trf2 mutation showed that TRF2 has essential functions during embryonic Drosophila development, and that the Trf2 function is essential for differentiation of both male and female germ cells [START_REF] Kopytova | Two isoforms of Drosophila TRF2 are involved in embryonic development, premeiotic chromatin condensation, and proper differentiation of germ cells of both sexes[END_REF]. Intensive studies has shown TRF2 is involved in many different transcriptional programs.

First of all, TRF2 associates with DNA replication-related element (DRE)-binding factor (DREF) and activates the transcription of target genes involved in DNA replication and cell proliferation through the binding of DREF to DRE motifs in promoters.

Secondly, TRF2 is required for transcription from TCT-dependent core promoters (Wang et al., 2014;Isogai et al., 2007a). Earlier ChIP-on-chip and RT-PCR analysis showed that TRF2 is required for expression of a cluster of ribosomal protein genes (Isogai et al., 2007a).

As already described before in section 2.3.1.1, the TCT core promoter motif is present in most ribosomal protein (RP) genes in Drosophila and human. Later, in vitro experiments showed that purified TRF2 activates TCT-containing promoters and that in Drosophila S2 cells, TCT-dependent transcription shows increase or decrease upon overexpression or depletion of TRF2, respectively (Wang et al., 2014). Consistently, ChIP-seq experiments showed that TRF2 is enriched at TCT-dependent promoters in vivo (Wang et al., 2014). Altogether, these data indicates a specialized TRF2-based Pol II transcription machinery driving the expression of RP genes.

Thirdly, TRF2 is required for transcription from DPE-dependent core promoters [START_REF] Hsu | TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription[END_REF][START_REF] Kedmi | Drosophila TRF2 is a preferential core promoter regulator[END_REF]. It has been shown that RNAi depletion of TBP decreases TATA-dependent but not DPE-dependent transcription, and that TBP overexpression increases TATA-dependent transcription but decreases DPE-dependent transcription [START_REF] Hsu | TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription[END_REF]. Upon ecdysone induction, TBP occupancy does not increase at DPE-containing promoter while Pol II occupancy does. These observation suggest that DPE-dependent transcription may occur in a TBP-independent manner [START_REF] Hsu | TBP, Mot1, and NC2 establish a regulatory circuit that controls DPE-dependent versus TATA-dependent transcription[END_REF]. Later on, it was found that TRF2 is enriched in protein fractions supporting DPE transcription, and that short TRF2 isoform preferentially activates DPE-containing promoters [START_REF] Kedmi | Drosophila TRF2 is a preferential core promoter regulator[END_REF].

Moreover, RNAi depletion of TRF2 reduces DPE-dependent but not TATA-dependent transcription, and genes induced by TRF2 overexpression are enriched for Inr and DPE motifs [START_REF] Kedmi | Drosophila TRF2 is a preferential core promoter regulator[END_REF]. In vitro affinity analysis also showed the enrichment of Inr and DPE motifs in DNA oligos bound by TRF2-containing complexes [START_REF] Kedmi | Drosophila TRF2 is a preferential core promoter regulator[END_REF]. Taken together, these findings suggest that TRF2 is functionally important for DPE-dependent transcription.

Fourthly, TRF2 selectively regulates the TATA-less Histone H1 gene promoter (Isogai et al., 2007a), apparently, by a DRE, TCT and DPE motifs independent mechanism (Vo Ngoc et al., 2017b).

Lastly, TRF2 is involved in piRNA cluster transcription [START_REF] Andersen | A heterochromatin-dependent transcription machinery drives piRNA expression[END_REF] (Figure 3456).

In Drosophila, TFIIA-L (homolog of human TFIIAαβ precursor) has a ovary-specific paralog Moonshiner, which lacks the TBP interaction domain. Moonshiner interacts with TRF2 to form an alternative TFIIA-TRF2 complex at bidirectional piRNA clusters through its interaction with Deadlock, a binding partner of HP1 variant Rhino, thus facilitating the transcription of piRNA [START_REF] Andersen | A heterochromatin-dependent transcription machinery drives piRNA expression[END_REF].

TRF2 is in C. elegans

In C. elegans, TRF2 is required for zygotic transcription during embryogenesis, and RNAi depletion of TRF2 results in embryonic lethality (Dantonel et al., 2000;Kaltenbach et al., 2000). TRF2-deficient embryo arrest as clusters of 80-350 undifferentiated cells before gastrulation. Analysis of gene expression revealed that TRF2 is required to for the expression of differentiation makers and also the establishment of bulk transcription during early embryogenesis (Dantonel et al., 2000;Kaltenbach et al., 2000).

TBP2, the vertebrate-specific TBP-related factor

TBP2 (also known as TBPL2 and TRF3) is the last identified TBP-related factor. It shares more than 90% identity with the TBP core domain and is able to bind the TATA-box, TFIIA and TFIIB (Persengiev et al., 2003;[START_REF] Bartfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Jallow et al., 2004). Although early studies suggested a widespread expression of Tbp2 (Persengiev et al., 2003;[START_REF] Yang | Regulated expression of TATA-binding protein-related factor 3 (TRF3) during early embryogenesis[END_REF], it has become apparent that in mice, TBP2 is exclusively expressed in the oocytes [START_REF] Bartfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Xiao et al., 2006;Gazdag et al., 2007). In Xenopus and zebrafish, expression of Tbp2 is broader, but is also highly enriched in the ovaries [START_REF] Bartfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Jallow et al., 2004;Xiao et al., 2006;Akhtar et al., 2009), suggesting a fundamental role for TBP2 in the vertebrate ovary.

In this section, I will mainly describe the function of TBP2 in Xenopus and zebrafish, and the role of TBP2 during mouse oogenesis will be described in chapter 4.

TBP2 function in Xenopus

Knockdown studies in Xenopus embryos showed that TBP2 is required for embryonic transcription and gastrulation (Jallow et al., 2004). In embryos, TBP2 overexpression can partially rescue TBP knockdown and restores the transcription of many TBP-dependent genes, suggesting that TBP2 may function as a substitute for TBP (Jallow et al., 2004). Transcriptome analysis showed that TBP2 is linked to vertebrate-specific embryonic genes and involved in ventral specification (Jacobi et al., 2007).

In Xenopus oocytes, TBP2 but not TBP is present at the protein level. After meiotic maturation, TBP2 is actively degraded following global repression of transcription, and only residual levels of TBP2 remain in the eggs and in the early embryos. After fertilization, maternal TBP mRNA is translated and TBP starts to accumulate during cleavage stages of development. Both TBP and residual TBP2 contribute to zygotic transcription (Akhtar et al., 2009;[START_REF] Muller | TBP2 is a general transcription factor specialized for female germ cells[END_REF][START_REF] Akhtar | TBP-related factors: a paradigm of diversity in transcription initiation[END_REF] (Figure 34567). Interestingly, TBP2 is recruited to transcriptionally active loops of 'lampbrush' chromosomes in oocytes (Akhtar et al., 2009).

TBP2 can promote transcription from TATA-containing promoters, and this function can be replaced by TBP when ectopically expressed in oocytes. Moreover, analysis of TBP2 occupancy in oocytes revealed that TBP2 is associated with active Pol II promoters. However, TBP can also promote transcription for these promoters when ectopically expressed in oocytes.

Furthermore, TBP2 is also recruited to 5S rRNA Pol III promoter, suggesting that TBP2

probably can also participate in Pol III transcription in Xenopus (Akhtar et al., 2009). Altogether, these findings indicates that TBP2 is more than a substitute for TBP. In

Xenopus, the TBP2-containing basal transcription machinery has specialized functions in oocytes, early embryogenesis and even nuclear reprograming by Xenopus oocytes.

TBP2 function in zebrafish

In zebrafish, knockdown studies showed TBP2 is required for embryonic development and expression of some differentiation maker genes [START_REF] Bartfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]. A later study reported that TBP2-depleted zebrafish embryos exhibit multiple developmental defects, in particular, fail to undergo haematopoiesis [START_REF] Hart | Initiation of zebrafish haematopoiesis by the TATA-box-binding protein-related factor Trf3[END_REF]. Expression profiling for TBP2-dependent genes revealed that mespa [murine orthologues (Mesp1 and Mesp2) of mespa are required for mesoderm specification] is the target gene required for embryonic development [START_REF] Hart | Initiation of zebrafish haematopoiesis by the TATA-box-binding protein-related factor Trf3[END_REF]. mespa further targets cdx4, a caudal-related gene required for haematopoiesis. Thus it was proposed that TBP2 initiated the transcription factor pathway for commitment of mesoderm to haematopoietic lineage [START_REF] Hart | Initiation of zebrafish haematopoiesis by the TATA-box-binding protein-related factor Trf3[END_REF].

Study of TSS usage during zebrafish early embryonic development by cap analysis of gene expression (CAGE) revealed that there is a widespread switch in TSS usage throughout maternal to zygotic transition [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF]. Moreover, maternal TSS selection is associated with the presence of an A/T-rich motif (referred as W-box). The similarity of the W-box with the TATA-box suggests that transcription initiation in the oocyte may be mediated by the oocyte-enriched TBP-related factor TBP2 [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF].

Controversial function of TBP2 in myogenesis

It has been reported that, during skeletal muscle differentiation, canonical TFIID is replaced by a TBP2-TAF3 complex for the activation of muscle genes [START_REF] Deato | Switching of the core transcription machinery during myogenesis[END_REF][START_REF] Deato | MyoD Targets TAF3/TRF3 to Activate Myogenin Transcription[END_REF]. However, further studies revealed that Tbp2 is not expressed in muscles, and Tbp2 null mice do not display any skeletal muscle phenotype (Gazdag et al., 2009).

Moreover, it has been shown that TBP, but not TBP2, is the essential component of the PIC that promotes muscle gene expression in differentiated skeletal muscles [START_REF] Malecova | TBP/TFIID-dependent activation of MyoD target genes in skeletal muscle cells[END_REF].

TFIIA and its paralog ALF

As mentioned in section 2.1.3, metazoan TFIIA contains three subunits, TFIIAα, TFIIAβ and TFIIAγ. TFIIAα and TFIIAβ are produced by Taspase1 cleavage of the TFIIAαβ precursor, and TFIIAαβ has a cell type-specific paralogue, called TFIIA-like factor (ALF).

TFIIAαβ, cleave or not make a difference

Initially, it was thought that TFIIA(α+β), the cleaved form, was the functional form. Later, it was reported that TFIIAαβ, the uncleaved form, together with TFIIAγ, interact stably with TBP and form a functional TBP-TFIIA(αβ+γ)-containing complex (TAC) in P19 embryonal carcinoma cells [START_REF] Mitsiou | TAC, a TBP-sans-TAFs complex containing the unprocessed TFIIAalphabeta precursor and the TFIIAgamma subunit[END_REF][START_REF] Mitsiou | p300 is involved in formation of the TBP-TFIIA-containing basal transcription complex, TAC[END_REF]. Taspase1-/-MEF cells with only uncleaved TFIIA are viable, although with cell cycle defects, indicating that the uncleaved TFIIA is functional (Zhou et al., 2006). In addition, uncleavable TFIIA mutant can rescue TFIIA knock-down in Xenopus (Zhou et al., 2006). Uncleavable TFIIA is more stable, as processed TFIIA can be more efficiently degraded by the ubiquitin-proteasome pathway [START_REF] Hoiby | Cleavage and proteasome-mediated degradation of the basal transcription factor TFIIA[END_REF].

Moreover, Taspase1 knockout mice are viable, although the majority die after birth and the survivors are smaller [START_REF] Takeda | Proteolysis of MLL family proteins is essential for taspase1-orchestrated cell cycle progression[END_REF]. Surprisingly, analysis of the surviving Taspase1 null mice shows that males are infertile, while Taspase1-/-females are fertile.

ALF

ALF is a paralogue of TFIIAαβ precursor (Upadhyaya et al., 1999;[START_REF] Ozer | A testis-specific transcription factor IIA (TFIIAtau) stimulates TATA-binding protein-DNA binding and transcription activation[END_REF]. Like TFIIA, the majority of ALF is cleaved into α and β subunits, which can form TFIIA with TFIIAγ.

ALF is only found in the gonads (Xiao et al., 2006). In testis, it is expressed in late pachytene spermatocytes and round spermatids [START_REF] Catena | Proteolytic cleavage of ALF into alpha-and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells[END_REF], and in ovary, it is expressed in the oocytes (Xiao et al., 2006). Immunoprecipitation with testis extracts revealed that full combination of hybrid TFIIA complexes exist [START_REF] Catena | Proteolytic cleavage of ALF into alpha-and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells[END_REF]

TBP2 and transcription regulation during oocyte growth

Oogenesis is a complex process with dynamic changes in gene expression that are regulated by a vast number of well-coordinated transcription factors (reviewed in (Jagarlamudi et al., 2012;[START_REF] Sanchez | Molecular control of oogenesis[END_REF])) (Figure 4-1). Following their colonization by PGCs, the female gonad continues its differentiation mostly under the influence of somatic cell expressed transcription factors LHX9, FOXL2, WT1, SF1 and GATA4 (reviewed in [START_REF] Sanchez | Molecular control of oogenesis[END_REF]).

In the embryonic ovary, the oogonia form into germ cell cysts, which later break apart as individual oocyte surrounded by several pre-granulosa cells, forming primordial follicles after birth [START_REF] Pepling | From primordial germ cell to primordial follicle: mammalian female germ cell development[END_REF]. Transcription factors such as FIGLA [START_REF] Soyal | FIGalpha, a germ cell-specific transcription factor required for ovarian follicle formation[END_REF], NOBOX [START_REF] Rajkovic | NOBOX deficiency disrupts early folliculogenesis and oocyte-specific gene expression[END_REF], FOXO3 [START_REF] Castrillon | Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a[END_REF][START_REF] Hosaka | Disruption of forkhead transcription factor (FOXO) family members in mice reveals their functional diversification[END_REF], SOHLH1 [START_REF] Pangas | Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8[END_REF], SOHLH2 (Choi et al., 2008b) and LHX8 [START_REF] Pangas | Oogenesis requires germ cell-specific transcriptional regulators Sohlh1 and Lhx8[END_REF]Choi et al., 2008a) that are expressed in the oocytes, as well as transcription factors such as ZGLP1 [START_REF] Li | GLP-1: a novel zinc finger protein required in somatic cells of the gonad for germ cell development[END_REF][START_REF] Strauss | GATA-like protein-1 (GLP-1) is required for normal germ cell development during embryonic oogenesis[END_REF] and FOXL2 [START_REF] Schmidt | The murine winged-helix transcription factor Foxl2 is required for granulosa cell differentiation and ovary maintenance[END_REF][START_REF] Uda | Foxl2 disruption causes mouse ovarian failure by pervasive blockage of follicle development[END_REF], that are expressed in the soma, have been revealed as crucial transcriptional regulators for follicle formation and early folliculogenesis. Further progression of follicle development requires YY1 [START_REF] Griffith | Yin-yang1 is required in the mammalian oocyte for follicle expansion[END_REF], TAF4b [START_REF] Freiman | Requirement of tissue-selective TBP-associated factor TAFII105 in ovarian development[END_REF] and TBP2 (Gazdag et al., 2009). Here I mainly focus on the role of TBP2 in transcription regulation during mouse oocyte growth. In the mouse, TBP2 is exclusively expressed in the oocytes (Xiao et al., 2006;Gazdag et al., 2007). During folliculogenesis, expression of TBP protein is decreasing, becoming undetectable during oocyte growth and reappearing only after fertilization. To the contrary, TBP2 is highly expressed in growing oocytes, declining to very low levels by the preovulatory follicle stage, only some traces of TBP2 persist up to the 2 cell-stage (Gazdag et al., 2007;[START_REF] Muller | TBP2 is a general transcription factor specialized for female germ cells[END_REF] Moreover, it has been show that Tbp2-defecient female mice are infertile due to a defect in folliculogenesis: ovaries from Tbp2 -/-mice have an increased number of primordial and primary follicles, but diminished number of secondary follicles compared to wild-type (Gazdag et al., 2009). TBP2 depletion impairs Pol II activity and chromatin structure in oocytes, and results in the downregulation of many oocyte-specific genes are downregulated, including Gdf9, Bmp15

and Zp3 (Gazdag et al., 2009). TBP2 binds to the promoters of several active genes in oocytes that have been tested (Gazdag et al., 2009). In addition, TBP2 is not able to fully compensate the depletion of TBP in MEFs [START_REF] Gazdag | Functional studies of TBP2 (TATA-binding protein 2) : The vertebrate specific homolog of TBP[END_REF], and TBP2 overexpression in zygotes altered pre-implantation embryo development (Gazdag et al., 2009).

Altogether, these observations indicate that there is a switch between TBP and TBP2-mediated transcription during oocyte growth and that TBP2 plays important role in oocyte transcription regulation.

Aims of the project

I discussed in the introduction that diversified basal transcription machineries can direct cell type-specific transcription. In agreement with this concept, it has been shown that TBP is replaced by TBP2 during oocyte growth. Moreover, Tbp2 -/-females are sterile due to defective folliculogenesis, altered chromatin organization and misregulation of key oocyte-specific genes.

These data indicate that TBP2 is essential for oocyte development, and that it plays a critical and specialized role in the regulation of oocyte-specific gene expression program.

In line with this, the aims of my PhD thesis were: Material & Methods

Mouse lines

Tbp2 knockout mouse line (Gazdag et al., 2009), Zp3-Cre Tg/Tg mouse line (Lewandoski et al., 1997), Taf10 f/f mouse line (Mohan et al., 2003), Taf7 f/f mouse line (Gegonne et al., 2012), Uprt transgenic mouse line [START_REF] Gay | Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA[END_REF], Rosa26 Cre and Rosa26 Flp [START_REF] Birling | Highly-efficient, fluorescent, locus directed cre and FlpO deleter mice on a pure C57BL/6N genetic background[END_REF] have already been described. Tbp-Tbp2 swap knock-in mouse line was generated at the Mouse Clinical Institute/Institut Clinique de la Souris.

Oocyte-specific Taf7 and Taf10 depletion was achieved by crossing Taf7

Cell culture

TBP2 stable overexpression cell line (3T3-II10) and mock cell line (3T3-K2) has already been described (Gazdag et al., 2007). NIH 3T3 cells were routinely cultured in DMEM (4.5g/l glucose) with 10% newborn calf serum (P122207N) at 37 °C and 5 % CO2.

Whole cell extracts

Whole cell extracts of II10 and K2 cells

Cells cultured in 15cm dish were washed twice with 1xPBS, subsequently harvested by scrapping on ice. Harvested cells were centrifuged 2000rpm at 4°C for 5min and then resuspended in 1 packed cell volume of whole cell extraction buffer consisting of 20mM TrisCl pH7.5, 2mM DTT, 20% Glycerol, 400mM KCl and 1x Protease inhibitor cocktails (PIC, Roche).

Cell lysates were froze in liquid nitrogen and thawed on ice for 3 times, followed by centrifuge at 13000rpm, 4°C for 15 min. The supernatant was collected and protein concentration was measured by Bradford protein assay (see 3.3). The cell extract was used directly for immunoprecipitations (see 5.1), or western blot (see 4), or stored at -80°C.

Whole cell extracts from ovaries

P13.5 ovaries collected from CD1 mice were homogenized in whole cell extraction buffer consisting of 20mM TrisCl pH7.5, 2mM DTT, 20% Glycerol, 400mM KCl and 5x Protease inhibitor cocktails. Cell lysates were froze in liquid nitrogen and thawed on ice for 3 times, followed by centrifuge at 14000rpm, 4°C for 15 min. The supernatant extracts were used directly for immunoprecipitations (see 5.2).

Bradford protein assay

1ul of a whole cell extract was mixed with the 1ml 1/5 th diluted dye reagent (Bio-rad protein assay, Bio-Rad), and the absorbance at 595 nm was measured. The concentration of whole cell extracts was calculated from a standard curve determined by six standard BSA samples with different concentration.

Western blot

Protein samples (15ug-25ug cell extracts or 15ul of IP elution) were mixed with 1/4 th volume of loading blue (100 mM Tris pH 6.8, 30% glycerol, 4% SDS, 0.2% bromophenol blue and fresh added 100 mM DTT) and boiled for 10 min. Samples were then resolved on 10 % SDS-PAGE and transferred to nitrocellulose membrane (Protran, Amersham). Membranes were blocked in 3% non-fat milk in 1xPBS at room temperature (RT) for 30 min, and subsequently incubated with the primary antibody (see 6.3) overnight at 4°C. Membranes were washed three times (10 min each) with 1xPBS plus 0.05% Tween20. Membranes were then incubated with HRP-coupled secondary antibodies for 1h at RT, followed by ECL detection (Thermo Fisher). The signal was acquired with the Chemidoc imaging system (Bio-Rad).

Immunoprecipitations

f) Elution

Add 100ul IP Glycine 0.1M pH=2.8, incubate 5 min at RT with gentle agitation, then centrifuge 3min at 600g/2500rpm at 4℃, take the SN to a new tube with 6.7ul Tris (Tris-base, 1.5M, pH8.8, adding before) to neutralize it quickly. Wash the beads with 20ul IP 100mM buffer, centrifuge 3min at 600g/2500rpm at 4℃, take the SN and mix with elution, add 5ul 100×PIC.

Centrifuge the elution at 13000prm for 10min at 4℃, then pipet the SN to a new tubes as ELUTION, and keep the beads at 4℃ for until finished the IP-WB. Do the western immediately or freeze it in liquid N2 and stored in -80℃.

Ovaries WCE IP with Dynabeads a) Washing/Preparation of Dynabeads

Resuspend Dynabeads Protein A (100.01D) by vorexing for 10-15 seconds, and transfer 200ul beads to a 1.5 EP tube (For ovaries WCE IP, using 150ul beads for IP and 50ul beads for pre-clean, and make sure the stock bead suspension is homogenous before pipetting). Put on the magnet for 20-30s and remove the supernatant, then remove the tube from the magnet and quick wash the beads three times with 1ml IP 100mM KCL buffer, and remove the supernatant. Add 200ul IP 100mM KCL buffer to the beads, resuspend, put on ice.

b) Antibody coupling to Dynabeads-Pro A

Place 200ul PCR tubes on ice and add 150ul pre-washed beads-Pro A, and add 10ul TBP2 (3024) antibody, place at RT on shaker for 30min, then 10-20 rpm on rotator for 2h at 4℃.

Place the tubes on magnet and remove the supernatant, add 150ul IP 500mM KCL buffer, transfer to a 500ul tube, wash the 150 tubes twice with 150ul IP 500mM KCL buffer make sure all the beads are transferred, followed by three times wash with 500ul IP 500mM KCL buffer (5min wash on rotator), remove the supernatant, and wash twice with 500ul IP 100mM KCL buffer (5min wash on rotator). Remove the supernatant, and leave beads on ice.

c) Pre-clean of INPUT

Ovary WCE sample was incubated with 50ul dynabeads @ cold room for 1h, then centrifuge at 14000rpm for 5min at 4℃, keep the supernatant as INPUT sample.

d) Protein fixation/Binding IP

Add the INPUT sample to the beads-antibody complex, and incubate at 4℃ overnight. Put on the magnet, keep the supernatant as SN for WB. Wash the beads with 500ul IP 500mM buffer three times on rotator (5min each), followed by two washes with 500ul IP 100mM buffer.

Before add Glycine, remove all the IP buffer.

e) Elution

Add 80ul IP Glycine 0.1M pH=2.8, vortex to mix, incubate 5 min at RT with gentle agitation, vortex, incubate 5min again, then put on the magnet, take the SN to a 200ul tube with 5.4ul

Tris (Tris-base, 1.5M, pH8.8, adding before) to neutralize it quickly. Add 20ul IP Glycine 0.1M pH=2.8 to the beads, mix, incubate 5 min at RT with gentle agitation, take the supernatant to the tube and add another 1.33ul Tris. Mix the elution, and put it on the magnet, transfer the supernatant to a low bind 1.5 EP tube, add 2ul 100xPIC, as ELUTION, totally around 100ul.

Take 15ul to do the western blot immediately, the left 85ul was used for mass-spectrometry analysis, which was described in the manuscript.

Antibodies

Rabbit polyclonal antibody generation from protein

Antibody generation is performed with the help of IGBMC animal facility. Two months old female rabbit (White New-Zealand strain, 2-2,5 kg) were used for immunization. Rabbit is anesthetized by intramuscular injection of Xylazine/Ketamine solution, and blood sample collected to produce the pre-immune serum as negative control. 1 ml of antigen solution (containing ∼200 ug of recombinant protein or ∼300 μg peptide) is mixed with 1 ml of "Complete Freund Adjuvant" and was injected intradermally to rabbit on 40 to 60 different locations for immunization. One month after, blood sample is collected every week for one month to obtain antibody-containing antiserums. Another injection of antigen (containing ∼100ug recombinant protein or ∼200 μg peptide) were performed and twelve days later, rabbit is anesthetized and then sacrificed for the boosted antisera. The antiserums were tested and purified to obtain polyclonal antibodies.

Antibody purification

Affinity purification of rabbit polyclonal antibodies was performed with SulfoLink™

Coupling Resin from Thermo Fisher Scientific according to the user manual.

List of antibodies

RNA preparation and sequencing

Oocytes collected above were washed through several M2 drops, and total RNA was isolated using NucleoSpin RNAXS kit from Macherey-Nagel according to the user manual.

RNA quality and quantity were evaluated by Bioanalyzer. RNA-sequencing and data analyses (including RNA-seq analyses, repeat element analyses and core promoter motif analyses)

were described in the manuscript. Measure the concentration of chromatin, aliquot 200ug-500ug per tubes to avoid de-freezing.

ChIP-seq

Save 15ug chromatin to reverse crosslink and check sonication efficiency, freeze the chromatin at Liquid Nitrogen and kept in -80°C. 15ug chromatin were add up to ∼300ul with L2 buffer. Add 12.5ul 5M NaCl to 0.2M final and 1.5ul of RNase A (10mg/ml) to final 50ug/ml, incubate at 37°C for 1h, then add 20ug Proteinase K (1ul of 20mg/ml Proteinase K) and heat with 400 rpm shaking at 65°C for 5 hours or overnight in a thermomixer to reverse crosslink.

DNA extraction by phenol/chloroform, and resuspend in 40ul TE buffer. Check the quality of the chromatin by running 10ul and 20ul on 1.5% agarose gel, the fragment size should be between 250 to 750 bp.

c) Pre-block of protein A/G-Sepharose beads

Take protein A/G-sepharose 50% beads slurry (100ul per ChIP ) into a 2ml tube, and wash twice with 1ml TE (Vortex, spin 2 min at 600g/2500rpm). For 1 ml beads (50% slurry), add 100ul (final 1ug/ul) of denatured tRNA (10mg/ml, denature 5min at 95°C) and 50ul (final 1ug/ul) of BSA (20mg/ml) in 1ml beads slurry solution. Incubate for 3 hours with rotation at 4°C, followed by two washes with 1ml TE. Add 500ul TE final and keep as 50% slurry at 4°C

(saturated beads are stable for 1 week in the fridge).

d) Immunoprecipitation

Use

e) Elution

Elute twice with 160ul freshly prepared elution buffer (10 minutes overhead shaking at RT, centrifuge at 600g/2500 rpm for 2mins). Pool the elution, centrifuge at 8000rpm for 3 min, take ∼300ul SN as final elution.

f) Reversal of crosslink and isolation of DNA

For ChIP-qPCR, add 12.5ul 5M NaCl (0.2M final conc.) to 300ul elution as well as input sample, then add 50ug/ml of RNase A (1.5ul 10mg/ml), and incubate for 30min at 37°C, then add 1ul 20mg/ml Proteinase K, incubate at 65°C O/N with 400 rpm shaking in a thermomixer.

For ChIP-seq, add 12.5ul 5M NaCl (0.2M final conc.) to 300ul elution, then add 50ug/ml of RNase A (1.5ul 10mg/ml) and incubate at 65°C O/N with 400 rpm shaking in a thermomixer.

Next day add 20ug Proteinase K, 20ul of tris pH7.9 (1M),

Sequencing and data analysis

ChIP-seq libraries were prepared and sequenced on an Illumina Hi-seq4000 as single-end 50-base reads. After sequencing, peak calling and quantitative comparisons were performed using the MACS, seqMINER and R.

DamID 10.1 DamID in cells

Dam-Tbp2 cDNA and Dam-only cDNA were cloned into pX vector, under the control of the TRE inducible promoter. Dam-Tbp2-NIH3T3 stable cell lines and Dam-only-NIH3T3 stable cell lines were obtained after transfection, FCAS sorting and puromycin selection. In the stable cell lines, leakage expression of Dam-Tbp2 and Dam could methylate the 'GATC' site to 'G m ATC'.

Thus, genomic DNA was extract from both Dam-Tbp2-NIH3T3 and Dam-only-NIH3T3 cells, and used for 'G m ATC'-methylation-specific PCR amplification [START_REF] Vogel | Detection of in vivo protein-DNA interactions using DamID in mammalian cells[END_REF]Marshall et al., 2016). PCR products were purified by column (MACHEREY-NAGEL #REF740609.50) and sonicated. Libraries were prepared with the sonicated PCR products and sequenced on an Illumina Hi-seq4000 as single-end 50-base reads. DamID-seq data was analysed using the published pipeline [START_REF] Marshall | damidseq_pipeline: an automated pipeline for processing DamID sequencing datasets[END_REF].

Oocyte DamID

Dam-Tbp2 cDNA and Dam-only cDNA were cloned into pRN3P vector, and capped mRNA of Dam-Tbp2 and Dam-only were obtained by in vitro transcription with mMESSAGE mMACHINE® Kit (AM1344) from Life Technologies. mRNA of Dam-Tbp2 (50ng/ul) was injected into oocytes (20-50 per group) collected from P7 and P13 mice, while oocytes injected with Dam-only mRNA (20ng/ul) are controls. After injection, oocytes were cultured in αMEM [START_REF] Pfender | Live imaging RNAi screen reveals genes essential for meiosis in mammalian oocytes[END_REF] with penicillin G, streptomycin, ITS, 5% fetal bovine serum and 0.01 mg/ml oFSH for ∼24 hours. Then the positively injected oocytes were collected and subsequently used for 'G m ATC'-methylation-specific PCR amplification. PCR products were purified by column. Libraries were prepared with purified PCR products and sequenced on an Illumina Hi-seq2500 as single-end 50-base reads.

CUT&RUN

Oocyte CUT&RUN were performed following the published protocol [START_REF] Hainer | Profiling of pluripotency factors in individual stem cells and early embryos[END_REF].

Briefly, oocyte nuclei prepared from P7 and P14 oocytes (500 oocytes used per experiment) was immobilized on Concanavalin A coated beads. After the incubation of primary antibody (TBP2 and Pol II antibodies were used), protein A-micrococcal nuclease (pA-MN) was added.

pA-MN binds specifically to the primary antibody, and upon the addition of Ca 2+ , pA-MN can cleave the DNA that bound by primary antibody. Cleaved DNA fragments were released from nuclei to the supernatant, and recovered by Phenol/chloroform/isoamyl alcohol extraction.

Libraries were prepared from these recovered DNA fragments and sequenced on Illumina Hi-seq4000. (reviewed in [START_REF] Choi | Genetics of early mammalian folliculogenesis[END_REF]). Shortly after birth, some primordial follicles enter folliculogenesis and undertake a growth phase during which oocytes increase their size until the pre-antral follicular stage (Pedersen and Peters, 1968). Later, in antral, preovulatory follicles, the fully-grown germinal vesicle (GV) stage oocyte is ready for ovulation. Maturation follows with the resumption of meiosis induced by ovulatory stimulus and proceeds until the metaphase II, at which stage the oocyte awaits for fertilisation (reviewed in (Li and Albertini, 2013)).

List of primers

Gene name Sequence(5' to 3') Application

Genes specifically expressed in the oocyte are necessary for either growth or for communication with follicular cells and a remarkable feature of oocyte is the high expression of the retrotransposons driven by Pol II transcription. These elements are interspersed repetitive elements that can be mobile in the genome. They represent a threat for the integrity and functionality of the genome and suppression mechanisms have been selected throughout evolution in the soma and in germ cells (reviewed in [START_REF] Crichton | Defending the genome from the enemy within: mechanisms of retrotransposon suppression in the mouse germline[END_REF]). There are 3 major classes of retrotransposons in mammals: long interspersed nuclear elements (LINE), short interspersed nuclear elements (SINE) and long terminal repeat (LTR) retrotransposons derived for retroviruses, also known as endogenous retroviruses (ERVs). There are 3 main sub classes of LTRs: ERV1, ERVK and endogenous retrovirus like (ERVL)-MaLR (mammalian apparent LTR retrotrosposons) defined by their phylogenetic relationships. Remarkably, some retrotransposons, LTRs in particular, are actively expressed in female germ cells. The class III MaLR family is highly expressed in oocytes and can regulate oocyte gene expression by acting for example as alternative promoters or first exons for somatic genes [START_REF] Peaston | Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos[END_REF].

Although transcription factors regulating oocyte-specific gene expression have been characterized (reviewed in (Jagarlamudi and Rajkovic, 2012)), little is known about the role of general transcription factors (GTFs) in regulating transcription initiation in female germ cells.

Pol II transcription requires the stepwise assembly of protein complexes on core promoters of genes forming the preinitiation complex (PIC) (reviewed in (Goodrich and Tjian, 2010)). A functional PIC consists of the Pol II and several multi-protein complexes called general transcription factors (GTFs). The evolutionary conserved TFIID complex plays a major role in transcription initiation as it is the first GTF to initiate the assembly of the PIC by recognizing the promoter. TFIID is a large multi-protein complex composed of the TATA boxbinding protein (TBP) and 13 TBP-associated factors (TAFs) in metazoans (Tora, 2002).

Among the 13 metazoan TAFs, TAF9, TAF10 and TAF12 are also shared by the Spt Ada Gcn5 acetyl transferase (SAGA) activator complex conserved in metazoans (reviewed in (Spedale et al., 2012)). The traditional text-book model suggesting that transcription is always regulated by the same transcription complexes has been challenged in metazoans in the last decade by the discovery of cell-type specific complexes containing specialized TAF paralogs (reviewed in (Goodrich and Tjian, 2010;[START_REF] Ho | Chromatin remodelling during development[END_REF][START_REF] Müller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF]) and of the contextdependent requirement of different TAFs for transcription (Bardot et al., 2017;Gegonne et al., 2012;Indra et al., 2005;Mohan et al., 2003;Tatarakis et al., 2008). Moreover, in the absence of TBP, TBP paralogues are able to mediate Pol II transcription of developmentally important genes in vertebrate embryos [START_REF] Ferg | The TATA-binding protein regulates maternal mRNA degradation and differential zygotic transcription in zebrafish[END_REF]Jacobi et al., 2007;Martianov et al., 2002b;[START_REF] Müller | TBP is not universally required for zygotic RNA polymerase II transcription in zebrafish[END_REF][START_REF] Veenstra | Distinct Roles for TBP and TBP-Like Factor in Early Embryonic Gene Transcription in Xenopus[END_REF]. Three metazoan TBP paralogs have been identified. The TBP related factor (TRF) TRF1 is insect specific (Crowley et al., 1993). The TBP like factor (TLF, also known as TRF2 or TBPL1) has been identified in several metazoan species (Dantonel et al., 2000;Kaltenbach et al., 2000;Ohbayashi et al., 1999;Teichmann et al., 1999) while the TBP-related factor 3 (TRF3, also known as TBPL2, here after called TBP2) has only been described in vertebrates [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Persengiev et al., 2003). While TLF and TBP2 share significant identity with the highly conserved saddle-like-C-terminal DNA binding domain (Hernandez, 1993) (78% and 92-93%, respectively [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Crowley et al., 1993;Persengiev et al., 2003)), TRF2 is more distant with only 42% identity [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]. A consequence of this difference is that TBP2, but not TRF2, is able to bind the TATAbox [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Ohbayashi et al., 1999;Rabenstein et al., 1999). TFIIA and TFIIB are two GTFs that are important for the stabilization of TFIID on the DNA. While TFIIB is composed of only one protein, TFIIA is a complex formed by 3 polypeptides α and β produced by taspase 1 cleavage of the TFIIA-αβ precursor, and TFIIA-γ (reviewed in [START_REF] Høiby | A facelift for the general transcription factor TFIIA[END_REF]). Variability is also present in the TFIIA complex, as a TFIIAα-β homolog called ALF (TFIIA-like factor or TFIIAτ), has been identified in male germ cells (Upadhyaya et al., 1999).

Similarly to TBP, all three TBP related factors are able to interact with TFIIA and TFIIB to mediate Pol II transcription initiation in vitro [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Hansen et al., 1997;Jallow et al., 2004;Rabenstein et al., 1999). In mice, Tbp2 is exclusively expressed in the oocytes [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Xiao et al., 2006). In zebrafish and xenopus, expression of Tbp2 is broader, but is enriched in the ovary, suggesting a fundamental role for TBP2 in the vertebrate ovary [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF]Jallow et al., 2004;Xiao et al., 2006).

We have previously shown in the mouse ovary that TBP is expressed in the oocytes in primordial follicles and becomes undetectable during oocyte growth whereas TBP2 is highly expressed in the growing oocytes, strongly suggesting that TBP2 is replacing TBP during folliculogenesis (Gazdag et al., 2007). The crucial role of TBP2 for oogenesis was demonstrated by the absence of phenotype of Tbp2 -/-mice, except female sterility due to defect in secondary follicles production (Gazdag et al., 2009). TBP2 binds to actively transcribed genes promoter and in its absence, transcription is perturbed in the oocyte, primarily at the primary follicle stage, and leads to altered transcriptional profile of oocyte-specific genes in a transcriptome analysis of 2 weeks old ovaries (Gazdag et al., 2009). Altogether, these data strongly suggest that TBP2 is playing a specialized role during oocyte development.

To understand whether and how TBP2 is controlling transcription initiation during oogenesis, we carried out RNA-seq analyses from wild-type and Tbp2 -/-oocytes from primary and secondary follicles. These analyses show an important impairment of gene expression, with a strong tendency for down regulation of the most abundantly expressed genes, as well as specific down-regulation of the expression of the MaLR (mammalian apparent LTR retrotransposons)-type endogenous retroviral elements. To identify the nature of the complex associated with TBP2 in the oocytes, we carried out immunoprecipitation followed by mass spectrometry. We demonstrate that, contrary to TBP, TBP2 associates in the oocytes with TFIIA, but does not assemble into a TFIID-type complex. Altogether, our data show that a specific TBP2-TFIIA containing transcription machinery, different from canonical TFIID, drives transcription in mouse oocytes.

Results

RNA polymerase II transcribed genes are affected in Tbp2 -/-mouse primary and secondary follicle oocytes

To analyse whether oocyte specific transcription is affected by the ablation of the mouse Tbp2 gene (official symbol Tbpl2), we have isolated primary and secondary follicles [post-natal days (P) 7 and P14], isolated polyA+ RNA and carried out RNA-seq analyses of wild-type and Tbp2 -/-oocytes. Each RNA sample was derived from three independent biological replicates and sequenced at 50 nucleotides single ends. Specific sequence-reads were mapped to the mouse genome, and unique reads were considered for further analyses. Next, we verified the expression profile of the Tbp2 gene on the IGV genome browser in wild type (WT) and knockout P7 and P14 oocytes with the data coverage normalization parameter (Fig. 1A). As expected the expression of the Tbp2 gene was hardly detectable, with no signal in the deleted exon 4, while Tbp2 expression was readily detected in the WT controls, validating the RNA-seq experiment. Besides Tbp2, we observed the down regulation of a number of oocyte specific genes, such as for example Ooep (Fig. 1B).

For the comparative analyses, all datasets were normalized across samples with the median-of-ratio method [START_REF] Anders | Count-based differential expression analysis of RNA sequencing data using R and Bioconductor[END_REF] and differential expression was accessed using DESeq2 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. To analyse the RNA-seq data more precisely a principal component analysis (PCA) and a hierarchical clustering were carried out (Fig. 2A and 2B, Supplementary Fig. 1A). Both analyses showed that the four distinct RNA samples separated in individual groups, but the triplicate samples belonging to the same group clustered together, indicating that the main explanation for the variance is the genotype, and not the stage. These analyses further validated the high throughput sequencing and the knock-out strategy. Next, we analysed how Tbp2 loss of function influences mRNA expression in both P7 and P14 oocytes. At both of these oocyte development stages we found that in WT P7 and in WT P14 about 10 4 of genes were expressed, (10622 and 10697 reads divided by the median length of transcripts in kb, respectively) although many of these transcripts maybe maternally deposited in the oocytes.

Out of these transcripts using an absolute log2 fold change cut off of 1, slightly more genes were downregulated (1720 in P7 and 1794 in P14) than up regulated (1577 in P7 and 1358 in P14) in the Tbp2 -/-oocytes (Fig. 2C and 2D, Supplementary Fig. 1B-C). Interestingly, the most highly expressed genes were down regulated in the two oocyte developmental stages (Fig. 2C and 2D, Supplementary Fig. 1B-C). When either the down regulated or the upregulated genes were compared between the two developmental stages in the Tbp2 -/-cells, we found that in each category about 50% or more genes of the TBP2 regulated genes were also influenced the same way in the two oocyte developmental stages (Fig. 2E). Next, we carried out a direct fold-change comparison between P7 and P14. This analysis indicated that loss of TBP2 has a major effect on the expression of the most abundantly expressed genes in oocytes at P7 and P14 (Fig. 2F).

These results together suggest that TBP2 has an important role in gene expression regulation in the growing oocytes. However, as TBP2 is suggested to play a role in Pol II transcription it is surprising that large proportion of genes are upregulated following Tbp2 ablation. As in the above analyses we measured steady state mRNAs changes, it is possible that the direct transcript synthesis effects have been masked by transcript buffering mechanisms (reviewed in (Timmers and Tora, 2018)).

To analyse and find potential differential gene function categories in transcripts down regulated in P7 and P14 oocytes, we carried out Gene Ontology (GO) analyses on the identified common down regulated categories of genes (Supplementary Table 1, carried out with DAVID [START_REF] Huang | Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources[END_REF]). Interestingly, many down regulated genes were classified in GO categories linked to chromatin binding, transcription factor activity, RNA Pol II activity, Pol II binding, DNA binding, core promoter specific binding (Fig. 3A), which are in good agreement with the hypothesis that TBP2 functions as a Pol II specific promoter binding factor in growing oocytes. One of the GO categories that came out with the highest significance was "polyA specific ribonuclease activity" containing many genes coding for two exonuclease complexes contributing to the majority of the deadenylation activity in eukaryotes: CCR4-NOT and PAN2-PAN3. This finding suggests that the activities of the CCR4-NOT and PAN2-PAN3 complexes are impaired, and thus mRNA decay is down regulated in oocytes lacking TBP2. Importantly, mRNAs stored in the oocytes undergo general decay during maternal-zygotic transition and their stability is tightly regulated (reviewed in [START_REF] Walser | Transcript clearance during the maternal-to-zygotic transition[END_REF]). In addition, it was

shown that an ERK1/2 triggered Btg4-mRNA translation is a key step in oocyte maturation and revealed that BTG4-CNOT7 and BTG4-CNOT8 mediated mRNA decay is required for the successful development of the oocytes [START_REF] Yu | BTG4 is a meiotic cell cycle-coupled maternal-zygotic-transition licensing factor in oocytes[END_REF]. Thus, next we analysed the expression of factors regulating mRNA decay in the oocytes. Interestingly, many genes coding for factors participating in the oocyte specific mRNA decay were significantly down regulated in the Tbp2 - /-mutant oocytes (Figure 3B, Supplementary Table 2), including Btg4 and Cnot8. Thus, it is possible that many transcripts, which were found upregulated in Tbp2 -/-mutant oocytes are indirectly regulated by the depletion of TBP2, due to the lack of the decay of maternal mRNAs.

Several core promoter motifs are enriched in TBP2 regulated genes

Several elements such as the TATA box, the initiator (Inr), the Motif Ten element (MTE) and the distal promoter element (DPE) are bound by TFIID (reviewed in [START_REF] Danino | The core promoter: At the heart of gene expression[END_REF]).

However, there are no individual core promoter sequence elements, or sequence element combinations that can be used to define TSSs, and the structure-function relationship of core promoters remains poorly understood. Nevertheless, as the C-terminal DNA binding domains of TBP2 and TBP are highly similar, TBP2 is able to bind the TATA box [START_REF] Bártfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF].

We hypothesized that the down regulated genes may be direct targets of TBP2-mediated transcription and thus, we carried out analyses to uncover the potential enrichment of different individual core promoter elements of the down regulated genes within a -50/+50 genomic sequence using regulatory sequence analysis tools (RSAT) [START_REF] Turatsinze | Using RSAT to scan genome sequences for transcription factor binding sites and cis-regulatory modules[END_REF]. We tested the presence of TATA box, Inr, MTE, DPE and two core promoter motifs localized around the TSS initially identified in the promoter of the hepatitis B virus X gene [START_REF] Danino | The core promoter: At the heart of gene expression[END_REF].

We analyzed the enrichment of predicted motifs between the common down regulated genes at P7 and P14 (down) compared to an array of random sequences (random), all the annotated genes (all) or to the protein coding genes of the analysis (CDS) (Fig. 4A-F). While our pipeline of analysis did not detect significant enrichments using the Inr element, we observed a small enrichment of all the other core promoter elements that was statistically significant for the MTA-Mm-Int are highly expressed and highly affected in P7 and P14 Tbp2 -/-mutant oocytes (Fig. 5E and 5F, and Supplementary Fig. 2C). In order to get a better mapping precision, we resequenced one sample of each triplicates in 100 nucleotides paired-ends and similar results

were obtained (Supplementary Fig. 3A-H). MaLRs encode no known proteins, but it has been shown that MaLR-dependent transcription is key in initiating synchronous developmentally regulated transcription to reprogram the oocyte genome [START_REF] Peaston | Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos[END_REF]. As TBP2 depletion is reducing MaLR transcription by 4-fold in both P7 and P14 oocytes, it could thus seriously deregulate oocyte specific transcription of neighbouring genes and consequently stop oocyte growth.

TBP2 is interacting with TFIIA in TBP2 in mouse oocytes

To characterize TBP2-containing transcription complexes, first we have carried out immunoprecipitations (IPs) from NIH3T3 cells artificially overexpressing TBP2 (NIH3T3-II10 cells) (Gazdag et al., 2007). To be able to use antibodies for anti-TBP2 IPs that recognize mouse (m) TBP2 with high affinity we have generated novel anti-mTBP2 rabbit polyclonal antibodies (pAb) and affinity purified them (Materials and Methods, Supplementary Fig. 4). Using the two of the best generated anti-TBP2 pAbs and the previously published anti TBP2 monoclonal antibody (mAb) (Gazdag et al., 2007), TBP2-containing complexes were IP-ed from NIH3T3

whole cell extracts, IP-ed complexes were eluted by pH 2.5 buffer and proteins were identified by mass spectrometry. In parallel, as a control we have also carried out an anti-TBP IP and the eluted TBP-containing complexes were also analysed. To be able to compare the composition of the IP-ed complexes, normalized spectral abundance factor (NSAF) values were calculated [START_REF] Zybailov | Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae[END_REF]. All 3 tested anti-TBP2 antibodies gave very similar results (Supplementary Fig. 5A). All the known TBP-containing complex subunits [Pol II TAFs (1)(2)(3)(4)(5)(6)(7)(8)[START_REF] Fuchs | The p400 complex is an essential E1A transformation target[END_REF][START_REF] Samuelson | p400 is required for E1A to promote apoptosis[END_REF][START_REF] Park | The SANT domain of p400 ATPase represses acetyltransferase activity and coactivator function of TIP60 in basal p21 gene expression[END_REF][START_REF] Kim | A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs[END_REF][START_REF] Hu | Homozygous disruption of the Tip60 gene causes early embryonic lethality[END_REF], Pol I TAF1s (A-D) and Pol III subunit BRF1/TF3B) subunits were identified by mass spectrometry analysis (Fig. 6A and Supplementary Fig. 5B). In contrast, the anti-TBP2 immunoprecipitations showed that the artificially expressed TBP2 can incorporate in TFIIDlike complexes, as TFIID TAFs were co-IP-ed with TBP2, however with lower stoichiometry (NSAF values) than that of TBP (Fig. 6A, Supplementary Fig. 5A). Interestingly, in spite of the high similarity between the core domains of TBP2 and TBP, we have not identified any of the Pol I or Pol III TBP-associated factors (Fig. 6A and Supplementary Fig. 5A). In addition, in the anti-TBP2 IPs, while TFIIB was not detected, we have identified TFIIA-αβ and TFIIA-γ as TBP2 partners (Fig. 6A and Supplementary Fig. 4A). To analyse whether the TBP2 associates with TFIID TAFs and TFIIA in same complex, NIH3T3-II10 whole cell extract was purified on a Superose 6 gel filtration column that separates proteins and complexes with molecular weights between 5 MDa and 5000 Da. This separation indicated that the most of TBP2 and TFIIA isolated from NIH3T3-II10 cells could be found in the same fractions [START_REF] Ye | seqMINER: an integrated ChIP-seq data interpretation platform[END_REF][START_REF] Adelman | Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans[END_REF][START_REF] Wang | Genomewide mapping of HATs and HDACs reveals distinct functions in active and inactive genes[END_REF][START_REF] Wang | Combinatorial patterns of histone acetylations and methylations in the human genome[END_REF][START_REF] Cai | Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex[END_REF] eluting around 150 kDa, while TBP2 protein was below the detection threshold of the western blot assay in the TFIID-containing fractions 9-15 (Fig. 6B). To verify whether in the fractions 22-26 TBP2 and TFIIA would form a complex, we have IP-ed TBP2 from fractions 23-25 pooled and subjected to mass spectrometric analysis. This anti-TBP2 IP confirmed that in the fractions eluting from the Superose 6 around 150 kDa TBP2 and TFIIA form a complex, with a calculated molecular weight of about 110-120 kDa (Fig 6C and Supplementary Fig. 5C).

To investigate whether TBP2 would form an endogenous complex with TFIID TAFs and/or TFIIA, we prepared mouse ovary whole cell extracts (WCE) from 120 P14 stage ovaries, where TBP2 expression is the highest in the growing oocytes. Three independent anti-TBP2

IPs identified TFIIA-α or unprocessed TFIIA-αβ and TFIIFA-γ subunits, as well as in one of the triplicate experiments we also detected one peptide corresponding to TAF5 and TAF10 (Fig. 7A). Nevertheless, in a control anti-TBP IP we have detected all TFIID subunits with high NSAF values, as well as background levels of TFIIFA-γ and TFIIB (Fig. 7B). As TBP2 is only expressed in the growing oocytes present in the ovaries, we wanted to further verify whether TBP2 could associate with TFIID TAFs. To this end we have designed a triple IP strategy (Fig. 7C). First, we have carried out an anti-TAF7 IP, to deplete all TAF7-containing TFIID, from the ovary WCE and analysed whether in the IP-ed complexes we would detect TBP2. All the TFIID subunits, except TAF7L, were identified in the 3 technical replicates with high confidence (Fig. 7D and Supplementary Fig. 6A). In contrast, TBP2 was detected with only one TBP2-specific peptide with a medium score in only 1 of the 3 technical replicates (data not shown) indicating that this detection is not significant. Remarkably, no TFIIA was detected in the TAF7-associated complexes (Fig. 7D and Supplementary Fig. 6A). As in the crude anti-TBP2 IPs (Fig. 7A) we detected background levels (one peptide) of TAF10 (a subunit of both TFIID and SAGA complexes), the anti-TAF7 IP flow through was re-immunoprecipitated with an anti-TAF10 antibody. This second IP demonstrated the anti-TAF7 depletion of TFIID was very efficient, as in the second anti-TAF10 IP only the shared SAGA-TFIID TAFs were detected (Supplementary Fig. 6B). Again, in the anti-TAF10 IP we did not detect any TBP2 nor TFIIA, but SAGA subunits (Fig. 7D and Supplementary Fig. 6B). Next from these TAF7 and TAF10 double depleted IP flow through extracts we have carried out an anti-TBP2 IP. The analysis of this third consecutive IP indicated that TBP2 forms a complex with TFIIA-αβ and TFIIFA-γ subunits (Fig. 7E and 7F and Supplementary Fig. 6C). Interestingly, we did not detect the germ cell specific TFIIA-αβ paralog ALF (Upadhyaya et al., 1999) despite its high level of expression in oocytes (Xiao et al., 2006). Thus, in conclusion we demonstrate that in mouse oocytes TBP2 forms a TFIID TAFs-free stable complex with TFIIA.

TAF7 is not required for oocyte growth and maturation

In order to functionally validate that TFIID TAFs, TAF7 in particular, are not required for transcription during oocyte growth, we carried out a conditional deletion of Taf7 during oocyte growth using the Zp3-Cre transgenic line (Lewandoski et al., 1997). We chose TAF7 since it is a TFIID specific subunit. We obtained Tg(Zp3-Cre/+);Taf7 flox/∆ females (Fig. 8A) and performed superovulation and histological analyses. As shown in Table 1, the oocyte specific deletion of Taf7 did not affect the numbers of collected mature oocytes after superovulation. Histological analyses of 6 weeks-old female ovaries confirmed the presence of antral follicles (Fig. 8C compared to 8B, magnification in Fig. 8D and8E). Altogether, these data further suggest that expression of Taf7 in the oocyte is not necessary for oocyte growth and is in good agreement with our finding that the oocyte specific functional TBP2-containing transcription complex does not contain TFIID TAFs.

Discussion

Three genes encoding different TBP-type factors, TBP, TLF/TRF2/TBPL1 and TBP2/TRF3/TBPL2 exist in vertebrate genomes (reviewed in [START_REF] Müller | Developmental regulation of transcription initiation: more than just changing the actors[END_REF]). TBP-type factors are bipartite proteins with variable N-terminal domains and a relatively well conserved shared C-terminal domain forming a saddle-like structure with a concave surface that is known to bind to DNA. TBP2 proteins from different vertebrates show a high degree of similarity in their C-terminal core domains amongst themselves, but also with TBP. On the other hand, the N-terminal domains of the TBP2s are highly variable amongst the different vertebrate homologues of TBP2s and also very different from TBPs. While TBP has a rather ubiquitous expression pattern in mammalian cells and tissues, the expression of TBP2 and TLF/TRF2 are highly specialized. TBP2 is expressed in growing oocytes and thus, essential for female germ cell differentiation in mice (Gazdag et al., 2007;Xiao et al., 2006). In a mirroring situation, TRF2 expression is enriched during spermatogenesis and male germ cells lacking TRF2 are blocked between the transition from late round spermatids to early elongating spermatids {Martianov:2001kk}. However, while TBP2 and TBP show contrasting expression patterns in the oocytes (Gazdag et al., 2007), TRF2 and TBP are co-expressed in spermatids (Martianov et al., 2002a;[START_REF] Schmidt | Developmental testis-specific regulation of mRNA levels and mRNA translational efficiencies for TATA-binding protein mRNA isoforms[END_REF]). Nevertheless, it seems that during gonad development specific transcription programs driven by either TBP2 during oocyte growth, or TRF2 during spermatogenesis, are required, and that these specified gonad specific transcription programs cannot be carried out by TBP.

In this study, we show that TBP2 forms a stable complex with TFIIA in mouse oocytes, but does not associate with either the oocyte expressed TFIIA-αβ paralogue, ALF protein (Upadhyaya et al., 1999;Xiao et al., 2006). While we detected the presence of TFIIA in all our TBP2-IPs from ovaries, the detection of TAF5 and TAF10 was observed only in 1 out of 3 biological replicates. In the same line, the detection of TBP2 in the anti-TAF7 IP has been observed in only 1 out 3 technical replicates with only a medium score. Altogether, these data strongly support the idea that the main TBP2-associated complex in the growing oocytes contains TFIIA but not TFIID TAFs. This conclusion is supported by our IPs experiments in the NIH3T3-II10 cell line that overexpresses TBP2. While TFIID is present in these cells and TBP2 able to interact with TFIID TAFs, our gel filtration experiment indicates that the vast majority of the TBP2-associated complex does not elute with TFIID and mass spectrometry experiment confirmed the absence of any TFIID TAFs in the 120 kDa fractions. Our oocyte specific Taf7 KO experiments, showing no phenotypic and functional problems following TAF7 depletion (Fig. 8), is in accordance with the lack of functional TFIID in mouse oocytes.

A possible explanation of the absence of significant interactions between TFIID TAFs and TBP2 in the oocyte could be the absence of TFIID TAFs proteins. Interestingly, we found that all TFIID Tafs, except Taf7l, are expressed at the mRNA level in oocytes (see Supplementary Table 4), however, whether they are also expressed in oocytes at the protein level is not known, except for TAF4B that has been detected in female neonate' oocytes (Falender et al., 2005).

We did not detect any TAFs proteins in crude oocyte extracts by proteomic analyses (data not shown) but this could be due to their potential relative low abundance. It could be conceivable that, similarly to Tbp mRNA that is transcribed but not translated in oocytes (Akhtar and Veenstra, 2009), TFIID Taf mRNA translations are also inhibited and as a result the canonical TFIID is not present in oocytes. Another reason why TBP2 does not interact with TAFs nor ALF, but rather interacts with TFIIA could be its N-terminal domain that is very different from TBP.

TFIIA was initially classified as a GTF, when it was first purified [START_REF] Dejong | Human general transcription factor TFIIA: characterization of a cDNA encoding the small subunit and requirement for basal and activated transcription[END_REF][START_REF] Ma | Isolation of a cDNA encoding the largest subunit of TFIIA reveals functions important for activated transcription[END_REF][START_REF] Ozer | Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription[END_REF] but since it is dispensable in transcription [START_REF] Van Dyke | Physical analysis of transcription preinitiation complex assembly on a class II gene promoter[END_REF][START_REF] Wu | Properties of PC4 and an RNA polymerase II complex in directing activated and basal transcription in vitro[END_REF], it can be considered more as a general cofactor. TFIIA is the proteolytical cleavage of TFIIA-αβ into a TFIIA-α and TFIIA-β moiety [START_REF] Høiby | A facelift for the general transcription factor TFIIA[END_REF].

Recent studies have shown that TFIIA is cleaved by Taspase1 (Zhou et al., 2006). In our proteomic experiments, we cannot determine whether the anti-TBP2 IPs bring down uncleaved TFIIA-αβ or processed TFIIA-α and TFIIA-β. Preliminary in vitro interaction experiments suggest that TBP2 interacts preferentially with uncleaved TFIIA (I. Berger personal communication). However, cleavage of TFIIA does not appear to serve as a step required for its activation in oocyte as Taspase1 knock-out female mice are fertile (Oyama et al., 2013). It is interesting to note that while TRF2 can interact with unprocessed TFIIA, this complex is not functional (Oyama et al., 2013). The crystal structure between the human and yeast TFIIA/TBP/DNA complex revealed conserved architecture (reviewed in [START_REF] Hantsche | Conserved RNA polymerase II initiation complex structure[END_REF]) and indicated that the β-sandwich domain of TFIIA interacts with the N-terminal stirrup region of TBP and with the backbone of the TATA box, whereas the four-helix bundle of TFIIA projects away from the TFIIA/TBP/DNA complex and is available for additional interactions with transcription factors. Further structural studies will determine whether TBP2 interacts with uncleaved or processed TFIIA, how TBP2-TFIIA interact with DNA and whether TFIIA/TBP2/DNA complex is different from that of its counterpart with TBP or TRF2.

An interesting parallel between TBP2 and TRF2 is that both TBP-type factors form endogenous stable complexes with TFIIA. The beginning of TBP2 accumulation in the oocyte nuclei, or TRF2 accumulation in male germ cell nuclei coincides with the phase of meiosis I (Gazdag et al., 2007;Martianov et al., 2001). It is thus conceivable that TBP2-TFIIA in oocytes, or TRF2-TFIIA during spermatogenesis are involved in the control of gene expression in a meiotic context and that probably both transcription complexes function in a more compacted DNA environment in which TBP/TFIID cannot. Moreover, in Drosophila TLF/TRF2 has been suggested to bind to promoters harbouring a non-canonical, TCT-containing initiator (Wang et al., 2014). In the future genome-wide TBP2 binding analyses with sensitive low-cell techniques, such as TBP2-Dam-ID or CUT&RUN (Marshall et al., 2016;[START_REF] Skene | Targeted in situ genome-wide profiling with high efficiency for low cell numbers[END_REF], will be needed to map more precisely the sequence motifs to which TBP2 is binding in the oocytes.

Oocytes display remarkable post-transcriptional regulatory mechanisms that control mRNA stability and translation. During oogenesis, the oocyte genome is transcriptionally active, and the newly synthesized maternal mRNAs are either translated or stored in a dormant form (reviewed in [START_REF] Bettegowda | Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development[END_REF]). The newly synthesized transcripts receive a long poly(A) tail and subsequently undergo poly(A) shortening in the cytoplasm, preventing translation. Until resumption of meiosis, mRNAs with a short poly(A) tail are stored in the cytoplasm in a dormant form. Thus, poly(A) tail deadenylation, amongst other activities, coordinates post-transcriptional regulation of the oocyte mRNA pool and is critical for normal the progression of early embryonic development. In this respect, it is interesting to emphasize that in Tbp2 -/-mutant oocytes the activity of the two major deadenylation complexes, CCR4-NOT and PAN2-PAN3, seems to be impaired. This in turn would result in the increase of transcripts with long(er) poly(A) tails and their unwanted translation of dormant maternal mRNAs in the oocytes. In addition, TBP2 could also control other oocyte-specific negative post-transcriptional regulatory mechanisms, which target mRNAs for degradation. In addition to mRNA deadenylation, such negative regulatory mechanisms could be mediated by the interaction of transcripts with RNA-binding proteins in a nonspecific or sequence-specific fashion, and/or potentially via actions of microRNA and repeat-associated small interfering RNA, which degrade maternal RNA transcripts. It is this possible that in the absence of TBP2 most of the upregulated transcripts are those that escape such negative regulation.

LTR retrotransposons, also known as ERVs, constitute ~10% of the mouse genome (reviewed in [START_REF] Crichton | Defending the genome from the enemy within: mechanisms of retrotransposon suppression in the mouse germline[END_REF]). While their expression is generally suppressed by DNA methylation and/or repressive histone modifications, a subset of ERV subfamilies retain transcriptional activity in specific cell types ( [START_REF] Faulkner | The regulated retrotransposon transcriptome of mammalian cells[END_REF]. ERVs are especially active in germ cells and early embryos (reviewed in [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF]). Indeed, many genomewide many transcripts are initiated in LTRs, such as for example of MaLRs in mouse oocytes, which constitute ~5% of the genome [START_REF] Consortium | Initial sequencing and comparative analysis of the mouse genome[END_REF][START_REF] Mccarthy | Long terminal repeat retrotransposons of Mus musculus[END_REF].

Members of the MT subfamily of MaLRs are particularly active in oocytes and hundreds of MT LTRs have been co-opted as oocyte-specific gene promoters [START_REF] Franke | Long terminal repeats power evolution of genes and gene expression programs in mammalian oocytes and zygotes[END_REF][START_REF] Peaston | Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos[END_REF]. The fact that in the TBP2 ablated oocytes MaLR MTA_Mm, MTA_Mm-int and MT-int transcripts are four-fold down regulated in general strongly suggests that TBP2 can binding to many of these MaLR LTR-initiated transcription units to nucleate the imitation of Pol II transcription. However, further experimental and bioinformatics investigations would be required to understand the mechanisms by which TBP2 is able to regulate these LTR-initiated transcription units. As LTR-initiated transcription units shape also the oocyte methylome, it will be important to analyse also how TBP2 influences DNA methylation in oocytes.

Material and Methods

Animal experimentation

Animal experimentations were carried out according to animal welfare regulations and guidelines of the French Ministry of Agriculture and procedures were approved by the French Ministry for Higher Education and Research ethical committee C2EA-17 (project n°2018031209153651). The Tg(Zp3-Cre), Taf7 flox and Tbp2 -lines have already been described (Gazdag et al., 2009;Gegonne et al., 2012;Lewandoski et al., 1997).

Histology analyses of ovaries

Ovaries were collected from 6 weeks-old Tg(Zp3-Cre/+);Taf7 flox/+ and Tg(Zp3-Cre/+);Taf7 flox/∆ oocyte specific mutant females, fixed in 4% paraformaldehyde (Electron Microscopy Sciences) over-night at 4°C, washed 3 times in PBS at room temperature and embedded in paraffin. Five µm-thick sections were stained with hematoxylin and eosin and images were acquired using a slide scanner Nanozoomer 2.0HT (Hamamatsu Photonics).

Supervovulation

5 U of pregnant mare serum (PMS) was injected intraperitoneally in 4-week-old female mice between 2-4 pm. After 44-46 hours, GV oocytes were collected from the ovaries by puncturing with needles.

Oocytes collection

After dissection, ovaries are freed from adhering tissues in &x PBS. A pool od 6 ovaries was digested in 500 µL of 2 mg/mL Collagenase, 0.025% Trypsin and 0.5 mg/mL type IV-S hyaluronidase, on a thermomixer at 600 rpm for 20 minutes. The digestion was then stopped by the addition of 1 mL of 37°C pre-warmed αMEM -5% FBS. The oocytes were then sizeselected under a binocular.

Cell lines and cell culture

The NIH3T3-II10 line overexpressing TBP2 and the control NIH3T3-K2 have already been described (Gazdag et al., 2007) and were maintained in high glucose DMEM supplemented with 10% of newborn calf serum at 37°C in 5% CO2.

Whole cell extracts

Cells cultured in 15 cm dish were washed twice with 1x PBS, subsequently harvested by scrapping on ice. Harvested cells were centrifuged 1000 rcf at 4°C for 5min and then resuspended in 1 packed cell volume of whole cell extraction buffer (20 mM Tris HCl pH7.5, 2 mM DTT, 20% Glycerol, 400 mM KCl, 1x Protease inhibitor cocktail (PIC, Roche)). Cell lysates were frozen in liquid nitrogen and thawed on ice for 3 times, followed by centrifugation at 20817 rcf, at 4°C for 15 min. The supernatant was collected and protein concentration was measured by Bradford protein assay. The cell extracts were used directly for IP and western blot, or stored at -80°C.

Ovaries collected from P14 CD1 female mice were homogenized in whole cell extraction buffer (20 mM Tris HCl pH7.5, 2 mM DTT, 20% Glycerol, 400 mM KCl, 5x PIC (Roche)).

Cell lysates were frozen in liquid nitrogen and thawed on ice for 3 times, followed by centrifugation at 20817 rc, at 4°C for 15 min. The supernatant extracts were used directly for immunoprecipitations or gel filtration.

Antibodies and antibody purification

The 2B12 anti-TBP2, the 3TF13G3 anti-TBP and the 15TF21D10 anti-GST mouse monoclonal antibodies have already been described [START_REF] Brou | Distinct TFIID complexes mediate the effect of different transcriptional activators[END_REF]Gazdag et al., 2007;[START_REF] Nagy | The metazoan ATAC and SAGA coactivator HAT complexes regulate different sets of inducible target genes[END_REF]. The J7 rabbit polyclonal anti TFIIA was a gift of H.G. Stunnenberg. The IGBMC antibody facility generated several anti-TBP2 polyclonal antibodies with peptides (2481, 2482, 3017, 3018, 3019, 3022, 3023 and 3024) or with purified protein (3498 and 3499) (see Supplementary Table 5). The different peptides were synthetized and coupled to ovalbumin and injected into 2 or 3 rabbits and the first 140 amino-acids of the mouse TBP2 fused to a His tag were produced in BL21DE3 bacteria and purified with Ni-NTA beads (Qiagen) and injected in 2 rabbits. The resulting sera were affinity purified by using the Sulfolink Coupling Gel (Pierce) following the manufacturer's recommendations.

Gel filtration

A Superose 6 (10/300) column was equilibrated with buffer consisting of 25mM Tris HCl pH 7.9, 5 mM MgCl2, 150 mM KCl, 5% Glycerol, 1 mM DTT and 1x PIC (Roche). Five hundred µL of whole cell extracts containing ∼5 mg of protein were injected in an Akta Avant chromatography device and ran at 0.4 mL per min. Protein detection was performed by absorbance at 280nm and 260nm. Five hundred µL fractions were collected.

Immunoprecipitation

Immunoprecipiation were carried out as already described in (Bardot et al., 2017).

Western blot

Protein samples (15-25 µg of cell extracts or 15 µL of IP elution) were mixed with 1/4 th volume of loading buffer (100 mM TrisHCl pH 6.8, 30% glycerol, 4% SDS, 0.2% bromophenol blue and freshly added 100 mM DTT) and boiled for 10 min. Samples were then resolved on a10 % SDS-PAGE and transferred to nitrocellulose membrane (Protran, Amersham).

Membranes were blocked in 3% non-fat milk in 1x PBS at room temperature (RT) for 30 min, and subsequently incubated with the primary antibody overnight at 4°C. Membranes were washed three times (10 min each) with 1x PBS -0.05% Tween20. Membranes were then incubated with HRP-coupled secondary antibodies for 1 hour at RT, followed by ECL detection (Thermo Fisher). The signal was acquired with the Chemidoc imaging system (Bio-Rad).

RNA preparation

Oocytes collected were washed through several M2 drops, and total RNA was isolated using NucleoSpin RNAXS kit (Macherey-Nagel) according to the user manual. RNA quality and quantity were evaluated using a Bioanalyzer.

Mass spectrometry analyzes and NSAF calculations

Samples were TCA precipitated, reduced, alkylated and digested with LysC and Trypsin at 37°C overnight. After C18 desalting, samples were analyzed using an Ultimate 3000 nano-RSLC (Thermo Scientific, San Jose, California) coupled in line with a linear trap Quadrupole (LTQ)-Orbitrap ELITE mass spectrometer via a nano-electrospray ionization source (Thermo Scientific). Peptide mixtures were loaded on a C18 Acclaim PepMap100 trap column (75 μm inner diameter × 2 cm, 3 μm, 100 Å; Thermo Fisher Scientific) for 3.5 min at 5 μl/min with 2% acetonitrile (ACN), 0.1% formic acid in H2O and then separated on a C18 Accucore nanocolumn (75 μm inner diameter × 50 cm, 2.6 μm, 150 Å; Thermo Fisher Scientific) with a 240 minutes linear gradient from 5% to 50% buffer B (A: 0.1% FA in H2O / B: 80% ACN, 0.08% FA in H2O) followed with 10 min at 99% B. The total duration was set to 280 minutes at a flow rate of 200 nL/min.

an Illumina HiSeq 4000. One sample per condition (SNVT1, SNVT6, SNVT10 and SNVT14)

were also resequenced 100 pb paired end.

Reads were preprocessed in order to remove adapter, polyA and low-quality sequences (Phred quality score below 20). After this preprocessing, reads shorter than 40 bases were discarded for further analysis. These preprocessing steps were performed using cutadapt version 1.10 (Martin, 2011). Reads were mapped to spike sequences using bowtie version 2.2.8 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF], and reads mapping to spike sequences were removed for further analysis. Reads were then mapped onto the mm10 assembly of Mus musculus genome using TopHat2 version 2.0.1.4 [START_REF] Kim | TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions[END_REF]. Gene expression quantification was performed from uniquely aligned reads using htseq-count version 0.6.1p1 [START_REF] Anders | HTSeq--a Python framework to work with highthroughput sequencing data[END_REF], with annotations from Ensembl version 93 and "union" mode. Read counts were normalized across samples with the median-of-ratios method [START_REF] Anders | Count-based differential expression analysis of RNA sequencing data using R and Bioconductor[END_REF], to make these counts comparable between samples and differential gene analysis were performed using the DESeq2 version 1.16.1 [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. All the figures were generated using R software version 3.5.1.

Repeat element analyses

For the repeat element analyses, data were processed as already described [START_REF] Fadloun | Chromatin signatures and retrotransposon profiling in mouse embryos reveal regulation of LINE-1 by RNA[END_REF] using Bowtie1 [START_REF] Langmead | Ultrafast and memoryefficient alignment of short DNA sequences to the human genome[END_REF]

Core promoter motif analyses

Sequences were downloaded from the Ensembl data base using EnsDb.Mmusculus.v79 R package version 2.99.0 (Rainer J (2017) EnsDb.Mmusculus.v79: Ensembl based annotation package. R package version 2.99.0.). All the -50/+50 genomic sequences across the TSS of all transcripts were considered in this analysis and 3 set of sequences were obtained: 1/ the sequences for the common genes that were expressed above 100 reads/kb median length of transcript and were downregulated for a log2 fold change below -1 at P7 and P14 (963 genes present in the annotation), 2/ all the genes present in the annotation (43309 genes) and 3/ all the genes confiding for protein present in the annotation (21989). A random sample of 18000 100 nucleotides sequences was also generated as a control using RSAT [START_REF] Turatsinze | Using RSAT to scan genome sequences for transcription factor binding sites and cis-regulatory modules[END_REF].

We used the following consensus sequences: TATA box; TATAWAAR, Inr; YYANWYY, MTE; CSARCSSAAC, DPE; GNNNDSWYVY, XCPE1; DSGYGGRAS and XCPE2;

VCYCRTTRCMY (IUPAC nomenclature) [START_REF] Danino | The core promoter: At the heart of gene expression[END_REF]to design Transfac format matrix for each element. We used the matrix scan module of RSAT to detect the presence of these different element in the 4 different sets of sequences using the following parameters: Control (Tg(Zp3-Cre/+);Taf7 flox/+ ) and mutant (Tg(Zp3-Cre/+);Taf7 flox/∆ ) females were stimulated for oocyte maturation and GV oocytes were counted. Mann and Whitney statistical analysis did not detect a significant difference between the control and the mutant data. Genomic views of the Tbp2 (A) and Ooep (B) genes whose expression is reduced in Tbp2 -/- mutant oocytes. As expected no reads map to the exon 4 of Tbp2 (gray dashed box) which is deleted in Tbp2 -/-mutant mice (A). Data were derived from three independent biological replicates sequenced in single ends. gene sequence using RSAT pattern motif search. The proportion of the different predicted core promoter elements between the coding genes (CDS) and the genes downregulated at P7 and P14 (down) was compared using Pearson's Chi 2 test with Yate's continuity correction. down; common down-regulated genes in Tbp2 -/-mutant oocytes (adjusted p value < 0.05 and log2 fold change ≤ -1, 963 genes in the annotation), CDS; genes annotated as protein coding genes (21989 genes), all; all annotated genes (43309 genes), random, 18000 100 nucleotides random sequences generated using RSAT., ns; non-significant, *; p value ≤0.05, ***; p value ≤0.001. Comparison of immunoprecipitated (IP-ed) protein from II10 cells whole cell extracts (WCE) using a TBP antibody and one of our specific mouse anti TBP2 antibody indicating that compared to TBP, TBP2 is strongly interacting with TFIIA but is able to interact with some TAF subunits. B. Western blot of a Superose 6 gel filtration analysis of NIH3T3-II10 WCE indicating that TBP2 is associated within a complex with TFIIA-α but does not co-purify with TAF6. C. LC/LC-MS analysis of the fraction highlighted in red (B) compared to an anti-TBP2-IP from II10 WCE, confirming that TFIIA, but no TAF proteins, is detected in the fraction.

LC/LC-MS data were derived from three technical replicates. NSAF; normalized spectral abundance factor, error bars indicate sem.

Results

Unpublished data

Unpublished results

Technical optimizations 1.1 Antibody generation and validation

As we planned to perform anti-TBP2 IP and ChIP on mouse oocytes that are very limited in numbers, high affinity anti-TBP2 antibodies are required. Besides the published 2B12 mouse monoclonal antibody, there were already 8 rabbit polyclonal anti-TBP2 sera in the lab, all generated with different TBP2 peptides, but they were not purified nor validated. I generated a new anti-TBP2 sera against the N-terminal part of the TBP2 protein, and purified and validated the anti-TBP2 sera.

Antibody generation

As it was thought that immunization of rabbit with protein fragments rather than peptides will have a better chance to obtain ChIP grade antibodies, I generated 2 new anti-TBP2 sera.

The N-terminal part of TBP2 (1-140aa) was cloned into the pET-15b vector, and recombinant protein produced in bacteria was used for rabbit immunization to generate anti-TBP2 sera, 3498 and 3499 (Figure 1-1 B). In total, besides 2B12, 10 anti-TBP2 sera have been raised

(Figure 1-1 C).

Results

Unpublished data

151 Localization of the peptides or protein fragments used to generate the new sera.

Antibody purification

All the anti-TBP2 sera (10ml boost sera used each) were purified by affinity purification with the corresponding TBP2 peptides or protein and purified antibody fractions were analyzed by 10% SDS polyacrylamide gel electrophoresis, followed by Coomassie-staining (Figure 12).

3498 was not purified as it was not good (Figure 1-1 B).

Antibody validation by WB

All the purified antibodies were then validated by western blots. TBP2 overexpressing NIH3T3-II10 cell extract and TBP2 negative K2 mock cell extract were used for WB. The

Results

Unpublished data 152 monoclonal 2B12 antibody was used as a positive control. The result showed that antibody 3023, 3024 and 3499 were good for WB, although with non-specific signal (Figure 123).

Antibody validation by IP

The anti-TBP2 antibodies were then validated by IP with II10 cell extract, and the IP elutions were analyzed by WB with the 2B12 antibody (Figure 1234), 2B12 being the most specific TBP2 antibody for WB. Results showed that 2481, 2482, 2B12, 3022, 3023 and 3024 were all good for IP, and that 3024 has the highest affinity.

Results

Unpublished data 153

Antibody validation by ChIP

Microarray analysis showed that, in the II10 cells, some genes such as Gadd45g and Mef2a, were upregulated and Cdca8 were downregulated [START_REF] Gazdag | Functional studies of TBP2 (TATA-binding protein 2) : The vertebrate specific homolog of TBP[END_REF]. TBP2 seemed to bind to the promoters of the upregulated genes [START_REF] Gazdag | Functional studies of TBP2 (TATA-binding protein 2) : The vertebrate specific homolog of TBP[END_REF]. Thus, I use Gadd45g and Mef2a as positive targets and Cdca8 and intergenic region as negative targets to validate the anti-TBP2 antibodies for ChIP using II10 cells.

Results

Unpublished data 154 Out of the 9 anti-TBP2 sera, 3023 and 3499 seemed to work for ChIP, but only 3023 has very high affinity (Figure 12345). 3023 and 3499 antibodies were further validated by IF with the help of Sascha Conic (Figure 123456). Altogether, I conclude that we have generated new TBP2 antibodies for different applications. The anti-TBP2 3023 is the best for ChIP and anti-TBP2 3024 is the best for IP.

Optimization of immunoprecipitation with low input amount

We aimed to identify the TBP2-containing complex/es by performing immunoprecipitation experiments coupled to mass spectrometry (IP mass-spec). TBP2 is only expressed in growing oocytes and a classical protocol for IP mass-spec requires more than 4mg protein extracts, which is not suitable for oocytes. Therefore, optimization of the IP protocol suitable for micrograms of starting material was needed. Based on the micro chromatin immunoprecipitation assay [START_REF] Dahl | A rapid micro chromatin immunoprecipitation assay (microChIP)[END_REF], I set up the micro immunoprecipitation (microIP) for low amount of starting material (Figure 1234567). With this protocol, I succeeded to carry out anti-TBP IP from only micrograms of HeLa nuclei extract input: for instance, in the elution of the anti-TBP microIP from 4ug of HeLa nuclear extract, TBP and TAF6 can be clearly detected by Western blot (Figure 1234567). The results confirmed that 3024 is the best anti-TBP2 antibody for IP and that anti-TBP2 IP can be performed with micrograms of whole cell extracts, which made it possible to carry out anti-TBP2 IP with ovaries.

Optimization of MOWChIP

To decipher the role and function of TBP2 during oocyte growth, initially, we planned to map TBP2 binding profiles in oocyte by carrying out anti-TBP2 ChIP-seq with oocytes.

As the sensitivity of conventional ChIP assays is a major obstacle for the study of low-abundance cells, to perform ChIP-seq with oocytes, we first need to setup a method that can be used for oocytes ChIP-seq.

After comparing the published low input ChIP methods (before 2015), such as uChIP [START_REF] Dahl | A rapid micro chromatin immunoprecipitation assay (microChIP)[END_REF]), iChIP (Lara-Astiaso et al., 2014), ULI-NChIP (Brind'Amour et al., 2015), ChIPmentation [START_REF] Schmidl | ChIPmentation: fast, robust, low-input ChIP-seq for histones and transcription factors[END_REF] and MOWChIP [START_REF] Cao | A microfluidic device for epigenomic profiling using 100 cells[END_REF], we decided to set up the MOMChIP, a microfluidic oscillatory washing-based ChIP-seq method that has been reported for epigenomic profiling with as few as 100 cells.

The major steps of MOWChIP contains: (i) formation of a packed bed of IP beads in the microfluidic chamber (∼0.71ul volume); (ii) performing ChIP by flowing the chromatin through the packed bed; (iii) oscillatory washing; (iv) removal of the unbound chromatin fragments by flushing the chamber; (v) collection of the IP beads [START_REF] Cao | A microfluidic device for epigenomic profiling using 100 cells[END_REF] (Figure 123456789).

In collaboration with Igor Kukhtevich (Robert Schneider Lab, Institute of Functional Epigenetics, Munich), we have tested many times with different microfluidic designs for H3K4me3 ChIP with chromatin prepared from 5000 NIH 3T3 cells (Figure 1 [START_REF] Cao | A microfluidic device for epigenomic profiling using 100 cells[END_REF]. (B) The first microfluidic design according to the published work [START_REF] Cao | A microfluidic device for epigenomic profiling using 100 cells[END_REF]. (C) The second microfluidic design, which has a narrow region with height of ∼2 um to help the packing and maintenance of beads (as the diameter of the dynabeads is ∼2.8 um).

TBP2 binds genome-widely to gene promoters

Before studying the binding profile of TBP2 in oocytes, we first analyzed its occupancy in TBP2 ectopically overexpressing NIH3T3 cells. We applied both antibody-independent (DamID-seq) and antibody-dependent (ChIP-seq) approaches.

Results

Unpublished data 159

TBP2 occupancy revealed by DamID-seq

Methyl-PCR products were purified by column and sonicated for DamID sequencing.

DamID-seq revealed that TBP2 binds to more than 13000 genes (Tao Ye, IGBMC) (Figure 2

TBP2 ChIP-seq in NIH3T3 cells

TBP2 ChIP-seq analysis was performed with II10 and K2 cells, and in parallel, TBP ChIP-seq was also performed with II10 cells. Astonishingly, TBP2 binds to more than 10000 genes, while TBP binds to less than 4000 genes, and more than 90% of the TBP bound genes are also bound by TBP2 (Figure 2-2 C). Consistently, ∼80% of TBP2 bound genes are overlapped with the DamID-seq peaks (Figure 2

RNA-seq analysis of TBP2 ectopically overexpressing NIH3T3 cells

To further analyze whether the promoter binding of TBP2 is functionally relevant or not, we performed RNA-seq analysis with II10 and K2 cells. Sequencing has been performed

(GenomEAST platform, IGBMC) and data analysis is ongoing.

TBP2 binding profiles in oocytes

After analysis of the TBP2 binding profiles in NIH3T3 II10 cells, we performed DamID-seq in growing oocytes, and also tested a new approach named CUT&RUN to map TBP2 occupancy in oocytes. PCR products were purified by column. 300ng of purified Methyl-PCR products were used for sequencing libraries preparation (Figure 34). Sequencing using an Illumina Hi-seq2500 (iGE3 Genomics Platform, Geneva) is ongoing.

TBP2 uliCUT&RUN with oocytes

Since the resolution of the TBP2-DamID-seq might not be optimal, I also tested a novel et al., 2017).

Results

Unpublished data 166

Analysis of the direct effect of TBP2 on active transcription

As mentioned in introduction 2.2.7, nascent RNAs are released from Pol II during transcription termination. They are rapidly exported to the cytoplasm, where their half-lives are generally determined by the cytoplasmic mRNA decay pathways. Emerging evidences have shown the existence of transcript buffering, a phenomenon that steady-state levels of mRNAs are ''buffered'' when mRNA synthesis or degradation is impaired by inactivation of either the transcription complexes or the enzymes responsible for cytoplasmic mRNA decay [START_REF] Shalem | Transcriptome kinetics is governed by a genome-wide coupling of mRNA production and degradation: a role for RNA Pol II[END_REF]Sun et al., 2012;[START_REF] Haimovich | The fate of the messenger is pre-determined: a new model for regulation of gene expression[END_REF][START_REF] Sun | Global analysis of eukaryotic mRNA degradation reveals Xrn1-dependent buffering of transcript levels[END_REF][START_REF] Rodriguez-Molina | Engineered Covalent Inactivation of TFIIH-Kinase Reveals an Elongation Checkpoint and Results in Widespread mRNA Stabilization[END_REF][START_REF] Baptista | SAGA Is a General Cofactor for RNA Polymerase II Transcription[END_REF][START_REF] Warfield | Transcription of Nearly All Yeast RNA Polymerase II-Transcribed Genes Is Dependent on Transcription Factor TFIID[END_REF]Timmers et al., 2018). Moreover, during folliculogenesis, oocyte stores large amount of RNAs [START_REF] Bettegowda | Mechanisms of maternal mRNA regulation: implications for mammalian early embryonic development[END_REF]. Therefore, in order to better understand TBP2's function in oocyte transcription, nascent RNA analysis is required.

Using an oocyte-specific expression of the uracil phosphoribosyl-transferase (UPRT) that allows specific 4-thiouracil (4TU) labeling of nascent RNAs in the oocyte (Lewandoski et al., 1997;[START_REF] Gay | Mouse TU tagging: a chemical/genetic intersectional method for purifying cell type-specific nascent RNA[END_REF], coupled to thiol(S)-linked alkylation for the metabolic sequencing of RNA (SLAM-Seq) [START_REF] Herzog | Thiol-linked alkylation of RNA to assess expression dynamics[END_REF], I performed nascent RNA analysis in both P14 WT oocytes and P14 Tbp2-deficient oocytes. 4TU was intraperitoneally injected in P14 Uprt Tg/+ ;Tg(Zp3-cre/+) and P14 Tg(Uprt/+);Tg(Zp3-cre/+);Tbp2 -/-female pups. Six hours after 4TU injection, the oocytes were collected and total RNA were isolated (see 8.1) for SLAM-seq [START_REF] Herzog | Thiol-linked alkylation of RNA to assess expression dynamics[END_REF] (Figure 345).

Total RNA samples from ∼300 oocytes of both WT and Tbp2-deficient oocytes were prepared in triplicates. Libraries are prepared using the QuantSeq 3'mRNA-Seq Library Prep Kit, as published [START_REF] Herzog | Thiol-linked alkylation of RNA to assess expression dynamics[END_REF].

In addition, total RNA from in vitro cultured oocytes were also prepared, including one sample from ∼600 P14 WT oocytes as a background control, one sample from ∼600 P14 WT oocytes cultured with 4sU that can be directly incorporated into the RNA, one sample from ∼600 P14 Tg(Uprt/+);Tg(Zp3-cre/+) oocytes cultured with 4sU and one last sample from ∼600

P14 Tg(Uprt/+);Tg(Zp3-cre/+) oocytes cultured with 4TU. TU-tagging coupled to SLAM-seq. Adapted from [START_REF] Cordeiro | Geography of Follicle Formation in the Embryonic Mouse Ovary Impacts Activation Pattern during the First Wave of Folliculogenesis[END_REF][START_REF] Herzog | Thiol-linked alkylation of RNA to assess expression dynamics[END_REF].

Results

Unpublished data 168

Potential TBP2-TFIIA interacting factors

In order to identify potential oocyte specific partners of the TBP2-TFIIA complex, we further analyzed the mass-spec results of the anti-TBP2 IP from ovary extracts and we found that among the triplicates, there are 247 common proteins (Figure 5

General coactivator SAGA might be required in oocytes

In parallel with the conditional deletion of Taf7 during oocyte growth (see in the manuscript) that show that the TFIID-specific TAF7 is not required for transcription in the oocyte, I also carried out a conditional deletion of Taf10 during oocyte growth using the Tg(Zp3-Cre) transgenic line (Figure 6-1 A). Interestingly, the ovaries size of 6-week-old Tg(Zp3-Cre/+);Taf10 flox/ △ female is much smaller compared to Tg(Zp3-Cre/+);Taf10

△ /+ littermates (n≥3) (Figure 6-1 B). While control ovaries (Tg(Zp3-Cre/+);Taf10 △/+ ) show obvious mature follicles, such structures are absent in Tg(Zp3-Cre/+);Taf10 flox/ △ ovaries. Moreover, after superovulation, no mature oocytes were obtained (n>6). As TAF10 is not only part of TFIID but also of the SAGA co-activator complex [START_REF] Helmlinger | Sharing the SAGA[END_REF], this defect may arise from SAGA specific function, as it has been shown that SAGA assembly is severely affected when Taf10 is deleted in the embryo (Bardot et al., 2017). According to our knock-in strategy, after or recombination, either the cDNA of Tbp2 or the cDNA of Tbp will be expressed in the growing oocytes under the control of the Tbp2 regulatory elements. Moreover, to facilitate the detection of the expression from the diferent modified alleles, Tbp and Tbp2 cDNA were fused to the cDNA of mCherry and Venus, respectively. In order to avoid any dominant negative effects from the fusion, we used a T2A linker to produce separate proteins. The plasmids were built by Jean Marie Garnier (IGBMC).

However, after recombination, neither Venus signal from Tbp2 TBP2:Venus mouse nor mCherry signal from Tbp2 TBP:mCherry mouse could be detected. More strangely, control homozygous Tbp2 TBP2:Venus:TBP2:Venus female are infertile (more than 5 breeding cages).

I analyzed expression of Tbp, Tbp2, Tbp-mCherry and Tbp2-Venus by qPCR using cDNA synthesized with mRNA obtained from WT, Tbp2 TBP:mCherry/+ , Tbp2 TBP:mCherry/TBP:mCherry , Tbp2 TBP2:Venus/+ and Tbp2 TBP2:Venus/TBP2:Venus oocytes. While Tbp mRNA is present in all the samples, Tbp-mCherry and Tbp2-Venus are not expressed (Figure 7-2). Moreover, the expression of Tbp2 is also abolished in both Tbp2 TBP2:Venus/TBP2:Venus and Tbp2 TBP:mCherry/TBP:mCherry oocytes (Figure 7-2), indicating that Tbp2 TBP:mCherry/TBP:mCherry and Tbp2 TBP2:Venus/TBP2:Venus mice are Tbp2 null mice. from WT, Tbp2 TBP:mCherry/+ , Tbp2 TBP:mCherry/TBP:mCherry , Tbp2 TBP2:Venus/+ and Tbp2 TBP2:Venus/TBP2:Venus oocytes. Expression was normalized to 18s rRNA.

I then asked the question why this knock-in strategy was not working as expected. An hypothesis was that Tbp2-Venus mRNA is degraded by RNA non-sense mediated decay (NMD) pathway. Therefore I cultured P14 Tbp2 TBP2:Venus/+ oocytes with or without emetine, a NMD inhibitor. After overnight incubation, the oocytes were collected for mRNA and cDNA

Results

Unpublished data 172 preparation, followed by qPCR analysis (Figure 7-3). cDNA samples prepared from both WT oocytes and nontreated Tbp2 TBP2:Venus/+ oocytes were used as control. Expression of Tbp2-Venus cannot be detected in any of the samples apart from the genomic DNA (Figure 7-3), indicating that the absence of Tbp2-Venus mRNA is not due to non-sense mediated decay, suggesting that Tbp2 TBP2:Venus is not transcribed. Expression of Tbp, Tbp2, Tbp-mCherry and Tbp2-Venus were analyzed by RT-qPCR using mRNA obtained from emetine treated Tbp2 TBP2:Venus/+ and control oocytes. Genomic DNA from Tbp2 TBP2:Venus/+ was used as a positive control for the TBP2-Venus primer. Expression was normalized to 18s rRNA.

Proteomics with oocytes

In order to study whether the TAFs are present at protein levels in growing oocytes, we performed proteomic analysis with P14 oocyte whole cell extracts from ∼4000 oocytes in duplicates and carried out an anti-TBP2 IP. We then performed proteomic analysis of both the IP elution and the flow through by mass-spec. Unfortunately, as no TBP2 peptide was detected, the experiment was not conclusive.

General discussion and perspectives 1. A TBP2-TFIIA-containing transcription machinery drives transcription in oocytes

The results presented in this thesis demonstrate that a TBP2-TFIIA-containing transcription machinery, different from TFIID, drives transcription in growing oocytes.

Importantly, a detailed discussion can be found in the Manuscript in Results section, where the results are discussed in light of the current knowledge. The following discussion section aims to present the results in a general context and to reveal questions that are still not addressed.

1.1 Which form of TFIIA is associated with TBP2?

As described in introduction 2.1.3 and 3.3.1, two of the three TFIIA subunits, TFIIAα and

TFIIAβ are encoded by a single gene and derived from the proteolytic cleavage of the TFIIAαβ precursor protein by Taspase1 (Zhou et al., 2006;[START_REF] Hoiby | A facelift for the general transcription factor TFIIA[END_REF]. Based on our proteomic results, we cannot determine whether the TBP2 associated TFIIA contains uncleaved TFIIAαβ, or processed TFIIAα and TFIIAβ. However, the cleavage of TFIIA is not necessary for its function in oocytes as Taspase1-deficient female mice are fertile (Oyama et al., 2013), indicating indirectly that TBP2 is probably associated with the uncleaved form of TFIIAαβ.

Further structural studies are required to determine which form of TFIIA (uncleaved or processed) interacts with TBP2 and how TBP2-TFIIA interacts with DNA.

1.2 Why are there genes upregulated following Tbp2 ablation?

Our RNA-seq analysis has shown that many genes are downregulated in the Tbp2 -/- oocytes. However, surprisingly, expression of a large number of genes (1577 at P7 and 1358 at P14) is upregulated following Tbp2 ablation. As TBP2 is supposed to play a general role in Pol II transcription initiation, then how can this upregulation be explained?

Detailed analyses revealed that most of the upregulated genes are expressed at low levels. Moreover, among the downregulated genes, many are involved in mRNA decay. Thus, it is possible that the upregulated genes are indirectly regulated following Tbp2 loss of function, and that the upregulation is due to mRNA decay defects in Tbp2 -/-oocytes.

Is TBP2 generally required for Pol II transcription in oocytes?

In growing mouse oocytes, all three TBP-type factors are expressed at mRNA levels (Xiao et al., 2006). However, at the protein level, TBP is undetectable, whereas TBP2 is highly expressed (Gazdag et al., 2007). Although it is not known whether TRF2 protein is present in the oocytes or not, Trf2-deficient mice do not show defects in female infertility (Martianov et al., 2001), indicating that TRF2 does not play crucial roles during oocyte growth, or at least its function can be compensated by TBP2. Altogether these data strongly suggest that TBP2 is required for most, if not all, Pol II transcription in oocytes.

In agreement with this hypothesis, we found that, in NIH3T3 cells ectopically overexpressing TBP2 (II10 cells), TBP2 binds to the promoter of more than 10000 genes. It is important to mention that in II10 cells, TBP2 can also be incorporated into a TFIID-like, complex. However, our RNA-seq analyses have shown that, in Tbp2 -/-oocytes, many genes (1720 at P7 and 1794 at P14) are downregulated, but they do not represent the majority of the genes that are expressed in the oocytes. A possible explanation could be that TRF2 is also involved in RNA pol II transcription, at least as a compensatory mechanism. Another possibility is that, since our RNA-seq analysis only gives a snapshot view of the steady state RNAs, transcript buffering mechanisms (Timmers et al., 2018) could mask more profound effect on transcription initiation. The ongoing experiments of TBP2 chromatin occupancy mapping and nascent transcripts analysis in oocytes will address this question.

How is TBP2 recruited to promoters without TAFs?

TAFs have been suggested to be important for recognition and binding to specific promoter elements, interaction with GTFs, as well as with activators and recognizing specific chromatin marks through their chromatin reading domains (Introduction 2.1.2.2 and 2.3.1).

However our results show that in oocytes TBP2 is associated with TFIIA, but not with TAFs.

TBP2 shares more than 90% identity with the TBP core domain and is able to bind the TATA-box (Persengiev et al., 2003;[START_REF] Bartfai | TBP2, a vertebrate-specific member of the TBP family, is required in embryonic development of zebrafish[END_REF], however, although TATA-box is enriched in TBP2 regulated genes, the majority of which do not contain a TATA-box (Manuscript figure 4A), raising the question how is TBP2 recruited to TATA-less promoters in oocytes?

Possible involvement of histone H3.3 in TBP2 recruitment to promoters

It has been shown that after nuclear transfer in Xenopus oocytes, molecular events of nuclear reprogramming take place in a sequential manner [START_REF] Jullien | Hierarchical molecular events driven by oocyte-specific factors lead to rapid and extensive reprogramming[END_REF]: firstly, a widespread binding of oocyte linker histone B4 replaces somatic linker histone H1; secondly, B4 around TSS is evicted due to the binding of H3.3 [START_REF] Braunschweig | Histone H1 binding is inhibited by histone variant H3.3[END_REF], finally, it has been suggested that transcription may be driven by TBP2-containing transcriptional machinery [START_REF] Jullien | Hierarchical molecular events driven by oocyte-specific factors lead to rapid and extensive reprogramming[END_REF] (Introduction Figure 34567). Interestingly, in mouse, double ablation of H3f3a

and H3f3b in folliculogenesis leads to early primary oocyte death, demonstrating a crucial role for H3.3 in oogenesis [START_REF] Tang | Contribution of the Two Genes Encoding Histone Variant H3.3 to Viability and Fertility in Mice[END_REF]. Moreover, ablation of H3.3 chaperone Hira in developing mouse oocytes results in increased DNA accessibility and aberrant transcription, specifically, decreased expression of highly expressed genes and increased expression of low or non-expressed genes [START_REF] Nashun | Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis[END_REF].

Taken together, either H3.3 itself or the dynamic DNA accessibility generated by H3.3 deposition plays crucial roles for TBP2-mediated transcription in oocytes. Perhaps in the more compacted 4N oocyte genome, DNA accessibility is more difficult and thus becoming a key determining factor for transcription machinery recruitment. Nevertheless, further investigations will be necessary on genome-wide chromatin accessibility in growing oocytes.

Recruitment through other factors in the TBP2-containing PIC

All the GTFs except TFIID should be present in the TBP2-containing PIC. It is known that TFIIB can bind to BRE [START_REF] Lagrange | New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB[END_REF][START_REF] Deng | A core promoter element downstream of the TATA box that is recognized by TFIIB[END_REF], it is thus possible that TBP2 can be recruited to the promoter though TFIIB. Besides, it is possible that TBP2-TFIIA complex contains other factors (unpublished results 5) which may help for the recruitment of TBP2.

Possible reason for the failure of TBP-TBP2 swap mouse model

In our TBP-TBP2 swap mouse model, the Tbp and Tbp2 cDNA constructions have been knocked into Tbp2 locus right after the start codon of endogenous Tbp2. However, neither

Tbp-mCherry nor Tbp2-Venus is transcribed. Since the knock-in strategy does not change any 5' regulatory sequence of Tbp2, a possible explanation would be the existence of promoter-proximal regulatory elements in the introns, especially the first intron [START_REF] Park | Conservation in first introns is positively associated with the number of exons within genes and the presence of regulatory epigenetic signals[END_REF]. An experiment to test this would be to clone the promoter region and first intron of Tbp2 with a reporter, followed by injection into growing oocytes.

Pol I and Pol III transcription in oocytes

TBP plays a crucial role in transcription initiation of all three RNA polymerases (Hernandez, 1993). Therefore, in growing oocytes, replacement of TBP by TBP2 (Gazdag et al., 2007;[START_REF] Muller | TBP2 is a general transcription factor specialized for female germ cells[END_REF], raises the question: how is Pol I and Pol III transcription regulated in oocytes?

In Xenopus, it has been reported that TBP2 is recruited to 5S rRNA Pol III promoter, suggesting that TBP2 may participate in Pol III transcription (Akhtar et al., 2009)

2.

Why is there a switch between TBP and TBP2-mediated transcription during oocyte growth?

TAFs proteins seem not be expressed in growing oocytes

Our RNA-seq data have shown that all the Taf genes (except Taf7l) are expressed in the oocytes at the mRNA levels. However, we were unable to detect TAF proteins in our proteomic analysis carried out on extracts prepared from growing oocytes (unpublished data 7.2), raising the question whether these proteins are indeed expressed in oocytes or whether their protein expression is under the detection limit of the experimental setup.

We have shown that in 3T3-II10 cells, besides the TBP2-TFIIA complex, TBP2 can also incorporate into a TFIID-type complex (Manuscript Figure 6), indicating that TBP2 can associate with TAFs. This is in good agreement with the high homology between the core domains of TBP and TBP2. However, in oocytes TBP2 associates only with TFIIA, but does not form a TFIID-type complex (Manuscript Figure 7). If the TAF proteins were expressed in oocytes, TBP should be able to interact with them, like in the TBP2 ectopically overexpressing cells. Thus, these results further suggest (however indirectly) that TAF proteins may not be expressed in oocytes, and thus this could be one possible reason for the basal transcription machinery switch.

The non-canonical epigenetic patterns require a different transcription machinery

As discussed in Introduction 1.3.2.3, oocytes possess non-canonical pattern of H3K4me3 (ncH3K4me3) that is enriched at low levels across large genomic regions (Zhang et al., 2016;[START_REF] Dahl | Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition[END_REF][START_REF] Liu | Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos[END_REF]Hanna et al., 2018b). In non-growing oocytes,

where TBP is still present, H3K4me3 is restricted to active promoters (Hanna et al., 2018b).

However, at further stages of oogenesis, when TBP is replaced by TBP2 (Gazdag et al., 2007), H3K4me3 accumulates at intergenic regions, putative enhancers and silent promoters through the recruitment of MLL2 to unmethylated CpG-rich regions in a transcription-independent manner (Hanna et al., 2018b), leading to the formation of ncH3K4me3 pattern. ncH3K4me3 pattern is maintained until early 2-cell stage and removed after zygotic genome activation (ZGA) at the late 2-cell stage [START_REF] Zhang | Allelic reprogramming of the histone modification H3K4me3 in early mammalian development[END_REF][START_REF] Dahl | Broad histone H3K4me3 domains in mouse oocytes modulate maternal-to-zygotic transition[END_REF][START_REF] Liu | Distinct features of H3K4me3 and H3K27me3 chromatin domains in pre-implantation embryos[END_REF].

Interestingly, at the same time TBP protein reappears in zygotes at low level and accumulates by 2-cell stage, allowing Pol II transcription initiation (Gazdag et al., 2007;[START_REF] Muller | TBP2 is a general transcription factor specialized for female germ cells[END_REF].

Importantly, it has been shown that TAF3 binds to H3K4me3 and facilitates PIC assembly [START_REF] Vermeulen | Selective anchoring of TFIID to nucleosomes by trimethylation of histone H3 lysine 4[END_REF][START_REF] Van Ingen | Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3[END_REF][START_REF] Lauberth | H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation[END_REF], and that H3K4me3, through interaction with TAF3, can direct PIC formation either independently or cooperatively with the TATA box [START_REF] Lauberth | H3K4me3 interactions with TAF3 regulate preinitiation complex assembly and selective gene activation[END_REF] 3). Thus, the TAF3-H3K4me3 interaction provides a way to recruit TFIID to the core promoter regions.

Moreover, it was proposed that ncH3K4me3 could function as 'sponges' to absorb and sequester transcription factors and regulators, therefore diluting transcription resources away from promoters [START_REF] Zhang | Allelic reprogramming of the histone modification H3K4me3 in early mammalian development[END_REF]. It is thus conceivable that, with the presence of ncH3K4me3 in oocytes, the canonical TBP-containing TFIID that contains TAF3, would not be able to find its targets efficiently, and that oocytes would require a TAF3-free transcription machinery to drive gene expression. This machinery could then be TBP2-TFIIA.

Lack of enhancer function in oocytes

It has been reported that mouse oocytes and fertilized eggs are not able to utilize enhancers [START_REF] Lawinger | Lack of enhancer function in mammals is unique to oocytes and fertilized eggs[END_REF], and recent studies suggest that that there may be enhancer function at the onset of oogenesis, which may be progressively eroded thereafter (Hanna et al., 2018b). Given the important role of enhancers in the transcription activation in the canonical transcription cycle, it is conceivable that lack of enhancer function in oocytes would require a different, a canonical TFIID-lacking, transcription machinery.

Other potential functions of TBP2

Different TSS usage of in oocytes

Our results also show that after its ectopic expression, TBP2 can bind to gene promoters genome-widely, however, the average binding profile of TBP2 shifts to the downstream of the canonical TBP-dependent TSS (unpublished data 2.2), suggesting that TBP2 would use a different TSS than TBP.

Interestingly, it has been shown that in zebrafish there is a switch in TSS usage throughout maternal to zygotic transition [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF], and it has been suggested that transcription initiation in the zebrafish oocyte may be mediated by TBP2 [START_REF] Haberle | Two independent transcription initiation codes overlap on vertebrate core promoters[END_REF].

With the new published SLIC-CAGE method [START_REF] Cvetesic | SLIC-CAGE: high-resolution transcription start site mapping using nanogram-levels of total RNA[END_REF], it is possible to analyze TSS usage from as little as 5-10 ng total RNA. Thus, it would be interesting to investigate whether TBP2-mediated transcription uses different TSSs in mouse growing oocytes or not.

Possible involvement of TBP2 in de novo DNA methylation

Several studies have shown that de novo methylation in mouse oocytes, at least at imprinted gDMRs, is coordinated with oocyte growth [START_REF] Obata | Maternal primary imprinting is established at a specific time for each gene throughout oocyte growth[END_REF][START_REF] Lucifero | Gene-specific timing and epigenetic memory in oocyte imprinting[END_REF][START_REF] Hiura | Oocyte growth-dependent progression of maternal imprinting in mice[END_REF], and acquisition of DNA methylation in oocytes is known to be positively correlated with transcription [START_REF] Chotalia | Transcription is required for establishment of germline methylation marks at imprinted genes[END_REF][START_REF] Smith | Transcription is required to establish maternal imprinting at the Prader-Willi syndrome and Angelman syndrome locus[END_REF][START_REF] Veselovska | Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape[END_REF].

Given the transcription defects in Tbp2 knockout oocytes, it would be interesting to analyze how TBP2 influences de novo DNA methylation in the oocyte.

Possible function of TBP2 in activation of zygotic genes

It has been shown that, in Xenopus and zebrafish embryos, TBP and TBP2 are both indispensable for embryonic development and are both required for activation of zygotic genes (reviewed in [START_REF] Muller | TBP2 is a general transcription factor specialized for female germ cells[END_REF]). In mouse oocyte, TBP2 accumulation gradually decreases after ovulation, is almost undetectable after fertilization by the two-cell stage (Gazdag et al., 2007), however, it is not known whether the residual TBP2 has any function in activation of zygotic genes before it totally disappears.

It has been shown that DUX-family transcription factors regulate ZGA in mammals [START_REF] De Iaco | DUX-family transcription factors regulate zygotic genome activation in placental mammals[END_REF][START_REF] Hendrickson | Conserved roles of mouse DUX and human DUX4 in activating cleavage-stage genes and MERVL/HERVL retrotransposons[END_REF][START_REF] Whiddon | Conservation and innovation in the DUX4-family gene network[END_REF], and that Dppa2 and Dppa4 directly regulate the Dux-driven zygotic transcription program [START_REF] Eckersley-Maslin | Dppa2 and Dppa4 directly regulate the Dux driven zygotic transcriptional programme[END_REF].

Interestingly, Dppa2 is downregulated more than 4-fold in Tbp2 -/-oocytes at both P7 and P14, and our preliminary data of TBP2 DamID-seq in oocytes showed that TBP2 binds to Dux gene.

Further study, for instance, by TBP2 Trim-Away in MII oocytes, could address this question.

Background

The histone acetyl trasferase (HAT), Tip60 (Tat interactive protein 60 kDa, also called KAT5) belongs to the MYST family of HATs that play key roles in acetylation of histones and other nuclear factors and thus influence chromatin structure and transcription regulation in the eukaryotic nucleus [1]. The defining feature of the MYST family of HATs is the presence of the highly conserved MYST domain, composed of an acetyl-CoA binding motif and a zinc finger [2]. The majority of cellular Tip60 exists in a stable nuclear multiprotein complex, called the mammalian Tip60 complex, consists of at least

Open Access

*Correspondence: laszlo@igbmc.fr 18 subunits and performs most transcription-and DNA damage-related Tip60 functions [3,4]. The yeast (y) homologue of Tip60 is the yEsa1 HAT that is a subunit of the yNuA4 complex [5]. This yNuA4 HAT complex, as well as the human Tip60-containing complex, contains a large number of homologue subunits [6,7]. In addition, the mammalian Tip60 complex seems to combine the functions of the yNuA4 HAT and the ySWR1 ATP-dependent chromatin remodelling complexes into a single complex [8]. The ATPase p400, belonging to the SWI2/SNF2 class of ATP-dependent chromatin remodelers [START_REF] Fuchs | The p400 complex is an essential E1A transformation target[END_REF], is an E1A-interacting protein essential for E1A-dependent apoptosis and cellular transformation [START_REF] Samuelson | p400 is required for E1A to promote apoptosis[END_REF]. The isolated mammalian Tip60 complexes were suggested to be heterogeneous, with a population that would contain p400 and another that would not, suggesting a dynamic assembly of the p400-containing Tip60 complex [7,[START_REF] Fuchs | The p400 complex is an essential E1A transformation target[END_REF][START_REF] Park | The SANT domain of p400 ATPase represses acetyltransferase activity and coactivator function of TIP60 in basal p21 gene expression[END_REF]. Tip60 complexes have three enzymatic functions: (1) a histone H2A/H4 acetlytransferase activity, (2) an ATPdependent H2AZ.H2B dimer exchange activity and (3) a helicase activity [7,8]. Several studies have shown that Tip60/NuA4-type complexes are involved in diverse cellular processes including transcription, cell cycle control, apoptosis, cell proliferation and DNA repair [4]. Mammalian Tip60 has been described as a transcriptional co-activator complex that is supposed to mediate the action of large variety of transcription factors, including nuclear receptors, c-Myc, STAT3, NF-kappaB, E2F1, p53 and others [4]. Importantly, a mass-spectrometry based study demonstrated that the intact Tip60-p400 (NuA4) HAT complex interacts with Myc and suggested that histone 3 and 4 acetylation patterns may be generated in part by interactions of Myc with the Tip60-p400 complex through Tip60 in mouse embryonic stem cells (mESCs) [START_REF] Kim | A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs[END_REF].

Homozygous knockout of the Tip60 gene in mouse results in pre-implantation lethality at embryonic day 3.5 [START_REF] Hu | Homozygous disruption of the Tip60 gene causes early embryonic lethality[END_REF]. Additionally, seven subunits of the Tip60 complex, including Tip60 and p400, have been further identified in an RNAi screen to be required for mESC maintenance [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF]. Moreover, siRNA down-regulation of six other components of the Tip60-complex exhibited the same phenotypic defects in alkaline phosphatase activity, embryonic body formation and teratoma formation as Tip60. This indicates that the whole Tip60 complex is necessary for mESC maintenance and normal mESC identity [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF]. Interestingly, siRNA-based depletion of Tip60 and p400 in mESCs resulted in an impaired expression of developmental regulators and expression of these affected genes significantly overlapped with that regulated by Nanog in mESCs [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF]. Chromatin immunoprecipitation (ChIP) linked to hybridization to promoter tiling arrays indicated that p400 localization correlates with H3K4me3 at both active and silent genes in mESCs [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF], though no anti-Tip60 ChIP or ChIP-seq was carried out in this study. Surprisingly, mRNA expression analyses identified that only about 800 genes were differentially regulated in both Tip60 and p400 knock-down mESCs [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF][START_REF] Chen | Hdac6 regulates Tip60-p400 function in stem cells[END_REF]. Moreover, a recent study demonstrated that Flag-Tip60containing complexes bind to active and developmental genes in mESCs [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF][START_REF] Chen | Hdac6 regulates Tip60-p400 function in stem cells[END_REF].

Interestingly, an additional HAT, Mof (males absent on the first or KAT8) was shown to be required for early mouse development and mESC pluripotency [START_REF] Gupta | The mammalian ortholog of Drosophila MOF that acetylates histone H4 lysine 16 is essential for embryogenesis and oncogenesis[END_REF][START_REF] Thomas | Mof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture[END_REF]. Recently, it has been shown that Mof-associated complexes have overlapping and distinct roles in mESCs [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF][START_REF] Chelmicki | MOFassociated complexes ensure stem cell identity and Xist repression[END_REF]. We hypothesise that there is a complex interplay between different transcriptional co-factors and that both Tip60-and Mof-containing complexes have distinct role in mESCs. To better characterize the genome-wide action of the Tip60 complex, we carried out an anti-Tip60 ChIP experiment coupled to high-throughput sequencing (ChIP-seq) in mESCs. Our data demonstrate that the Tip60 complex is present at all active promoters and a subset of well-defined mESC-specific enhancer sites, suggesting that mouse Tip60 complex plays a very broad role in regulating the gene expression programmes necessary for mESC maintenance.

Results

The Tip60 complex acts mainly in large molecular complexes and is enriched at active promoters in mESCs

In order to investigate whether Tip60 acts mainly in large molecular complexes in mESCs, nuclear extracts were prepared and subjected to gel filtration that allows separation of macromolecules of different sizes. The analysis of the gel filtration by western blot indicated that Tip60 is present mainly in fractions eluting around 2 MDa that may correspond to endogenous Tip60 complex (about 1.3 MDa) (Fig. 1a). Moreover, in these fractions Tip60 is present together with two other Tip60 complex subunits, Tip48 (or RuvBl2) and Baf53a [7]. Note that the three subunits are also present in smaller size fractions, but less abundantly (Fig. 1a). Importantly, Tip60 is only detectable at very low levels in fractions eluting around 60 kDa, suggesting that there is very little 'free' Tip60 in mESCs. These results indicate that Tip60 binding profiles will mainly represent the genome-wide binding of Tip60containing complexes.

To gain more insights into the genome-wide function of Tip60 in mESCs, we have generated high quality ChIPseq data using previously characterized purified polyclonal anti-Tip60 antibodies that specifically recognize endogenous Tip60 [START_REF] Frank | MYC recruits the TIP60 histone acetyltransferase complex to chromatin[END_REF]. Using MACS14 algorithm [START_REF] Zhang | Model-based analysis of ChIP-Seq (MACS)[END_REF], we determined high-confidence binding sites for Tip60. The validation of several randomly selected ChIP-seq positive sites by ChIP-qPCR indicated specific Tip60 enrichments at these sites, when compared to control IgG ChIP signals and to background enrichment at an intergenic region negative for Tip60 binding (Fig. 1b).

Next, we verified our genome-wide ChIP-seq Tip60 binding data at known Tip60-regulated genes [START_REF] Park | The SANT domain of p400 ATPase represses acetyltransferase activity and coactivator function of TIP60 in basal p21 gene expression[END_REF][START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF]. We also compared Tip60 binding at these genes with available data for DNase I hypersensitive sites (DHSs), H3K4me3 and RNA polymerase II (Pol II) profiles that are markers of open chromatin and active transcription. Importantly, Tip60 is enriched at these previously described target gene promoters (Rps9, Nodal and Cdkn1 [START_REF] Park | The SANT domain of p400 ATPase represses acetyltransferase activity and coactivator function of TIP60 in basal p21 gene expression[END_REF][START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF]), together with DHSs, Pol II binding and histone H3K4me3 mark (Fig. 1c-e). These results, together with the ChIP-qPCR validation (Fig. 1b), indicate that the obtained anti-Tip60 ChIP-seq signal is specific.

To analyze Tip60 binding genome-wide around all mESC transcription start sites (TSSs), we compared the binding of Tip60 and Pol II, and the appearence of the H3K4me3 mark at all ENSEMBL TSSs [START_REF] Ye | seqMINER: an integrated ChIP-seq data interpretation platform[END_REF] by k-means clustering. Interestingly, the resulting heatmap shows that Tip60 is enriched at virtually all Pol II and H3K4me3 positive promoters (11719) in mESCs (Fig. 2a).

To better characterize the genome-wide Tip60 binding around promoters, we selected all Pol II positive genes and analysed the global distribution of Tip60 around annotated TSSs (Fig. 2b). Surprisingly Tip60 peaks at about 200 bp downstream of the TSSs. Note that this Tip60 peak is slightly more downstream than the Pol II enrichment peak, which is known to be around 40-50 bps downstream of the TSSs (reviewed in [START_REF] Adelman | Promoter-proximal pausing of RNA polymerase II: emerging roles in metazoans[END_REF]).

The distinct binding profiles of Tip60, MSL and NSL complexes at TSSs suggest specific roles for these HAT complexes in transcription regulation

Genome-wide binding studies in differentiated human cells show a global co-localization of HATs and acetylated histones at transcriptional active promoters [START_REF] Wang | Genomewide mapping of HATs and HDACs reveals distinct functions in active and inactive genes[END_REF][START_REF] Wang | Combinatorial patterns of histone acetylations and methylations in the human genome[END_REF]. The mammalian HAT Mof is the catalytic subunit of the NSL (non-specific lethal) and MSL (male-specific lethal) complex [START_REF] Cai | Subunit composition and substrate specificity of a MOF-containing histone acetyltransferase distinct from the male-specific lethal (MSL) complex[END_REF][START_REF] Mendjan | Nuclear pore components are involved in the transcriptional regulation of dosage compensation in Drosophila[END_REF]. We have recently analysed the genome-wide binding of two complex specific subunits Nsl1 (NSL) and Msl1 (MSL) in mESCs [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF]. Since both Mof and Tip60 deletions affect mESC pluripoteny [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF][START_REF] Thomas | Mof (MYST1 or KAT8) is essential for progression of embryonic development past the blastocyst stage and required for normal chromatin architecture[END_REF][START_REF] Li | The histone acetyltransferase MOF is a key regulator of the embryonic stem cell core transcriptional network[END_REF] we were further interested in the genome-wide comparison of Tip60, NSL and MSL binding at promoters. Thus, we isolated 12304 Pol II positive ENSEMBL TSSs and conducted k-means clustering. The resulting heatmap in Fig. 2c indicates Tip60, Nsl1 and Msl1 enrichment in a subset of active promoters (upper cluster), while only Tip60 and Msl1 co-localize at the second subset of promoters (lower cluster). These results suggest that the function of Tip60, NSL and MSL complexes may overlap at certain, but not all promoters.

To better dissect the function of these complexes, we compared the binding distribution of Tip60, Nsl1 and Msl1 at all Pol II positive promoters (Fig. 2d). These analyses show that Nsl1 binds directly to the TSSs, Tip60 peaks about 200 bps downstream and Msl1 even more downstream of the TSSs (in the gene bodies [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF]) showing that all three HAT complexes have distinct binding profiles at promoters. These data suggest that the Tip60and Mof-containing (NSL and MSL) complexes may not have only redundant, but also specific roles in histone acetylation, histone variant exchange and/or transcriptional regulation. Additional genome-wide comparisons between Tip60 binding and available acetylated histone H3 and H4 profiles (H3K9ac, H3K27ac and H4K16ac) show that Tip60 overlaps with these marks, but peaks slightly upstream of the analysed acetylated H3 and H4 marks (Fig. 2e).

The large majority of Tip60 binding sites overlap with that of c-Myc

Tip60 is known to interact with and regulate various transcription factors as a transcriptional co-factor [4]. c-Myc is a transcription factor of the basic helix-loop-helix leucine zipper (bHLH-LZ) family, which dimerizes with another bHLH-LZ protein, Max [START_REF] Amati | Oncogenic activity of the c-Myc protein requires dimerization with Max[END_REF]. Importantly, the oncoprotein c-Myc recruits Tip60 [START_REF] Frank | MYC recruits the TIP60 histone acetyltransferase complex to chromatin[END_REF] and is regulated by the catalytic activity of Tip60 [START_REF] Patel | Analysis of genomic targets reveals complex functions of MYC[END_REF]. c-Myc is a somatic cell reprogramming factor (together with Oct4, Sox2, Klf4, Nanog and others) and a member of the so-called Myc regulatory module (together with n-Myc, Rex1, Zfx and E2f1) that is known to be involved in mESC self-renewal and cell metabolism [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Kim | An extended transcriptional network for pluripotency of embryonic stem cells[END_REF][START_REF] Yeo | The transcriptional regulation of pluripotency[END_REF]. Protein-protein interaction (See figure on next page.) Fig. 1 Tip60 binds to promoters as a complex in mESCs. a Gel filtration of mESC nuclear extracts. Every second fraction eluted from a Superose 6 column was analysed for the presence of Tip60, together with Tip49 and Baf53α by Western Blot. Native molecular weight markers eluting in the corresponding fractions are indicated on the top of the panel. b ChIP-qPCR validation of the ChIP-seq data in mESCs using purified anti-Tip60 antibodies [START_REF] Frank | MYC recruits the TIP60 histone acetyltransferase complex to chromatin[END_REF] and a negative control IgG antibody. Primers were designed at randomly selected MACS14 peaks with different tag densities (t), as indicated. An intergenic region (IR) without Tip60 binding was selected as an additional negative control. The graph represents the results obtained in two biological replicates (with three technical qPCR replicates each). Standard deviations are indicated. c-e Tip60 binding profiles (GSE69671) together with DNAse I hypersensitive sites (DHS) (GSM1014154), H3K4me3 (GSM307618) and Pol II (GSM307623) binding are shown at three selected genes (Rps9, Nodal and Cdkn1a) as demonstrated by the UCSC genome browser. The input (GSM798320) serves as control Ravens et al. Epigenetics & Chromatin (2015) networks revealed that Tip60 complex interacts with c-Myc in mESCs [START_REF] Kim | A Myc network accounts for similarities between embryonic stem and cancer cell transcription programs[END_REF]. Moreover, c-Myc binds predominantly active genes in mESCs [START_REF] Kidder | Stat3 and c-Myc genome-wide promoter occupancy in embryonic stem cells[END_REF] and c-Myc is known to recruit Tip60 to target promoters [START_REF] Martinato | Analysis of Myc-induced histone modifications on target chromatin[END_REF]. To better understand the genome-wide interactions between Tip60 and c-Myc at the chromatin, we analysed the overlap between Tip60 and c-Myc high-confidence binding sites, which were identified by MACS14 peak calling algorithm [START_REF] Zhang | Model-based analysis of ChIP-Seq (MACS)[END_REF].

When either all high-confidence (7693) Tip60 peaks are compared to published c-Myc sites (Fig. 3a), or when all 5318 c-Myc peaks are compared to Tip60 sites (Fig. 3b) using k-means clustering, about 50-65 % of all Tip60 e Average binding profiles of Tip60, H4K16ac (GSM1056596), H3K9ac (GSM775313) and H3K27ac (GSM594578) in a region of ∓1 kb around active TSSs was calculated. Reads were normalized to input. To be able to better compare the datasets, the H3K27ac tag densities were divided by five. The Input (GSM798320) serves as control Ravens et al. Epigenetics & Chromatin (2015) 8:45 binding sites are co-bound by c-Myc and vice versa. Importantly, other mESC pluripotency factors, such as Oct4, Sox2 and Nanog, are not enriched at the c-Myc and Tip60 co-bound sites (Fig. 3b), which is in agreement with the finding that the Myc-cluster appears to function independently from the core pluripotency network [START_REF] Kim | An extended transcriptional network for pluripotency of embryonic stem cells[END_REF][START_REF] Kidder | Stat3 and c-Myc genome-wide promoter occupancy in embryonic stem cells[END_REF]. These results suggest that the Tip60 complex is co-bound at about 50-65 % of Myc/Max sites in mESCs and that it is directly involved in regulating the c-Mycdependent transcriptional network.

Tip60 locates to transcriptional active genes in mESCs

To further characterize Tip60 function, we categorized the binding of Tip60 to different genomic regions.

When the MACS identified 7693 Tip60 binding peaks were annotated to promoter-TSS, 5′-or 3′-untranslated regions (UTRs), exons, introns and intergenic regions, about 42 % of all high-confidence Tip60 binding peaks were found at promoter-TSS regions (Fig. 4a). Interestingly, about 35 % of the binding sites were localized at either intronic (24 %) or intergenic regions (11 %), suggesting that Tip60 may also play a role in regulating enhancer activity (see below). Next, we were interested in expression levels of Tip60 enriched genes and how Tip60 binding correlates with gene expression. To isolate Tip60 bound ENSEMBL genes, each Tip60 peak was annotated to its closest gene in a region of 1 kb up-or downstream of TSSs. Thus, we defined 6193 genes bound by the Tip60 complex. Importantly, the average expression level of all Tip60 positive genes is significantly higher compared to that of all ENSEMBL genes (Fig. 4b), suggesting that Tip60 complexes bind predominantly to active genes. As it is known that the Pol II binding strength at promoters reflects the gene expression levels [START_REF] Barski | Highresolution profiling of histone methylations in the human genome[END_REF] (Fig. 4c), we wanted to analyse whether this would be the case for Tip60 complexes. Similarly to Pol II, the Tip60 peak tag density positively correlates with the gene expression level of bound genes (Fig. 4d). Altogether our data suggest that Tip60 locates to Pol II positive and transcriptional active genes.

Tip60 binds also to bivalent genes and active enhancer elements

To further address the global distribution and function of the Tip60 complex in mESCs, we compared high-confidence Tip60 binding sites with marks, which are either associated with active transcription at promoters (Pol II and H3K4me3) or enriched at enhancer sites (H3K4me1 and H3K27ac) [START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF][START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]. We also analysed H3K27me3, which establishes, together with H3K4me3, a bivalent chromatin state in mESCs at developmental genes [START_REF] Bernstein | A bivalent chromatin structure marks key developmental genes in embryonic stem cells[END_REF]. After conducting k-means clustering using seqMINER against all identified Tip60 peaks [START_REF] Ye | seqMINER: an integrated ChIP-seq data interpretation platform[END_REF], a large colocalization with Pol II, H3K27ac and H3K4me3 enriched sites was found (6136 peaks in Fig. 5a). Our GO term analyses of Tip60 enrichment at Pol II positive genes show that these Tip60 positive genes are involved not only in biological functions such as 'metabolic processes' and 'gene expression' , but also in 'cell cycle and 'cellular response to stress' (Fig. 5b, upper panel). Importantly, some of these GO categories, i.e. metabolic processes' , are identical than the ones defined for c-Myc [START_REF] Kim | An extended transcriptional network for pluripotency of embryonic stem cells[END_REF], in agreement with our finding that at 50-65 % Tip60-and c-Myc-bound loci these factors cooperate in gene regulation.

The heatmap in Fig. 5a further indicates that Tip60 binds to about 400 sites, which are positive for H3K4me3 and H3K27me3 and are thus defined as bivalent sites (Fig. 5a). Tip60 binding at a representative bivalent gene is further illustrated in Fig. 5c. GO term analyses of all Tip60-bound bivalent genes resulted in a significant enrichment of GO terms with developmental functions (see Fig. 5b lower panel). In agreement with observations that bivalent genes are very weakly transcribed (reviewed in [START_REF] Voigt | A double take on bivalent promoters[END_REF]), Tip60 tag density at bivalent sites (green line) was lower than Tip60 enrichment observed at 'Pol II positive peaks' (blue line) (Fig. 5d). Therefore, our analyses show that Tip60 complexes locate also to bivalent or developmental genes, as suggested previously [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF][START_REF] Chen | Hdac6 regulates Tip60-p400 function in stem cells[END_REF].

Interestingly, two of the last Tip60 positive clusters within the heatmap (Fig. 5a) show low Pol II enrichment levels. One of these Tip60 positive clusters, comprising 476 sites, contains high levels of H3K4me1 and H3K27ac marks, suggesting that these sites correspond to active enhancers [START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF][START_REF] Heintzman | Distinct and predictive chromatin signatures of transcriptional promoters and enhancers in the human genome[END_REF]. The comparison of H3K4me1/ K3K27ac positive Tip60 peak distances to TSSs of annotated genes revealed that these peaks are located at distal regulatory regions, such as active enhancers (Additional file 1: Figure S1a,b). These findings are further illustrated at a known enhancer region, (see UCSC genome browser tracks at Additional file 1: Figure S1c). The second 'very low Pol II' cluster may represent less active enhancers and other not well-characterized genomic regions. Nevertheless, clustering of Tip60 with the above-described well-known chromatin marks and Pol II allowed us to suggest that Tip60-containing complexes act mainly at active Pol II promoters, at bivalent genes and at active enhancers.

Tip60 defines a subset of mESC-specific enhancer

As our above analyses suggested that Tip60 complexes could bind to enhancers; we wanted to examine the total enrichment of Tip60 at all known enhancer sites. To this end, we have taken annotated enhancer sites from mESCs [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF]. Since active enhancers often have high H3K27ac levels [START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF], enhancers were sorted for H3K27ac signal intensity and analysed for enrichment of Tip60, p300, H3K4me1 and DHS. Interestingly, on these enhancer sites Tip60 binding showed a co-occurence with p300, H3K4me1 and DHSs and a partial overlap with a subset of sites having high H3K27ac signals (Fig. 6a). Next, we divided these H3K27ac labelled enhancers into four equal clusters (from high to low H3K27ac signals) and average tag densities for Tip60, as well as for DHS profiles, around enhancer mid points of each cluster were calculated (Fig. 6b,c). Importantly, Tip60 and DHS enrichment have a positive correlation with each other and with that of H3K27ac. To define the number of enhancers that are positive for Tip60 and all the other enhancer defining marks (H3K4me1, H3K27ac and DHS), k-means clustering was performed to analyse the presence of Tip60, p300, H3K4me1, H3K27ac and DHS at these defined enhancer sites [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF]. The heatmap analysis in Fig- ure S2A clearly shows that 2305 enhancers defined in cluster 1 are positive for Tip60 and all the other enhancer chromatin marks (p300, H3K27ac and H3K4me1). In contrast, the 6489 enhancers in cluster 2 show almost no Tip60 enrichment (Additional file 2: Figure S2b,c). Moreover, GO terms of genes in the vicinity of the 2305 Tip60 bound enhancers are associated with developmental processes (Fig. 6d). Thus, it seems that Tip60 is recruited to about 26 % of all mESC-specific enhancers. As two of the so-called 'super enhancer' regions of the Nanog gene and the enhancer of the Klf4 gene are bound by Tip60, but not by Nsl1 and Msl1 (Fig. 7a,b), we further characterized Tip60 binding at enhancer regions in detail by analysing Tip60 presence at the previously defined 231 'super enhancer' regions [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF]. We observed Tip60 enrichment (compared to the Input control) between the start and end positions of these 231 'super-enhancers' (Fig. 7c; Additional file 2: Figure S2d). However, global analyses of Mof-containing complexes at these 'super-enhancer' regions displayed Msl1, but no or very weak Nsl1 enrichment (Fig. 7d,e). Altogether our data show that Tip60 is recruited to a subset of active enhancers out of which certain have been defined as super enhancers in mESCs.

Discussion

In this study, we analysed the genome-wide binding of Tip60-containing complexes to understand their role in transcription regulation in mESCs. Our gel filtration analyses show that Tip60 incorporates into large molecular complexes in mESCs, as previously described in other systems, and that heterogeneous populations of Tip60containing complexes exist, which might dynamically change their association with p400 and/or other subunits. However, our observations that there is very little free Tip60 within the nuclei of mESCs indicates that the ChIP binding profiles obtained with anti-Tip60 antibodies represent mostly the behaviour of the corresponding endogenous Tip60 complexes. 50 -chr6:122,596,311-122,672,445 (76,135 bp) The 231 super enhancers [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF] were divided into 80 bins and Tip60 (b), Nsl1 (c) and Msl1 (d) enrichment was calculated per bin between their start and end positions as well as up to 4 kb up-and downstream. Signals were normalized to the input (GSM798320)

Our study demonstrates that the majority of genomewide Tip60 binding occurs at promoter regions, where it co-localizes with Pol II, H3K4me3 and DHS sites. Importantly, Tip60 bound genes are expressed and the Tip60 enrichment positively correlates with gene expression levels. Interestingly, using an unbiased clustering method of all mapped Tip60 reads against ENSEMBL TSS, and we observed that all transcriptionally active genes (Pol II positive) show Tip60 enrichment. Genome-wide binding and knock-down studies of Tip60-p400, NSL and MSL HAT complexes reported the binding of these complexes at Pol II positive genes and in mESCs [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF][START_REF] Chelmicki | MOFassociated complexes ensure stem cell identity and Xist repression[END_REF]. When directly comparing binding profiles of Tip60-and Mof-containing complexes around Pol II positive promoter regions, we have found a global overlap at Pol II positive mRNA coding genes, whereas each of the HAT complexes has a distinct binding profile around the TSS. Altogether, this suggests that (1) Tip60-and Mof-containing HAT complexes globally regulate gene expression though their presence at promoters (NSL and Tip60) and further downstream of promoters (i.e. MSL) and ( 2) that there might be a different function of each HAT complex in histone acetylation, histone exchange and transcriptional regulation.

The frequent overlap of Tip60 and c-Myc binding strongly indicates a role of Tip60 complexes within the Myc-centred regulatory pluripotency network in mESC [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF][START_REF] Yeo | The transcriptional regulation of pluripotency[END_REF]. Moreover, Tip60 complexes bind and possibly regulate a subset of active enhancers and super enhancers, revealing an additional layer of regulation by which Tip60 complexes influence mESC maintenance. Tip60 complexes preferentially acetylate histone H4 [8]. Acetylation of several histone H3 and H4 lysines was described to be up-regulated at the majority of Myc-target promoters [START_REF] Eberhardy | Direct examination of histone acetylation on Myc target genes using chromatin immunoprecipitation[END_REF]. Several studies in Drosophila, mESCs and human cells have revealed that c-Myc targets at least 10-15 % of all cellular promoters [START_REF] Martinato | Analysis of Myc-induced histone modifications on target chromatin[END_REF]. Nevertheless, it has been suggested that although Myc can bind to a large number of genes, it is critical for the regulation of a subset of those genes depending on (1) protein-protein interactions between Myc/Max dimers, such as chromatin-bound protein complexes [START_REF] Sabo | Genome recognition by MYC[END_REF] and (2) the cellular or physiological context [START_REF] Perna | Genome-wide mapping of Myc binding and gene regulation in serumstimulated fibroblasts[END_REF]. Our finding that at least half of the c-Myc binding sites are co-bound by the Tip60 complex suggests that Myc/Max and the Tip60 complex may cooperate to stabilize each other's binding. Moreover, at these co-bound sites the Tip60 complex is thought to be involved in acetylation of promoter-associated nucleosomes that can participate in transcriptional activation [START_REF] Martinato | Analysis of Myc-induced histone modifications on target chromatin[END_REF].

Interestingly, the genome-wide Tip60 complex binding profile peaks at regions that are about 200 bps downstream of TSSs (Fig. 2b). It is, therefore, conceivable that this downstream binding may reflect an additional function of the Tip60 complex that may play a role in the early steps of transcription elongation processes, as suggested also in Drosophila [START_REF] Kusch | Histone H3 lysine 4 trimethylation regulates cotranscriptional H2A variant exchange by Tip60 complexes to maximize gene expression[END_REF]. Interestingly, c-Myc is believed to control the release of Pol II from promoter proximal transcriptional pause [START_REF] Perna | Genome-wide mapping of Myc binding and gene regulation in serumstimulated fibroblasts[END_REF]. Thus, the Tip60 complexes bound 200 bps downstream of the TSS at many active genes may cooperate with c-Myc to co-regulate Pol II pause release and consequently, transcriptional elongation in mESCs.

Moreover, our results indicate that Tip60 complexes may be involved in gene expression regulation by occupying a set of previously identified ESC-specific enhancers [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF]. Tip60 enrichment correlates with H3K27ac levels and chromatin accessibility (DHS sites). Genes associated with enhancers with increased Tip60 binding (determined by k-means clustering) are predicted to be involved in regulation of development, metabolism and proliferation of mESCs. Based on high Mediator occupancy, some of these enhancers can be clustered to 231 super-enhancer regions [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF], which likely control important genes for stem cell maintenance (i.e. the Nanog or Klf4 gene). Importantly, these super-enhancers show Tip60 and Msl1 occupancy, but have only very little Nsl1 binding. The fact that Tip60 possibly regulates a subset of active enhancers and super enhancers reveals an additional layer of regulation by which Tip60 complexes influence mESC maintenance. Note that previously we have described enhancers, which were bound by the ATAC HAT complex but not by p300 in differentiated human cells [START_REF] Krebs | SAGA and ATAC histone acetyl transferase complexes regulate distinct sets of genes and ATAC defines a class of p300-independent enhancers[END_REF]. Thus, it is possible that different enhancers are bound by a given combination of HAT complexes to regulate their activity.

Human Tip60 is known to play a wide role in transcriptional regulation [START_REF] Sapountzi | Cellular functions of TIP60[END_REF]. The identified Tip60 bound genes belong to gene sets with housekeeping and developmental functions. Other studies further suggested that the Tip60 complex might have a repressive function at low expressed bivalent/developmental genes [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF][START_REF] Chen | Hdac6 regulates Tip60-p400 function in stem cells[END_REF]. Since the Tip60 complex has different catalytic activities by acetylating histones or exchanging H2A.Z, which further depend on each other, it will be challenging to understand the molecular mechanism by which the Tip60 complexes regulate transcription at these low-expressed genes in mESCs. Nevertheless, it is tempting to speculate that the Tip60 complex is recruited to these genes to poise these genes that will be important for their rapid activation during cellular differentiation.

Our genome-wide binding analyses suggest that the Tip60 complex has a very broad role in regulating transcription in mESCs, which is in agreement with the observation that Tip60 and several other Tip60-complex subunits are required for mESC maintenance [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF]. In contrast, Tip60 knock-down studies identified less than 900 differentially expressed genes when analysing steady-state mRNAs [START_REF] Fazzio | An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity[END_REF][START_REF] Chen | Hdac6 regulates Tip60-p400 function in stem cells[END_REF]. The potential reason for this apparent contradiction may be that the previous Tip60 knock-down studies analysed steadystate mRNAs instead of newly synthetized transcripts. Recently, it has been demonstrated that the sole analysis of polyA + mRNAs may not give any information on the direct transcriptional output of a given transcription factor/co-activator complex, as cells can buffer global transcription changes by adjusting mRNA decay in parallel ( [START_REF] Bonnet | The SAGA coactivator complex acts on the whole transcribed genome and is required for RNA polymerase II transcription[END_REF][START_REF] Sun | Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation[END_REF][START_REF] Helenius | Requirement of TFIIH kinase subunit Mat1 for RNA Pol II C-terminal domain Ser5 phosphorylation, transcription and mRNA turnover[END_REF] and refs therein). Thus, novel newly synthetized RNA detection methods are needed to address global effects of Tip60 on newly synthetized transcripts, such as in vivo RNA labelling with 4-thiouridine (4sU) [START_REF] Sun | Comparative dynamic transcriptome analysis (cDTA) reveals mutual feedback between mRNA synthesis and degradation[END_REF] or analyses of profiles of transcriptional active Pol II (i.e. NET-seq; [START_REF] Mayer | Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution[END_REF]) under ∓ Tip60 conditions. Furthermore, the understanding of how the different activities (acetylation, histone variant exchange and/or nucleosome remodelling activity) of the Tip60 complexes influence transcription at promoters and enhancers in mESCs will be an equally important task.

Conclusions

Overall, we establish that Tip60-complexes are present at promoter regions of active RNA polymerase II genes and that half of Tip60 binding sites overlap with binding of the somatic cell reprograming factor, c-Myc, known to regulate an ESC specific transcriptional module. Importantly, Tip60, NSL and MSL coactivator HAT complexes have a genome-wide overlap at many active genes, but their specific functions might be reflected in their distinct binding profiles around the TSSs. Moreover, Tip60 complexes define a subset of bivalent developmental genes and a subset of ESC-specific enhancers. Thus, our study suggests that the Tip60 complex is important for mESC pluripotency by acting as a global transcriptional co-factor at active genes and distal regulatory elements.

Methods

Cell culture

The ES cell line, E14 was derived from 129P2/OlaHsd strain blastocysts [START_REF] Hooper | HPRT-deficient (Lesch-Nyhan) mouse embryos derived from germline colonization by cultured cells[END_REF] and cells were cultivated on 0.1 % gelatine (Sigma, France) and CD1 feeder cells (37 °C, 5 % CO 2 ) in DMEM (4,5 g/L glucose/w-Glutamax) (Invitrogen, France), 15 % foetal calf serum (PAA), 5uM LIF, 50 mM ß-mercaptoethanol (Invitrogen, France), penicillin (10,000 U/ml) and streptomycin (10 mg/ml) (Invitrogen, France), 200 mM l-glutamine (Invitrogen, France) and 1× non-essential amino acids (GIBCO, France). To work under feeder-free conditions cells were treated with 1 mg/ml Collagenase and 2 mg/mL Dispase (GIBCO, France) and cultivated for one passage without feeder cells on 0.1 % gelatine plates. Experiments were conducted with E14.wt cells at passages 26-29.

Chromatin immunoprecipitation coupled to Solexa sequencing (ChIP-seq)

ChIP was carried out as described in [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF]. For 500 μg of chromatin 6 μg of the well-characterized rabbit anti-Tip60 antibody mixture (RLPV, CLGT and CLHF purified polyclonal sera) was used [START_REF] Frank | MYC recruits the TIP60 histone acetyltransferase complex to chromatin[END_REF]. 8 ng of precipitated DNA from Tip60 ChIP was used for Solexa sequencing. Rabbit anti-IgG antibody (ab37415) was purchased from Abcam. To create a genomic library the instructions of NEBNext protocol (E6240, Biolabs) was followed. The library was validated with the Agilent Bioanalyzer. Single reads run sequencing was done with Illumina HiSeq 2000. Image analysis and base calling were conducted with the Illumina pipeline (1.8.2). The July 2007 Mus musculus genome assembly (NCBI37/mm9) from NCBI was used for the sequence alignment by the software Bowtie (0.12.7) [START_REF] Langmead | Aligning short sequencing reads with Bowtie. Current protocols in bioinformatics/editoral board[END_REF]. The analysis was conducted with unique reads. Read density (wig) files were created out of bed files by extending reads to 200 bp length and creating 25 bp bins. To detect ChIP-seq peaks the MACS14 peak-calling algorithm was applied using default parameters [START_REF] Zhang | Model-based analysis of ChIP-Seq (MACS)[END_REF]. The Tip60 ChIP-seq data were deposited in the Gene Expression Omnibus (GEO) databank under the accession number GSE69671.

For further analyses the following ChIP-seq files were included from Gene Expression Omnibus(http://www. ncbi.nlm.nih.gov/geo/): Input (GSM798320) [START_REF] Karmodiya | H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells[END_REF], RNA polymerase II (GSM307623), H3K4me3 (GSM307618), c-Myc (GSM288356), Oct4 (GSM288347), Sox2 (GSM288346) and Nanog (GSM288345) [START_REF] Chen | Integration of external signaling pathways with the core transcriptional network in embryonic stem cells[END_REF], H3K4me1 (GSM594577), H3K27ac (GSM594578) [START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF], H3K9ac (GSM775313) [START_REF] Karmodiya | H3K9 and H3K14 acetylation co-occur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells[END_REF], H4K16ac (GSM1056596) [START_REF] Taylor | H4K16 acetylation marks active genes and enhancers of embryonic stem cells, but does not alter chromatin compaction[END_REF], p300 (GSM723018) [START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF], H3K27me3 (GSM307619) [START_REF] Mikkelsen | Genome-wide maps of chromatin state in pluripotent and lineage-committed cells[END_REF], Nsl1 (GSM1300940) and Msl1 (GSM1300939) [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF]. Tip60 data are deposited in GEO under the following accession number: GSE69671. Fastq files were generated from SRA lite format and aligned to the NCBI37/mm9 assembly using Bowtie (0.12.7) [START_REF] Langmead | Aligning short sequencing reads with Bowtie. Current protocols in bioinformatics/editoral board[END_REF]. DNAse I hypersensitive sites were taken from Encode/UW (GSM1014154). Detailed information summarizing all the used ChIP-seq files is presented in Additional file 3: Table S1.

ChIP-qPCR

The Tip60 ChIP-seq was validated by ChIP coupled to quantitative PCR (ChIP-qPCR). Identified MACS14 Tip60 peaks were randomly taken based on different tag densitites (t). SYBR Green (Roche) was used according to the manufacturers protocol. Following primers were designed: Ravens et al. Epigenetics & Chromatin (2015)

Gel filtration

The gel filtration experiment using a Superose 6 column was described in [START_REF] Ravens | Mofassociated complexes have overlapping and unique roles in regulating pluripotency in embryonic stem cells and during differentiation[END_REF]. Input nuclear extract and every second fraction eluting from the column were tested by western blot assays using the Tip60 antibody mixture [START_REF] Frank | MYC recruits the TIP60 histone acetyltransferase complex to chromatin[END_REF] at dilution 1:2000, the anti-Tip48 or anti-Baf53α antibodies [START_REF] Robert | The transcriptional histone acetyltransferase cofactor TRRAP associates with the MRN repair complex and plays a role in DNA double-strand break repair[END_REF] at dilution 1:500.

Bioinformatics analyses of Tip60 ChIP-seq in mESCs

Density profile calculation around TSSs as well as K-means linear clustering was conducted with seqMINER [START_REF] Ye | seqMINER: an integrated ChIP-seq data interpretation platform[END_REF]. K-means clustering was performed with normalized read densities, while resulting heatmaps show total number of reads. Obtained MACS14 peaks were annotated using the software HOMER [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cisregulatory elements required for macrophage and B cell identities[END_REF] based on the ENSEMBL 67 database (mm9).

To determine the Tip60 or Pol II enrichment at genes, the peak tag density of the nearest peak to the TSS (in a region of +2 kb) was taken and correlated with gene expression levels. We considered a total of 26,460 ENSEMBL TSSs based on the ENSEMBL 67 database (mm9). For this, raw RNA-seq data of mESCs from Gene Expression Omnibus (GSE34473) were processed using the software tools TopHat [START_REF] Trapnell | TopHat: discovering splice junctions with RNA-Seq[END_REF] and HTSeq with default parameters. FPKM (fragments per kilobase of exon per million fragments mapped) values were calculated with Cufflinks [START_REF] Roberts | Identification of novel transcripts in annotated genomes using RNA-Seq[END_REF]. FPKM values were correlated with Tip60 and Pol II enrichment and taken to analyse average expression levels of Tip60 bound genes.

Gene ontology (GO) term analyses of Pol II positive or bivalent peaks as well as enhancer sites were conducted with Manteia [START_REF] Tassy | Manteia, a predictive data mining system for vertebrate genes and its applications to human genetic diseases[END_REF]. Peaks were annotated to nearest promoters [START_REF] Heinz | Simple combinations of lineage-determining transcription factors prime cisregulatory elements required for macrophage and B cell identities[END_REF] prior GO term analysis.

ES-specific enhancers and super enhancer regions were taken from [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF]. ES-specific enhancers were ranked according to H3K27ac signal intensities. Enhancer-midpoints were calculated for further analysis. The 231 total super enhancers were divided into 80 bins from start to end positions and the mean Tip60 and Input read densities were calculated for each bin. Moreover, read densities of regions 4 kb down-or upstream of super enhancer start or end positions were determined for each 50 bp bin. Total Number of reads is normalized to the Input.

Changwei YU Analysis of the composition and the function of oocyte-specific TBP2-containing transcription machinery during mouse oogenesis

Résumé

La synthèse d'ARN au cours de la différenciation des ovocytes est essentielle à la fécondation et à l'initiation du développement précoce. La nature de la machinerie basale de transcription pendant la croissance ovocytaire n'est pas connue mais la protéine TBP est remplacée par une protéine semblable spécifique des vertébrés, TBP2. Pour comprendre le rôle de TBP2 dans l'initiation de la transcription, nous avons effectué un RNA-seq à partir d'ovocytes contrôles et Tbp2-/-et montré que l'expression des gènes les plus transcrits ainsi celle des éléments rétroviraux endogènes de type MaLR est diminuée. Par immunoprécipitation couplée à la spectrométrie de masse à partir d'ovaires, nous avons montré que TBP2 ne forme pas un complexe TFIID, mais est associé à TFIIA dans les ovocytes. Globalement nos données montrent qu'une machinerie d'initiation de la transcription spécifique différente du complexe canonique TFIID contrôle la transcription dans les ovocytes de souris.

Mots clés : TFIID, TBP2, RNA polymerase II, TFIIA, MaLR, oocytes

Résumé en anglais

Mammalian oocytes go through consecutive differentiation process, during which the synthesis and accumulation of RNAs are essential for oocyte growth, maturation, fertilization and early embryogenesis. Little is known about the nature and function of the oocyte Pol II transcription machinery. During oocyte growth TBP is replaced by a vertebrate specific paralog, TBP2, and Tbp2-/-females are sterile. To understand whether and how TBP2 is controlling transcription initiation during oogenesis, we carried out RNA-seq analyses from wild-type and Tbp2-/-oocytes from primary and secondary follicles. These analyses show a main decrease in the expression of the most abundant genes as well as specific down-regulation of the expression of the MaLR-type endogenous retroviral elements. To identify the nature of the complex associated with TBP2 in the oocytes, we carried out immunoprecipitation followed by mass spectrometry. We demonstrate that, in the oocytes, TBP2 associates with TFIIA, but does not assemble into a TFIID-type complex. Altogether, our data show that a specific TBP2-TFIIA-containing transcription machinery, different from canonical TFIID, drives transcription in mouse oocytes.

Keywords : TFIID, TBP2, RNA polymerase II, TFIIA, MaLR, oocytes

1 .

 1 Identification de complexes similaires à TFIID contenant TBP2 Des études antérieures menées dans le laboratoire ont montré l'existence d'une machinerie basale de la transcription spécifique aux ovocytes. Afin de caractériser cette machinerie, nous avons utilisé la technique d'immunoprécipitation couplée à la spectrométrie de masse. Dans un premier temps, nous avons généré et testé 10 anticorps polyclonaux de lapin dirigés contre la protéine TBP2 murine afin de sélectionner les meilleurs anticorps à utiliser pour les techniques de western-blot, d'immuno-précipitation (IP), d'immuno-fluorescence (IF) et d'immuno-précipitation de la chromatine (ChIP). Dans une lignée de cellules 3T3 surexprimant TBP2 (lignée II10), j'ai d'abord recherché les protéines de TFIID potentiellement associées à TBP2. Il se trouve que TBP2 est retrouvé dans un complexe similaire à TFIID mais TBP2, contrairement à TBP, n'interagit pas avec TFIIB, BTAF1 et les sous-unités des machineries transcriptionnelles associées avec l'ARN Pol I et Pol III. En revanche, d'après les analyses Résumé xiii d'immunoprécipitation couplées à la chromatographie d'exclusion, TBP2 interagit fortement avec TFIIA au sein d'un même complexe.

2 .

 2 Identification des gènes régulés par TBP2 dans les ovocytes Afin d'identifier les gènes régulés par TBP2 dans les ovocytes, j'ai comparé les profils d'enrichissement de TBP2 au niveau de la chromatine, obtenus avec les techniques de DamID et Cut&Run, avec les niveaux d'expression des gènes, obtenus par mRNA-seq et SLAM-seq. i. RNA-seq et SLAM-seq dans les ovocytes Les ARN collectés à partir d'ovocytes contrôles ou mutants pour Tbp2 chez des souris âgées de 7 ou de 4 jours ont été séquencés par RNA-seq. Les analyses ont révélé que les niveaux d'expression de plus d'un millier de gènes sont diminués de plus de deux fois à 7 et 14 jours après la naissance. Parmi ces gènes, de nombreux rétrovirus endogènes sont présents, dont notamment les MaLR. Afin de confirmer ces résultats, j'ai analysé la transcription naissante grâce à la technique de SLAM-seq. Les résultats sont en cours d'analyse.

Figure 1 - 1 :

 11 Figure 1-1: The hierarchical model of chromatin organization...................................................

Figure 1 - 2 :

 12 Figure 1-2: 3D genome organization. .........................................................................................

Figure 1 - 3 :

 13 Figure 1-3: Cycle of active DNA demethylation. ........................................................................

Figure 1 - 4 :

 14 Figure 1-4: Histone variants of human core histones. .............................................................

Figure 1 - 5 :

 15 Figure 1-5: Chromatin plasticity. ...............................................................................................

Figure 1 - 6 :

 16 Figure 1-6: Proposed mechanisms for PGC specification in mice. .........................................

Figure 1 - 7 :

 17 Figure 1-7: Scheme of mouse PGCs specification and migration. ..........................................

Figure 1 - 8 :

 18 Figure 1-8: Overview of mouse spermatogenesis. ..................................................................

Figure 1 - 9 :

 19 Figure 1-9: Schematic representation of oogenesis. ...............................................................

Figure 1 - 10 :

 110 Figure 1-10: Chromatin dynamics during gametogenesis and embryogenesis. .....................

Figure 2 - 1 :

 21 Figure 2-1: Pol II basal transcription machinery. ......................................................................

Figure 2 - 2 :

 22 Figure 2-2: Structure of Pol II. ..................................................................................................

Figure 2 - 3 :

 23 Figure 2-3: The composition and conservation of Pol II CTD. .................................................

Figure 2 - 4 :

 24 Figure 2-4: Structure of TBP core domain with / without TATA box. ........................................

Figure 2 - 5 :

 25 Figure 2-5: Schematic representation of human TFIID subunits. ............................................

Figure 2 - 6 :

 26 Figure 2-6: Core promoter recognition by TFIID. .....................................................................

Figure 2 - 7 :

 27 Figure 2-7: Structure of the human core-TFIID and model for TFIID assembly. .....................

Figure 2 - 8 :

 28 Figure 2-8: Structure of the yeast Pol II-TFIIB complex. ..........................................................

Figure 2 - 9 :

 29 Figure 2-9: TFIIE architecture and interactions. .......................................................................

Figure 2 - 10 :

 210 Figure 2-10: Structure of yeast TFIIH within PIC and DNA opening. .......................................

Figure 2 - 11 :

 211 Figure 2-11: The transcription cycle and its potentially regulated steps. .................................

Figure 2 - 12 :

 212 Figure 2-12: Subunit composition of the Mediator complex. ...................................................

Figure 2 - 13 :

 213 Figure 2-13: SAGA modular organization and the subunits shared with other complexes. ....

Figure 2 - 14 :

 214 Figure 2-14: Chromatin opening and PIC assembly. ...............................................................

Figure 2 - 15 :

 215 Figure 2-15: Schematic representation of PIC assembly and transcription initiation. .............

Figure 2 - 16 :

 216 Figure 2-16: Transcription initiation and Pol II promoter escape. ............................................

Figure 2 - 17 :

 217 Figure 2-17: Pol II promoter-proximal pausing. ........................................................................

Figure 2 - 18 :

 218 Figure 2-18: Paused Pol II release. ..........................................................................................

Figure 2 - 19 :

 219 Figure 2-19: PTMs of Pol II and histones .................................................................................

Figure 2 - 20 :

 220 Figure 2-20: Transcription termination. ....................................................................................

Figure 2 - 21 :

 221 Figure 2-21: Transcription regulatory interactions with Cis-acting elements. ..........................

Figure 2 -

 2 Figure 2-22: Pol II core promoter motifs...................................................................................

Figure 2 - 23 :

 223 Figure 2-23: Core promoter TSS patterns................................................................................

Figure 2 - 24 :

 224 Figure 2-24: Chromatin signals at the core promoter. .............................................................

Figure 2 - 25 :

 225 Figure 2-25: H3K4me enrichment at active genes. .................................................................

Figure 2 - 26 :

 226 Figure 2-26: Three types of core promoters. ...........................................................................

Figure 2 - 27 :

 227 Figure 2-27: Divergent transcription with unidirectional core promoters. ................................

Figure 2 - 28 :

 228 Figure 2-28: Features of active enhancer. ...............................................................................

Figure 2 - 29 :

 229 Figure 2-29: Models of enhancer-promoter communication. ...................................................

Figure 3 - 1 :

 31 Figure 3-1: TAF4b-containing transcription machinery for male germ cell maintenance in spermatogonia. .........................................................................................................................

Figure 3 - 2 :

 32 Figure 3-2: Roles of TRF2, TAF4b and TAF7L in germ cell differentiation. .............................

Figure 3 - 3 :

 33 Figure 3-3: TAF7L regulates spermatogenesis and adipogenesis. .........................................

Figure 3 - 4 :

 34 Figure 3-4: Dynamic expression of TRF2 during spermatogenesis. .......................................

Figure 3 - 5 :

 35 Figure 3-5: TRF2-containing PIC in spermatids. ......................................................................

Figure 3 - 6 :

 36 Figure 3-6: TRF2-dependent piRNA cluster transcription. .......................................................

Figure 3 - 7 :

 37 Figure 3-7: Switching of TATA-binding proteins between oocytes and embryos. ....................

Figure 3 - 8 :

 38 Figure 3-8: Basal transcription machinery exchange following nuclear transfer to Xenopus oocyte. ......................................................................................................................................

Figure 3 - 9 :

 39 Figure 3-9: Taspase1-TFIIA-TRF2 axis regulates spermatogenesis. ......................................

Figure 3 - 10 :

 310 Figure 3-10: Summary of TFIIA complexes exist in testis extracts. .........................................

Figure 4 - 1 :

 41 Figure 4-1: Crucial transcription factors during mouse oogenesis. .........................................

Figure 4 - 2 :

 42 Figure 4-2: Scheme of TBP-TBP2 switch and global transcription activity during oogenesis and early embryogenesis. ...............................................................................................................

Figure 1 - 1 :

 11 Figure 1-1: Generation of new anti-TBP2 sera. .....................................................................

Figure 1 - 2 :

 12 Figure 1-2: Affinity purification of polyclonal antibodies. ........................................................

Figure 1 - 3 :

 13 Figure 1-3: Antibody validation by WB with TBP2 overexpressed cell line. ..........................

Figure 1 - 4 :

 14 Figure 1-4: Antibody validation by IP. .....................................................................................

Figure 1 - 5 :

 15 Figure 1-5: TBP2 antibody validation by ChIP. ......................................................................

Figure 1 - 6 :

 16 Figure 1-6: Antibody validation for IF. .....................................................................................

Figure 1 - 7 :

 17 Figure 1-7: Optimization of micro immunoprecipitation. ........................................................

Figure 1 - 8 :

 18 Figure 1-8: Micro immunoprecipitation validation of TBP2 antibodies. ..................................

Figure 1 - 9 :

 19 Figure 1-9: Overview of the different microfluidic designs for MOWChIP. .............................

Figure 1 - 10 :

 110 Figure 1-10: Test of the MOWChIP setup. .............................................................................

Figure 2 - 1 :

 21 Figure 2-1: TBP2 DamID in NIH3T3 cells. .............................................................................

Figure 2 - 2 :

 22 Figure 2-2: TBP2 DamID-seq and qPCR validation. ..............................................................

- 3 :

 3 Figure 2-3: TBP2 binds to gene promoters. ...........................................................................

Figure 3 - 1 :

 31 Figure 3-1: Dam-Tbp2 and Dam-only mRNA synthesized by in vitro transcription. ..............

Figure 3 - 2 :

 32 Figure 3-2: TBP2 DamID in oocytes. .....................................................................................

Figure 3 - 3 :

 33 Figure 3-3: Methyl-PCR result of oocytes samples. ..............................................................

Figure 3 - 4 :

 34 Figure 3-4: Size distribution of oocytes TBP2 DamID sequencing libraries. .........................

Figure 3 - 5 :

 35 Figure 3-5: Schematic diagram of TBP2 CUT&RUN with oocytes. .......................................

Figure 4 - 1 :

 41 Figure 4-1: Oocyte nascent transcriptome analysis strategy. ................................................

Figure 5 - 1 :

 51 Figure 5-1: Potential TBP2-TFIIA interacting factors. ............................................................

Figure 6 - 1 :

 61 Figure 6-1: Conditional deletion of Taf10 in growing oocytes impairs oocyte growth............

Figure 7 - 1 :

 71 Figure 7-1: Mouse model of Tbp and Tbp2 cDNA knock-in at the Tbp2 locus. .....................

Figure 7 - 2 :

 72 Figure 7-2: Gene expression in Tbp2 TBP:mCherry and Tbp2 TBP2:Venus oocytes. .........................

Figure 7 - 3 :

 73 Figure 7-3: Gene expression in Tbp2 TBP2:Venus/+ oocytes after treatment with emetine. ........

Figure 7 - 4 :

 74 Figure 7-4: Oocytes collection for proteomics. .......................................................................

Figure 1 - 1 :

 11 Figure 1-1: The hierarchical model of chromatin organization. DNA is wrapped around nucleosomes to form chromatin chains and fibers, which can undergo further compaction to fit

Figure 1

 1 Figure 1-2: 3D genome organization. Nucleosome contacts form clusters and fibers, which could further engage in longer distance chromatin loops. Chromatin looping gives rise to TADs. Associations among TADs form A/B compartments, which generally reflect euchromatin and heterochromatin,

Figure 1 - 3 :

 13 Figure 1-3: Cycle of active DNA demethylation.

Figure 1 - 4 :

 14 Figure 1-4: Histone variants of human core histones. Light purple and green represent

  of DNA synthesis (Ray-Gallet et al., 2002;[START_REF] Ricketts | Ubinuclein-1 confers histone H3.3-specific-binding by the HIRA histone chaperone complex[END_REF][START_REF] Nashun | Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis[END_REF]

  modifiers, histone variants, histone chaperones and chromatin remodelers, together with methylated DNA binding proteins and histone PTMs readers, endow chromatin with plasticity and establish a sophisticated regulatory network to modulate chromatin structure and properties (Figure1-5). The chromatin dynamics could ultimately affect transcription by altering the accessibility of DNA to transcription factors and transcription machinery.

Figure 1 - 5 :

 15 Figure 1-5: Chromatin plasticity. Variations by dynamic combinations of DNA methylation, histone variants incorporation and histone PTMs, together with the modifiers, readers and remodelers, enable chromatin plasticity. From[START_REF] Yadav | Chromatin plasticity: A versatile landscape that underlies cell fate and identity[END_REF].

Figure 1 - 6 :

 16 Figure 1-6: Proposed mechanisms for PGC specification in mice. (A) Schematics of mouse embryo at the stage of germ cell specification. Red and blue dotted circles show the BMP and the WNT signals, respectively. VE, visceral endoderm; AVE, anterior visceral endoderm; ExE, extra-embryonic ectoderm; ExM, extra-embryonic mesoderm; EM, embryonic mesoderm. (B) Proposed model for mouse PGC specification[START_REF] Aramaki | A mesodermal factor, T, specifies mouse germ cell fate by directly activating germline determinants[END_REF]. Adapted

Figure 1 - 7 :

 17 Figure 1-7: Scheme of mouse PGCs specification and migration. Epi, epiblast; DVE, distal visceral endoderm; Sm, somite; Al, allantois. From(Saitou et al., 2012a)

Figure 1 - 8 :

 18 Figure 1-8: Overview of mouse spermatogenesis. Adapted from[START_REF] Schagdarsurengin | Epigenetics in male reproduction: effect of paternal diet on sperm quality and offspring health[END_REF]

Figure 1 - 9 :

 19 Figure 1-9: Schematic representation of oogenesis. Adapted from[START_REF] Pepling | From primordial germ cell to primordial follicle: mammalian female germ cell development[END_REF][START_REF] Racki | CPEB controls oocyte growth and follicle development in the mouse[END_REF]

Figure 1 - 10 :

 110 Figure 1-10: Chromatin dynamics during gametogenesis and embryogenesis. Adapted

Figure 2 - 1 :

 21 Figure 2-1: Pol II basal transcription machinery. (A) Purification of GTFs by fractionation of

Figure 2 - 2 :

 22 Figure 2-2: Structure of Pol II. (A) Structure of ten-subunit Pol II core with four modules, from (Cramer et al., 2001). (B) Ribbon model of mammalian (human) Pol II, from (Bernecky et al., 2016).

Figure 2 - 3 :

 23 Figure 2-3: The composition and conservation of Pol II CTD. From[START_REF] Harlen | The code and beyond: transcription regulation by the RNA polymerase II carboxy-terminal domain[END_REF]

Figure 2 - 4 :

 24 Figure 2-4: Structure of TBP core domain with / without TATA box. (A) Structure of TBP core domain, from (Davidson, 2003). (B) Structure of Human TBP core domain with TATA box,

Figure 2 - 5 :

 25 Figure 2-5: Schematic representation of human TFIID subunits. (a) TBP and TBP like factors. (b) Human TAFs, TAF paralogs and TAF-like proteins (except TAF5L and TAF6L, which are TAF-like proteins present in SAGA). TAFs present in the core TAF complex (see

Figure 2 - 6 :

 26 Figure 2-6: Core promoter recognition by TFIID. (A) TFIID binds core promoter elements, from (Goodrich et al., 2010). (B) Low-resolution cryo-EM structure of human TFIID bound to promoter DNA with TATA box (red), Initiator (Inr, purple), motif ten element (MTE, dark green)

2. 1 . 2 . 3

 123 Structure of the core-TFIID and holo-TFIID assembly TAFs contain several conserved structural domains (Figure 2-5 b), notably, histone fold domains (HFDs), which mediate heterodimerization of TAF3-TAF10, TAF8-10 (Gangloff et al., 2001

  enabling the remaining TAFs to assemble along the periphery[START_REF] Bieniossek | The architecture of human general transcription factor TFIID core complex[END_REF]

Figure 2 - 7 :

 27 Figure 2-7: Structure of the human core-TFIID and model for TFIID assembly. (A) cryo-EM structure of the human TFIID core complex. (B) Model for holo-TFIID assembly.

Figure 2 - 8 :

 28 Figure 2-8: Structure of the yeast Pol II-TFIIB complex. (A) TFIIB domain organization. (B) Ribbon model of TFIIB. (C) Ribbon model of Pol II-TFIIB complex. (D) Clash of RNA stand with the B-reader. Adapted from[START_REF] Kostrewa | RNA polymerase II-TFIIB structure and mechanism of transcription initiation[END_REF][START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF]

(

  [START_REF] Plaschka | Transcription initiation complex structures elucidate DNA opening[END_REF] (Figure2-9 B).

Figure 2 - 9 :

 29 Figure 2-9: TFIIE architecture and interactions. (A) Domain organization of yeast TFIIE. (B) TFIIE domain architecture. (C) TFIIE interactions within the closed complex. (D) TFIIE interactions within the open complex. (Closed complex and open complex see section 2.2.2).

  The eWH domain of TFIIEα anchors TFIIE to Pol II clamp, and it contacts DNA backbone at positions -13/-14 upstream of TSS. Moreover, TFIIEα eWH contacts the TFIIFβ WH domain above upstream DNA, together with other two WH domains from TFIIEβ, encircling and retaining promoter DNA (Grunberg et al., 2012; Plaschka et al., 2016) (Figure 2-9 C&D). The E-ribbon of TFIIEα interacts with the Pol II clamp, as well as stalk subunit RPB7 of Pol II and B-ribbon of TFIIB (Plaschka et al., 2016) (Figure 2-9 D).

  evidences support the translocase model for ATP-dependent DNA opening, according to which, XPB uses its ATP hydrolysis activity to track along the DNA template strand in the 3'-5' direction and translocates DNA away from Pol II, resulting in insertion and rotation of promoter DNA into the Pol II cleft, and leading to DNA unwinding[START_REF] Grunberg | Architecture of the RNA polymerase II preinitiation complex and mechanism of ATP-dependent promoter opening[END_REF][START_REF] Fishburn | Double-stranded DNA translocase activity of transcription factor TFIIH and the mechanism of RNA polymerase II open complex formation[END_REF][START_REF] Schilbach | Structures of transcription pre-initiation complex with TFIIH and Mediator[END_REF] (Figure 2-10 B).

Figure 2 - 10 :

 210 Figure 2-10: Structure of yeast TFIIH within PIC and DNA opening. (A) cryo-EM structure of yeast PIC (core PIC + TFIIH). (B) Schematic cross-section of the PIC with open and closed DNA, during DNA opening, Ssl2/XPB ATPase translocates to the right and DNA moves to the left. Adapted from[START_REF] Schilbach | Structures of transcription pre-initiation complex with TFIIH and Mediator[END_REF].

  complex (PIC) (Step 2). DNA is then opened (forming the 'transcription bubble') and RNA synthesis commences, termed initiation (Step3). Early elongating Pol II synthetizes only a short stretch of nascent RNA (~30-50 nucleotides) and then undergoes promoter-proximal pausing (Step 4). Positive transcription elongation factor b (P-TEFb) phosphorylates Ser2 of Pol II CTD, DRB sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), leading to the dissociation of NELF. Pol II escapes from the pausing (Step 5), and enters either premature termination or productive elongation. If not termination, Pol II then productively elongates through the gene body (Step 6). After transcribing the gene, Pol II undergoes termination (Step 7), and the released free Pol II can reinitiate to start a new round of transcription (Step 8). Apparently, all these steps are the targets for transcription regulation.

Figure 2 - 11 :

 211 Figure 2-11: The transcription cycle and its potentially regulated steps. Adapted from the cover of cold spring harbor transcription meeting 2017 "Mechanisms of eukaryotic transcription"

  comprises of 25 subunits in yeast and up to 30 subunits in humans, which are organized into four distinct modules: a head module, a middle module, a tail module and a transiently associated CDK8 kinase module (Soutourina, 2018) (Figure 2-12).

Figure 2 - 12 :

 212 Figure 2-12: Subunit composition of the Mediator complex. (a) Yeast Mediator complex, asterisks indicate the 10 subunits that are essential for yeast viability. (b) Mammalian Mediator complex. From[START_REF] Soutourina | Transcription regulation by the Mediator complex[END_REF].

Figure 2 - 13 :

 213 Figure 2-13: SAGA modular organization and the subunits shared with other complexes.

- 14 &

 14 Figure 2-15).

Figure 2 - 14 :

 214 Figure 2-14: Chromatin opening and PIC assembly. From[START_REF] Koster | Genesis of Chromatin and Transcription Dynamics in the Origin of Species[END_REF].

Figure 2 - 15 :

 215 Figure 2-15: Schematic representation of PIC assembly and transcription initiation.

2. 1 . 7 )

 17 , forming the 'transcription bubble', and leading to the formation of open PIC[START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] (Figure2-15). TFIIB and TFIIF contribute to the stabilization/maintenance of the transcription bubble[START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF]. In presence of NTPs, Pol II initiates transcription at TSS and RNA synthesis commences[START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF] (Figure2-15).To continue transcribing, Pol II need to dissociate from the promoter-binding GTFs (promoter escape), which is mediated by the phosphorylation of Pol II CTD at Ser5 and Ser7 through TFIIH with its CDK7 kinase[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] (Figure2-16). The activity of CDK7

Figure 2 - 16 :

 216 Figure 2-16: Transcription initiation and Pol II promoter escape. TFIIH mediated Pol II CTD Ser5 and Ser7 phosphorylation is accompanied by transcription initiation and Pol II promoter escape. From (Haberle et al., 2018).

Figure 2 - 17 :

 217 Figure 2-17: Pol II promoter-proximal pausing. From (Haberle et al., 2018).

  , leading to the dissociation of NELF and conversion of DSIF into a positive transcription elongation factor, thereby triggering paused Pol II release (Jonkers et al., 2015) (Figure 2-18). A detailed list of pausing-related factors seeTable 2-4.

Figure 2 - 18 :

 218 Figure 2-18: Paused Pol II release. From[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF]

  H2A-H2B dimers, facilitating the passage of Pol II(Chen et al., 2018a). During elongation, besides the rapid disassembly and reassembly of nucleosomes, histone PTMs also significantly change, H2Bub, H3K36me3, H3K79me2 and H3K79me3 are enriched on the gene body (Vakoc et al., 2006; Fuchs et al., 2014; Chen et al., 2018a) (Figure 2-19). Notably, in mammals, H3K36me3 is deposited by Pol II CTD associated SETD2, and DNMT3B can recognize this mark and methylate the associated DNA on gene body to help prevent cryptic transcription[START_REF] Baubec | Genomic profiling of DNA methyltransferases reveals a role for DNMT3B in genic methylation[END_REF][START_REF] Neri | Intragenic DNA methylation prevents spurious transcription initiation[END_REF].

Figure 2 - 19 :

 219 Figure 2-19: PTMs of Pol II and histones at gene bodies. Adapted from(Chen et al., 2018a)

  to the torpedo model, cleavage of the nascent RNA allows the entry of the 5'-3' exoribonuclease XRN2, which degrades the transcript downstream of the poly(A), subsequently leading to transcription termination. On the other hand, the allosteric model posits that poly(A) signal-dependent loss of elongation factors and/ or conformational changes of Pol II destabilizes the elongation complex, triggering termination (Porrua et al., 2015; Chen et al., 2018a) (Figure 2-20 C).

Figure 2 - 20 :

 220 Figure 2-20: Transcription termination. Adapted from[START_REF] Porrua | Transcription termination and the control of the transcriptome: why, where and how to stop[END_REF].

2. 3 . 1 . 1

 311 Core promoter elementsThe activity of the core promoter largely depend on its sequence motifs, which are diverse in terms of sequence differences and also their positions relative to TSS(Vo Ngoc et al., 2017b;[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] (Figure 2-22) (Table 2-).

Figure 2 - 22 :

 222 Figure 2-22: Pol II core promoter motifs. Typically, a core promoter might contain zero to three of the shown motifs, locations of which are roughly scaled. From (Vo Ngoc et al., 2017b).

  Transcription typically is initiated at a defined position, called transcription start site (TSS),

  cluster of TSSs probably derived from a single PIC, 'dispersed' core promoters have multiple weak TSSs with similar use frequency over an ∼50 to ∼100 bp region, and a variety of 'mixed' TSS patterns commonly exist (Vo Ngoc et al., 2017b) (Figure 2-23).

Figure 2 - 23 :

 223 Figure 2-23: Core promoter TSS patterns. From (Vo Ngoc et al., 2017b). Indeed, ∼70% of vertebrate genes have 'dispersed' (or mixed) promoters (Juven-Gershon et al., 2010), which add another layer of control for transcription through alternative TSSs selection/usage. Interestingly, it has been reported that during zebrafish early embryonic development, there is a widespread switch in TSS usage throughout maternal to zygotic transition (Haberle et al., 2014). 2.3.1.3 Chromatin signals at the core promoter

Figure 2 - 24 :

 224 Figure 2-24: Chromatin signals at the core promoter. From (Vo Ngoc et al., 2017b).First of all, a prominent feature of active gene promoter associated chromatin is the enrichment of specific histone PTMs, such as H3K4me3, H4ac and H3K27ac(Barski et al.,

  H3K27ac is not clear. Strikingly, in Drosophila, it has been reported that H3K9me3 could direct the transcription of PIWI-interacting RNA (piRNA) clusters in heterochromatin[START_REF] Andersen | A heterochromatin-dependent transcription machinery drives piRNA expression[END_REF] (discussed in more detail in section 3.2.2.4). Moreover, in plants, Pol IV (a variant of Pol II) is recruited to promoters containing methylated H3K9[START_REF] Law | Polymerase IV occupancy at RNA-directed DNA methylation sites requires SHH1[END_REF] Zhang et al., 2013a).

Figure 2 - 25 :

 225 Figure 2-25: H3K4me enrichment at active genes. Adapted from (Chen et al., 2018a).

2. 3 . 1 . 4

 314 Core promoter typesBase on their distinct features (including sequence motifs, TSS patterns and chromatin properties that were discussed above) and the function of their dominant genes, major core promoters have been grouped into three general functional classes, referred as types I, II and III[START_REF] Lenhard | Metazoan promoters: emerging characteristics and insights into transcriptional regulation[END_REF][START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] (Figure 2-26).

Figure 2 - 26 :

 226 Figure 2-26: Three types of core promoters. Adapted from (Haberle et al., 2018).

2. 3 . 1 . 5

 315 Divergent transcription with unidirectional core promoters Mammalian promoter regions frequently exhibit divergent transcription, a phenomenon in which transcription of downstream protein-coding genes is coupled with the transcription of short(50-2000 nucleotides) and unstable upstream antisense RNAs (uaRNAs) in the reverse direction[START_REF] Core | Nascent RNA sequencing reveals widespread pausing and divergent initiation at human promoters[END_REF][START_REF] Preker | RNA exosome depletion reveals transcription upstream of active human promoters[END_REF][START_REF] Seila | Divergent transcription from active promoters[END_REF][START_REF] Wu | Divergent transcription: a driving force for new gene origination?[END_REF][START_REF] Core | Analysis of nascent RNA identifies a unified architecture of initiation regions at mammalian promoters and enhancers[END_REF][START_REF] Andersson | Human Gene Promoters Are Intrinsically Bidirectional[END_REF] (Figure2-27).

Figure 2 - 27 :

 227 Figure 2-27: Divergent transcription with unidirectional core promoters. From (Haberleet al., 2018)

Figure 2 - 28 :

 228 Figure 2-28: Features of active enhancer. Left: Transcription of enhancer RNAs from active enhancer. Right: Histone PTMs at active enhancer. Adapted from(Chen et al., 2018a;[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF]

- 29 Introduction

 29 Diversity of basal transcription machinery 62

3. 1 . 1

 11 Differential requirement of several TAFs during development 3.1.1.1 Differential requirement of TAF10

  3.1.1.2 TAF8 mutation does not impair Pol II transcription Loss of TAF8 in mouse embryonic stem cells (ESCs) leads to cell death and a global decrease in Pol II transcription, however, in human patient cells with homozygous Taf8

  gonocyte proliferation is impaired from post-natal day 2 onward, and expression of spermatogonial stem cell markers (such as c-Ret, Plzf and Stra8 ) is diminished (Figure3-1).

Figure 3 - 1 :

 31 Figure 3-1: TAF4b-containing transcription machinery for male germ cell maintenance in spermatogonia. From (Levine et al., 2014).

Figure 3 - 2 :

 32 Figure 3-2: Roles of TRF2, TAF4b and TAF7L in germ cell differentiation. Adapted from(Goodrich et al., 2010)

Figure 3 - 3 :

 33 Figure 3-3: TAF7L regulates spermatogenesis and adipogenesis.

Figure 3 - 4 :

 34 Figure 3-4: Dynamic expression of TRF2 during spermatogenesis. SG, spermatogonia; PL, preleptotene; L, leptotene; Z, zygotene; P, pachytene; D, diakinetic; RS, round spermatids;

- 5 )

 5 . It is also possible that haploid gene expression is driven by two distinct set of PICs, one containing TRF2-TFIIA/ALF while the other containing TBP-TAF7L (Martianov et al., 2016) (Figure 3-5).

Figure 3 - 5 :

 35 Figure 3-5: TRF2-containing PIC in spermatids.In spermatid, TRF2 is proposed to act as a testis-specific subunit of TFIIA/ALF, cooperating with TBP and TAF7L to regulate haploidcell gene expression. From[START_REF] Martianov | TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression[END_REF].

Figure 3 - 6 :

 36 Figure 3-6: TRF2-dependent piRNA cluster transcription.

Figure 3 - 7 :

 37 Figure 3-7: Switching of TATA-binding proteins between oocytes and embryos. Adapted from[START_REF] Akhtar | TBP-related factors: a paradigm of diversity in transcription initiation[END_REF]

Figure 3 - 8 :

 38 Figure 3-8: Basal transcription machinery exchange following nuclear transfer to Xenopus oocyte. From (Jullien et al., 2014).

  Further analysis with noncleavable TFIIAαβ mutant mice revealed that both Taspase1-/-and noncleavable TFIIAαβ testes release immature germ cells with impaired transcription of Transition proteins (Tnp) and Protamines (Prm), exhibiting chromatin compaction defects, and recapitulating the observations in Trf2-/-testes (Oyama et al., 2013). Interestingly, although the noncleavable TFIIA(αβ+γ) can still interact with TRF2, they are not able to target and activate Tnp1 and Prm1 promoters, indicating Taspase1-mediated cleavage of TFIIAαβ is essential for TFR2-dependent testis-specific transcription (Oyama et al., 2013) (Figure 3-9).

Figure 3 - 9 :

 39 Figure 3-9: Taspase1-TFIIA-TRF2 axis regulates spermatogenesis. From (Oyama et al., 2013).

  . It is known ALF interacts with TRF2[START_REF] Catena | Proteolytic cleavage of ALF into alpha-and beta-subunits that form homologous and heterologous complexes with somatic TFIIA and TRF2 in male germ cells[END_REF][START_REF] Martianov | TRF2 is recruited to the pre-initiation complex as a testis-specific subunit of TFIIA/ALF to promote haploid cell gene expression[END_REF], however, its function is not clear yet.

Figure 3 - 10 :

 310 Figure 3-10: Summary of TFIIA complexes exist in testis extracts. From (Catena et al., 2005).

Figure 4 - 1 :

 41 Figure 4-1: Crucial transcription factors during mouse oogenesis. Transcription factors act at different stages of folliculogenesis as revealed by mouse knockouts. TFs expressed in germ cells, granulosa cells, or both germ cell and granulosa cells are marked in pink, greenand black, respectively. From(Jagarlamudi et al., 2012).

  . Interestingly, the global transcriptional activity in oocyte is increased during oocyte growth, reaches its peak at early antral follicles, subsequently declines and becomes quiescent by preovulatory follicle stage (De La Fuente, 2006) (Figure 4-2).

Figure 4 - 2 :

 42 Figure 4-2: Scheme of TBP-TBP2 switch and global transcription activity during oogenesis and early embryogenesis. Adapted from[START_REF] De La Fuente | Chromatin modifications in the germinal vesicle (GV) of mammalian oocytes[END_REF] Gazdag et al., 2007;[START_REF] Zhou | Dual functions of TAF7L in adipocyte differentiation[END_REF][START_REF] Clift | Restarting life: fertilization and the transition from meiosis to mitosis[END_REF].

  (a) To determine the role of TBP2 is controlling transcription initiation during oogenesis; (b) To map TBP2 occupancy in oocytes and to decipher the exact role of TBP2 in regulating oocyte gene expression program; (c) To characterize the oocyte specific basal transcription machinery, in particular, the TBP2-containing complex; (d) To understand what is the biological significance of the TBP replacement by TBP2 during oocyte growth

  initiation by RNA Polymerase II (Pol II) is central to any developmental process. Female germ cells develop during oogenesis leading to the formation of a highly differentiated and specialised cell, the oocyte. Oogenesis comprises a well-defined series of events, each of which must be regulated. In females, the oocytes enter meiosis during the embryonic life and quiescent primordial follicles composed of meiotically arrested oocytes at the late diplotene stage surrounded by granulosa cells, are formed perinatally in mice

  instead of Maq. The repeatMasker annotation was used to identified the different types of repeat elements (Smit, AFA, Hubley, R & Green, P. RepeatMasker Open-4.0. 2013-2015 http://www.repeatmasker.org). All the figures were generated using R software version 3.5.1.

  background model estimation method: Markov order 1, organism specific Mus musculus GRCm38 upstream-noorf, scanning options: single strand. The number of motifs detected per gene was then evaluated by a R script. All the figures were generated using R software version 3.5.1. facility and S. Falcone, M. Poirot and F. Memedov of the IGBMC animal facility for the animal care taking.

Figure 1 :

 1 Figure 1: Protein coding genes expression is affected in Tbp2 -/-mutant oocytes. A-B.

Figure 2 :

 2 Figure 2: TBP2 is responsible for the expression of the majority of the genes expressed in

Figure 3 :

 3 Figure 3: Affected expression of genes related to the RNA decay pathway at post-natal

Figure 4 :

 4 Figure 4: Analysis of the core promoter elements within the promoter of downregulated

Figure 5 :

 5 Figure 5: High expression of repeat elements in oocytes and specific down regulation of

Figure 6 :

 6 Figure 6: TBP2 is interacting with TFIIA in 3T3 cells overexpressing TBP2. A.

Figure 7 :

 7 Figure 7: TBP2 is interacting with TFIIA in the oocytes. A-B. TBP2 (A) and TBP(B)

Figure 1 - 1 :

 11 Figure 1-1: Generation of new anti-TBP2 sera. (A) TBP2 (1-140aa) expression and concentration measurement. (B) WB test of the new anti-TBP2 sera before purification. (C)

Figure 1 - 2 :

 12 Figure 1-2: Affinity purification of polyclonal antibodies. Affinity-purified antibody fractions were analyzed on 10% SDS-PAGE, followed by Coomassie-staining. Asterisk indicates the albumin contamination.

Figure 1 - 3 :

 13 Figure 1-3: Antibody validation by WB with TBP2 overexpressed cell line.

Figure 1 - 4 :

 14 Figure 1-4: Antibody validation by IP.

Figure 1 - 5 :

 15 Figure 1-5: TBP2 antibody validation by ChIP. Pol II ChIP was performed as a positive control. '3023 LiCl' means that an additional harsh wash with LiCl buffer was performed during the ChIP.

Figure 1 - 6 :

 16 Figure 1-6: Antibody validation for IF.

Figure 1 - 7 :

 17 Figure 1-7: Optimization of micro immunoprecipitation. (A) Experimental design. (B) Anti-TBP microIP with different amount of HeLa nuclear extract input. after IP, elution was analyzed by WB with the anti-TBP (3G3) antibody, the anti-TAF6 (2G7) antibody and the anti-TAF10 (2B11) antibody.

Figure 1 - 8 :

 18 Figure 1-8: Micro immunoprecipitation validation of TBP2 antibodies. Anti-TBP2 microIPs were performed with different amount of II10 WCE input, and the elution was analyzed by WB with the 2B12 anti-TBP2 antibody.

  seems promising, most of the tests are not working with huge variation between different experiments. Therefore, I also carried out alternative approaches to study TBP2 occupancy in oocytes (See Unpublished data 3.1 & 3.2).

Figure 1 - 9 :

 19 Figure 1-9: Overview of the different microfluidic designs for MOWChIP. (A) Illustration of the major steps of MOWChIP, from (Cao et al., 2015). (B) The first microfluidic design

Figure 1 - 10 :

 110 Figure 1-10: Test of the MOWChIP setup. (A) Conventional H3K4me3 ChIP with 50 ug chromatin, as control. (B) Best result obtained by H3K4me3 MOWChIP with chromatin from

1

 1 Generation of Dam-TBP2 NIH3T3 stable cell lines Inducible Dam-Tbp2-NIH3T3 and Dam-only-NIH3T3 stables cell lines were generated (Figure 2-1 A&B&C) as described in the Material and Methods (10.1). Genomic DNA was extracted from both Dam-Tbp2-3T3 and Dam-only-3T3 cells in the absence of doxycycline and used for 'G me ATC'-methylation-specific PCR amplification (Figure 2-1 D).

Figure 2 - 1 :

 21 Figure 2-1: TBP2 DamID in NIH3T3 cells. (A) DamID plasmid construction. (B) Generation of Dam-TBP2 and Dam-only NIH3T3 stable cell lines. (C) Validation of the stable cell lines by western blot. (D) Methyl-PCR with processed genomic DNA from Dam-TBP2 and Dam-only NIH3T3 stable cell lines.

- 2 D

 2 ). A screenshot from IGV of some of the TBP2 bound genes is shown below (Figure2-2 A). Some of the binding sites were validated by DamID-qPCR (Figure2-2 B).

Figure 2 - 2 :

 22 Figure 2-2: TBP2 DamID-seq and qPCR validation. (A) A screenshot (from IGV) of genes bound by TBP2. (B) DamID-qPCR validation of some genes.

  -2 C), which shows that TBP2 binds to gene promoters genome-widely. Moreover, although both TBP2 and TBP bind to gene promoters, the TBP2 average binding profile shifts downstream of the TSS indicating that TBP2 might be involved in a different TSS usage (Figure 2-2 A&B).

Figure 2 - 3 :

 23 Figure 2-3: TBP2 binds to gene promoters. (A) Plots of average TBP2 binding profiles relative to TSSs of Pol II genes. (B) Plots of TBP profiles relative to TSSs. (C) Overlap of TBP2 and TBP binding genes. (D) Overlap of TBP2 bound genes revealed by ChIP-seq and DamID-seq.

1

 1 Dam-Tbp2 and Dam-only capped mRNA synthesis As described in the Material & Methods (10.2), Dam-Tbp2 cDNA and Dam-only cDNA were cloned into the pRN3P vector (Figure 3-1 A), and capped Dam-Tbp2 and Dam-only mRNAs were obtained by in vitro transcription (Figure 3-1 B). Capped mRNAs were validated in GV oocytes (Figure 3-1 C): both Dam-Tbp2 and Dam-only mRNAs are translated in GV oocytes and methylate genomic DNA as shown by the signal of a 'G m ATC' tracer (a Dpn I truncation fused to eGFP).

Figure 3 - 1 : 3 . 1 . 2

 31312 Figure 3-1: Dam-Tbp2 and Dam-only mRNA synthesized by in vitro transcription. (A) Dam-Tbp2 cDNA and Dam-only cDNA in pRN3P vector. (B) Dam-Tbp2 and Dam-only capped

Figure 3 - 2 :

 32 Figure 3-2: TBP2 DamID in oocytes. (A) Oocyte cytoplasmic injection of Dam-Tbp2 and Dam-only mRNAs. (B) P7 oocytes after injection and incubation. (C) P13 oocytes after injection and incubation.

Figure 3 - 3 :

 33 Figure 3-3: Methyl-PCR result of oocytes samples.

Figure 3 - 4 :

 34 Figure 3-4: Size distribution of oocytes TBP2 DamID sequencing libraries. 300ng of purified PCR products were used for libraries preparation, and the size of the libraries mainly distribute between 150bp to 1500bp (GenomEAST platform, IGBMC).

  and more sensitive technique named CUT&RUN (Material & Methods 11) (Figure3-5). TBP2 CUT&RUN was performed with 500 oocytes, and sequencing libraries were prepared with the recovered DNA fragments, and the libraries are now under sequencing (GenomEAST platform, IGBMC).

Figure 3 - 5 :

 35 Figure 3-5: Schematic diagram of TBP2 CUT&RUN with oocytes. Adapted from (Skene

Figure 4 - 1 :

 41 Figure 4-1: Oocyte nascent transcriptome analysis strategy. Nascent RNA study using

  -1 A), among which, 161 proteins overlap with proteins pulled down by TBP2 after the triple sequential IP (see manuscript) (Figure5-1 B). We analyzed the 123 common non-ribosomal proteins by GO analysis, many proteins are linked to RNA binding, interestingly, there are also a few proteins are linked to transcription factor and cofactors, such as GATA4, SETDB2, TRIM28 and ZAR1.

Figure 5 - 1 :

 51 Figure 5-1: Potential TBP2-TFIIA interacting factors. (A) Common proteins in anti-TBP2 IP mass-spec results. (B). (C) Overlap between 'direct TBP2 IP' and 'sequential TBP2 IP'

Figure 6 - 1 : 7 . 7 . 1

 61771 Figure 6-1: Conditional deletion of Taf10 in growing oocytes impairs oocyte growth. (A) Scheme of the breeding and experiments. (B) Morphology of ovaries from 6-week-old Tg(Zp3-Cre/+);Taf10 △/+ (control) and Tg(Zp3-Cre/+);Taf10 flox / △ (mutant) females.

Figure 7 - 1 :

 71 Figure 7-1: Mouse model of Tbp and Tbp2 cDNA knock-in at the Tbp2 locus. (A) Map of the Tbp/Tbp2 cDNA knock-in at the Tbp2 locus. (B) After recombination, the Tbp2 cDNA is expressed as a control. (C) After recombination, the cDNA of Tbp is expressed under the control of the Tbp2 regulatory elements.

Figure 7 - 2 :

 72 Figure 7-2: Gene expression in Tbp2 TBP:mCherry and Tbp2 TBP2:Venus oocytes. Expression of Tbp, Tbp2, Tbp-mCherry and Tbp2-Venus were analyzed by RT-qPCR using mRNA obtained

Figure 7 - 3 :

 73 Figure 7-3: Gene expression in Tbp2 TBP2:Venus/+ oocytes after treatment with emetine.

Fig. 3 Fig. 2

 32 Fig. 3 Tip60 and c-Myc binding overlap. a-b Heatmap representing k-means clustering results of normalized Tip60 (GSE69671), Pol II (GSM307623), c-Myc (GSM288356), Oct4 (GSM288347), Sox2 (GSM288346) and Nanog (GSM288345) density profiles against all 7693 MACS14 Tip60 peaks (a), or against all 5318 MACS14 c-Myc peaks (b). Two main clusters are observed. Cluster 1 shows Tip60 and c-Myc overlap in both panels. The colour scale bars under each data set in a and b reflect the read densities between 1 and 25 of the given dataset. The number of reads of each data set is indicted in the colour scale bars in millions (m) of reads

Fig. 4

 4 Fig. 4 Tip60 is a positive transcriptional co-activator. a All the identified Tip60 peaks (100 %) were annotated to different genomic elements: Promoter-TSS, UTRs (untranslated regions), exons, introns, TSS and intergenic regions and are represented in the % of their presence at these elements. b Boxplots showing the log2 of RNA fragments per kilobase per million mapped fragments (FPKM) expression values from ESCs (GSE34473) of all analysed genes and Tip60 bound genes. c-d RNA expression values are ranked into five groups with equal number of genes from highest to lowest RNA expression level as indicated. Boxplots represent the Pol II densities (c) or the Tip60 peak density (d) around the TSSs. Only density values higher than zero were taken. The median is significantly different between groups, if the notches do not overlap

( 5

 5 See figure on next page.) Fig. Tip60 binding defines active gene sets and enhancer regions. a Heatmap showing k-means clustering of Tip60 (GSE69671), Pol II (GSM307623), H3K27ac (GSM594578), H3K4me3 (GSM307618), H3K4me1 (GSM594577) and H3K27me3 (GSM307619) using 7693 high-confidence Tip60 binding sites as reference coordinates. Densities are represented in region of ∓5 kb around Tip60 binding sites. Four clusters are defined as indicted by the dotted lines. The two "enhancer" peaks are divided into H3K4me1 positive and H3K4me1 negative peaks. The colour scale bars under each data set in A reflect the read densities between 1 and 20 of the given dataset. The number of reads of each data set is indicted in the colour scale bars in millions (m) of reads. b GO term analyses of Pol II positive or bivalent peaks after gene annotation using Manteia [62]. c UCSC genome browser profiles of DHS, Tip60 binding, presence of H3K4me3 and H3K27me3, Pol II binding and input (negative control) at a randomly chosen bivalent gene, Stc2, are shown. An arrow labels the gene orientation. d Density profiles of Tip60 binding at the different clusters as defined in panel a

Fig. 6

 6 Fig. 6 Tip60 correlates with p300, H3K3me1 and DHS presence and with a subset of H3K27ac signals at ESC-specific enhancer sites. a Enhancers [41] were sorted for H3K27ac (GSM594578) signal intensities. Signals of Tip60 (GSE69671), p300 (GSM723018), H3K27ac (GSM594578), H3K4me1 (GSM594577), H3K27me3 (GSM307619) and DHS (GSM1014154) were calculated around enhancer mid points. The colour scale bars under each data set in a reflects the read densities between 1 and 20 of the given dataset. The number of reads of each data set is indicted in the colour scale bars in millions (m) of reads. b, c Enhancers were divided into four categories having equal number of genes based on their H3K27ac signal intensity from high to low (I-IV) as indicated on the right of the panels. Graphs show Tip60 enrichment (b) or DHS signals (c) around enhancer mid points of the different categories. (d) GO term category analysis by Manteia [62] of the closest genes to the Tip60 positive enhancers [41] identified by k-means clustering

Fig. 7

 7 Fig.7Tip60 locates to super enhancer regions. a Tip60 (GSE69671) binding profiles together with DHS (GSM1014154), H3K4me1 (GSM594577), H3K27ac (GSM594578), Nsl1 (GSM1300940) and Msl1 (GSM1300939) are represented using the UCSC genome browser at the well-defined super enhancer regions (boxed in red) of the Nanog and Klf4 locus. The Input (GSM798320) serves as negative control. Data were uploaded as wig files. b-d The 231 super enhancers[START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF] were divided into 80 bins and Tip60 (b), Nsl1 (c) and Msl1 (d) enrichment was calculated per bin between their start and end positions as well as up to 4 kb up-and downstream. Signals were normalized to the input (GSM798320)

Table 1 -

 1 1. Overview of the transcription related histone PTMs. ................................................ 2: Function of histone variants. ...................................................................................

Table 1 -

 1 3: Histone chaperones involved in histone deposition. ...............................................

Table 2 -

 2 1: Subunits of Pol II. ....................................................................................................

Table 2 -

 2 2: Subunits of TFIIH. ....................................................................................................

Table 2 -

 2 3: Pioneer factors. .......................................................................................................

Table 2 -

 2 4: Pausing-related factors. .........................................................................................

Table 2 -

 2

5: Known core promoter motifs. ..................................................................................

Table 1 -3: Histone chaperones involved in histone deposition.

 1 Adapted from[START_REF] Gurard-Levin | Histone chaperones: assisting histone traffic and nucleosome dynamics[END_REF] and[START_REF] Hammond | Histone chaperone networks shaping chromatin function[END_REF].

	Histone chaperones	Histone preference	Complex(s)	Main function(s)
	ASF1A/B	H3.1-, H3.2-, H3.3-H4	Multiple	Histone donor for CAF-1 and HIRA

Table 2 -1: Subunits of Pol II. Adapted

 2

	from (Sainsbury et al., 2015)

.2.3), pause-release (section 2.2.6), elongation (section 2.2.6) and termination (section 2.2.7). Besides

	, thus playing important roles at different steps of transcription, including
	transcription initiation (see section 2those known canonical roles, Pol II CTD also
	undergoes cooperative liquid phase separation, helping Pol II to form clusters/hubs at active
	genes

(Figure 2-8 A&C). Fourthly

	, its B-linker domain
	binds to Pol II rudder and clamp coiled-coil domains, helping DNA opening and/or
	maintenance of the transcription bubble. Its B-reader domain contacts the DNA template
	strand, assisting in TSS selection and DNA positioning for the initiation of RNA synthesis

Table 2 -

 2

	TFIIH	Subunit	Gene name	Mass (kDa)	Copies
			Yeast	Human	Yeast	Human
		Subunit 1 (p62)	TFB1	GTF2H1	72.9	62	1
		Subunit 2 (p44)	SSL1	GTF2H2	52.3	44	1
		Subunit 3 (p34)	TFB4	GTF2H3	37.5	34.4	1
	TFIIH	Subunit 4 (p52)	TFB2	GTF2H4	58.5	52.2	1
	(core)	Subunit 5 (p8)	TFB5	GTF2H5	8.2	8.1	1
		XPD subunit	RAD3	ERCC2	89.8	86.9	1
		XPB subunit	SSL2	ERCC3	95.3	89.3	1
		7 subunits			414.5	377.3
	TFIIH	Cyclin H	CCL1	CCNH	45.2	37.6	1
	(kinase	CDK7	KIN28	CDK7	35.2	39.0	1
	module)	MAT1	TFB3	MNAT1	38.1	35.8	1
		3 subunits			118.5	112.4	1

2: Subunits of TFIIH. Adapted from

[START_REF] Sainsbury | Structural basis of transcription initiation by RNA polymerase II[END_REF]

.

Table 2 -

 2

	Pioneer factors	DNA binding domain	Refs
	AP-1	Basic leucine zipper	(Biddie et al., 2011)

3: Pioneer factors. Adapted from

[START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF]

  Moreover, some coactivators harbor more than one enzymatic activities, therefore they can target chromatin through chromatin remodeling and/or histone modifications. Altogether, coactivators can alter chromatin structure, open the promoter and facilitate the binding of GTFs and Pol II.

	1	Ets	(Barozzi et al., 2014)
	SOX2	HMG box	(Soufi et al., 2015)
	SOX9	HMG box	(Adam et al., 2015)
	TP53	p53	(Sammons et al., 2015)
	P63	p53	(Sammons et al., 2015)

RFX

Rfx-type winged helix

[START_REF] Masternak | Chromatin remodeling and extragenic transcription at the MHC class II locus control region[END_REF]

2.2.1.2 Recruitment of coactivators Following the binding of activators, coactivators are recruited to the regulatory elements by activators, chromatin modifications, DNA, and/or regulatory RNAs (Figure 2-14). Usually, coactivators are multi-subunit complexes that enhance transcription, through reorganizing nucleosomes, like chromatin remodelers (see section 1.2.3); or through modifying histone covalently, like histone-modifying complexes, especially the histone acetyltransferases; or through interacting directly with GTFs and Pol II, like the Mediator complex (see 2.2.1.3).

Table 2 -4: Pausing-related factors

 2

. From

(Chen et al., 2018a)

.

Table 2 -5: Known core promoter motifs

 2

	Motifs	Sequence logo	Consensus	Position
			sequence

. From

[START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF]

.

  Type I consists of adult tissue-specific promoters, which have TATA-box, Inr and 'sharp' TSS pattern, but lack CGIs. Type II promoters are associated with 'housekeeping' genes, and are TATA-less promoters with 'dispersed' TSS patter. In mammals, they overlap individual CGIs. Finally, Type III promoters are preferentially associated with developmental transcription factors. In mammals, they are associated with large CGIs or multiple CGIs(Lenhard et al.,

	2012; Haberle et al., 2018). TCT promoters are minor promoters not included in this
	classification. They have 'sharp' TSS pattern, but other features differ substantially from type I
	promoters (Lenhard et al., 2012).

  with the incorporation of H3.3/H2A.Z histone variants, relatively high DNA accessibility, TFs and coactivators binding, and enrichment of Pol II (Ser5P but not Ser2P).

, thus playing critical roles in spatial and temporal control of gene expression. Prior to activation, enhancers can exist in a primed state, which are associated with H3.3/H2A.Z incorporation, pioneer TFs binding and presence of H3K4me1. Upon activation, enhancers initiate bidirectional eRNAs transcription, and active enhancers are typically marked by H3K4me1 and H3K27ac (Figure 2-28), and are also associated

Antibody coupling to Dynabeads-Pro A/G

  IP 500mM buffer three times on rotator, 5min each. Wash the beads with 1ml IP 100mM buffer twice on rotator, 5min each. Keep 10ul of Beads as PBE (protein before elution, optional), and before add Glycine, remove all the IP buffer.

	5.1 IP with sepharose-beads
	a) IP buffers
	IP100mM KCl buffer 100ml	stock	IP500mM KCl buffer	100ml	Stock
	25 mM Tris Cl pH 7,9	2.5ml	1M	25 mM Tris Cl pH 7.9	2.5ml	1M
	0.1% NP40	1ml	10%	0.1% NP40	1ml	10%
	5mM MgCl2	0.5ml	1M	5mM MgCl2	0.5ml	1M
	∼10% Glycerol	10ml	87%	∼10% Glycerol	10ml	87%
	100mM KCl	3.33ml	3M	500 mM KCl	16.66ml	3M
	2mM DTT	200ul	1M	2mM DTT	200ul	1M
	1X PIC	2 tablets	1X PIC	2 tablets
	Water	82.7ml	Water	69.4ml
	Aliquot 100ul IP 100mM KCL buffer (1 beads volume) into 1.5ml tubes, add 100ul
	pre-washed beads-Pro G. Add 10ul/20-100ug antibodies to each tube, place at 10-30 rpm on
	rotator for at least 2h at 4℃ (approximately 1mg antibody per ml of protein G sepharose
	beads). 3 times wash with 500ul IP 500mM KCL buffer and 2 times wash with 500ul IP 100mM
	KCL buffer (5 min wash for each). Remove the supernatant, keep the beads and put it on ice.
	d) Pre-clean of INPUT
	4mg WCE + 1/10 volume sepharose beads, pre-clean at 4℃ 2h, then centrifuge 5min at
	600g/2500rpm at 4℃, keep the SN as INPUT sample.

b) Washing/Preparation of beads

Resuspend sepharose-beads by vorexing for 10-15 seconds, transfer 150ul 50% sepharose beads-Pro A/G slurryto a 1.5 EP tube (For each IP from 4mg WCE, 100ul 50% beads slurry + 10ul antibody was used, also take 1/10 volume of beads to pre-clean WCE). 3 times quick wash with 1ml 1×PBS and 2 times quick wash with 1ml IP 100mM KCL buffer (centrifuge 2min at 600g/2500rpm after each wash). Remove the SN and add IP 100mM KCL buffer to the beads to make it as 50% slurry, put on ice.

c) e) Protein fixation/Binding IP

Add the INPUT sample to the beads-antibody complex, incubate overnight at 4℃. Centrifuge 3min at 600g/2500rpm at 4℃, keep the supernatant as SN for WB. Wash the beads with 1ml

9.1 Chromatin immunoprecipitation a) Crosslinking and nuclei preparation

	1M glycine to a final concentration of 125mM. Incubate 10 min at RT while mixing by rotating
	agitation, then trash the solution in the formaldehyde waste. Wash the fixed cell twice with cold
	1xPBS. Scrape cells using scrapper in 5ml cold 1XPBS per 15cm-dish and collect in 15ml
	tubes, then spin at 2000 rpm or 600g for 5min at 4˚C to pellet cells. Wash once again with 5ml
	1XPBS/PIC and discard SN. Resuspend cells in 2 ml / large dish (about 1ml per 1.0×107cells,
	or 6 volumes to the packed pellet) of L1 buffer, and incubate for 10min on a rocker at 4°C. Spin
	down to pellet nuclei for 5 min at 800g or 2500 rpm at 4°C in 1.5ml Eppendorf centrifuge,
	discard the supernatant. Resuspend nuclei in L2 buffer at 2.0×10 7 cells / ml, incubate for 10min
	on a rocker at 4°C.
	b) Sonication and sonication check
	Samples (nuclei) were distributed 600ul per tube into tubes for covaris sonication,
	sonication conditions should be optimized for each cell type with Covaris E210 sonicator. For
	NIH3T3 cells, sonication was performed with Duty cycle 20%, Intensity 8 and cycle burst 200
	for 15min. After sonication transfer the sonicated chromatin into 1.5ml Eppendorf, and
	9ml of

When cells reache 80-90% confluence, remove the medium, wash the cells once with room temperature PBS, fix cells by adding 20ml 1xPBS with 1% concentration formaldehyde, incubate for 10min at RT while mixing by rotating agitation. Stop crosslink by adding 2.

centrifuge for 15min at 14000 rpm, take the supernatant and transfer into new Eppendorf tubes.

Table 1 : Taf7 deletion in oocytes does not impair the production of GV oocytes.

 1

  . However, in NIH 3T3 cells ectopically overexpressing TBP2, we did not detect any TBP2 association with respectively), in contrast to TBP, which interacts with these Pol I and Pol III cofactors. These results together suggest that TBP2 is not involved in Pol I and Pol III transcription in NIH 3T3 cells. Similarly in our anti-TBP2 ovary IPs we did not detect any Pol I and Pol III cofactors, further suggesting that TBP2 may not be involved in Pol I or Pol III transcription. Although in oocytes Trf2 is expressed at mRNA level(Xiao et al., 2006), it is not known if TRF2 protein is present . Besides, no evidence has been shown that TRF2 is involved in Pol I and Pol III(Vo Ngoc et al., 2017b), and Trf2-deficient mice do not display defects in oocytes(Martianov et al., 2001; Zhang et al., 2001b). To explain which TBP-type factor can drive Pol I or Pol III transcription in oocytes, one possibility could be that residual levels of TBP may play a role in Pol I and Pol III transcription, alternatively, Pol I and Pol III transcription may be inactive in oocytes, however, this hypothesis requires further investigations.

  1 Cellular Signalling and Nuclear Dynamics Programme, Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), CNRS UMR 7104, INSERM U964, Université de Strasbourg (UdS), BP 10142, 1 Rue Laurent Fries, CU de Strasbourg, 67404 Illkirch Cedex, France Full list of author information is available at the end of the article

Importance du remplacement de TBP par TBP2 pour la transcription dans les ovocytesDans le but de comprendre l'importance de TBP2 pour la transcription dans les ovocytes lorsque TBP est absente, nous avons utilisé un système permettant de forcer l'expression de TBP dans les ovocytes. La séquence codante de TBP a été insérée au niveau du locus de Tbp2 afin d'exprimer TBP à la place de TBP2 dans les ovocytes. Cette expérience a été infructueuse, les souris génétiquement modifiées avec cette construction étant stériles, indiquant que la régulation de l'expression de Tbp2 est complexe.ConclusionsDans cette étude, nous avons analysé la fonction de TBP2 pour l'initiation de la transcription au cours de l'ovogenèse. Premièrement, nous avons démontré ici que TBP2 n'est pas présent au sein du complexe TFIID in vivo. Deuxièmement, TBP2 régule l'expression de très nombreux gènes en contactant physiquement de nombreux gènes.Afin de confirmer ces résultats, nous avons aussi employé les techniques de Dam-ID-seq et de Cut&Run dont les résultats sont en cours d'analyse. Par ailleurs, afin de distinguer la contribution de TBP2 pour l'initiation de la transcription et la stabilité des transcrits, liée à la grande quantité de transcrits contenue dans les ovocytes, nous avons analysé la transcription naissante en utilisant la technique de SLAM-seq. Les résultats obtenus ici montrent l'existence d'une machinerie transcriptionnelle spécifique dans les ovocytes, différente de TFIID pour la transcription.

Figure 7-4: Oocytes collection for proteomics.

Acknowledgements i

Acknowledgements

First of all, I would like to thank Prof. Dr. Petra Hajkova, Dr. Irwin Davidson, Dr. Antoine H.F.M. Peters and Prof. Dr. Ferenc Mueller for accepting to be members of my thesis committee. Thank you for your time and effort to read and evaluate my thesis.

I would like thank Laszlo for being my supervisor and for giving me the opportunity to work

Acknowledgements

Acknowledgements

We thank D. Singer and A. Gegonne for the gift of the Taf7 flox mouse line and H. Stunnenberg for TFIIA antibodies. We would also like to thank all members of the Tora lab for thoughtful discussions and suggestions throughout the course of the work. We are grateful to M.E. Torres Padilla for advice, preliminary experiments, C. Hérouard from the GenomEAST platform for library preparation, P. Eberling for the generation of the peptides, G. Duval for the generation of the polyclonal sera, V. Hisler for help with analyses, the histology platform, the IGBMC cell culture

Acknowledgements

We are very grateful to B. Amati for providing the Tip60 antibodies. We would like to thank the IGBMC sequencing and bioinformatics platform for data generation and support, M. Hestin and G. Rossi from the IGBMC ES cell culture facility and F. Klein for the gelfiltration experiments. We thank J. Pellegrino, M. Gerard and D. Devys for critical reading the manuscript. SR was supported by a fellowship from Association pour la Recherche sur le Cancer (ARC). This work was supported by funds from CNRS, INSERM, Strasbourg University, the European Commission Marie Curie-ITN (NR-NET) and the Agence Nationale de Recherche (ANR-11-BSV5-010-02 Chromact; ANR-13-BSV6-0001-02 COREAC; ANR-13-BSV8-0021-03 DiscoverIID). LT is recipient of a European Research Council (ERC) Advanced Grant (ERC-2013-340551, Birtoaction).

Funding

This work was supported by funds from CNRS, INSERM, and Strasbourg University. This study was also supported by the European Research Council (ERC) Advanced grant (ERC-2013-340551, Birtoaction) and grant ANR-10-LABX-0030-INRT (to LT), a French State fund managed by the Agence Nationale de la Recherche under the frame program Investissements d'Avenir ANR-10-IDEX-0002-02.

Cis-acting elements, code behind the cycle

The regulatory DNA sequences plus the associated epigenetic modifications of a specific gene provide the code that dictates when, where and at which level the gene should be transcribed. Beside epigenetic modifications, the regulatory sequences code usually derives from three parts [START_REF] Fuda | Defining mechanisms that regulate RNA polymerase II transcription in vivo[END_REF] : the core promoter [START_REF] Juven-Gershon | Regulation of gene expression via the core promoter and the basal transcriptional machinery[END_REF][START_REF] Muller | Chromatin and DNA sequences in defining promoters for transcription initiation[END_REF], the transcription factor binding sites (TFBSs) proximal to the core promoter [START_REF] Ong | Enhancer function: new insights into the regulation of tissue-specific gene expression[END_REF][START_REF] Lenhard | Metazoan promoters: emerging characteristics and insights into transcriptional regulation[END_REF], and the more distant cis-regulatory elements (≥ 1 kb from TSS, including enhancers, silencers and insulators [START_REF] Ong | Enhancer function: new insights into the regulation of tissue-specific gene expression[END_REF]. The promoter-proximal TFBSs can also exist in clusters, forming cis-regulatory modules (CRMs) [START_REF] Lenhard | Metazoan promoters: emerging characteristics and insights into transcriptional regulation[END_REF]. [START_REF] Lenhard | Metazoan promoters: emerging characteristics and insights into transcriptional regulation[END_REF].

Promoter-proximal TFBSs and enhancers direct the binding of activators/repressors, which further mediate the recruitment of coactivators/repressors, followed by PIC assembly on the core promoter. In the following part, I will mainly focus on the core promoter and enhancer.

C) implies that enhancer-bound TFs and promoter-bound TFs physically interact with each other by looping out the intervening DNA. The first evidence for looping came from E. coli [START_REF] Lee | In vivo DNA loops in araCBAD: size limits and helical repeat[END_REF], and after, many proteins have been proposed to bridge chromatin looping, including Mediator, CTCF, TAF3, BRG1 [reviewed in [START_REF] Vernimmen | The Hierarchy of Transcriptional Activation: From Enhancer to Promoter[END_REF]]. The looping-tracking/linking model (Figure 2-29 D) proposes that long-range loops can bring enhancers close to (but not directly contacted with) the target promoter, tracking or linking would bridge the distance left [START_REF] Furlong | Developmental enhancers and chromosome topology[END_REF]. From [START_REF] Furlong | Developmental enhancers and chromosome topology[END_REF].

Results

Manuscript

Abstract

Mammalian oocytes go through consecutive differentiation process, during which the synthesis and accumulation of RNAs and proteins are essential for oocyte growth, maturation, fertilization and early embryogenesis. Little is known about the nature and function of the transcriptional machinery that is involved in RNA polymerase II (Pol II) transcription initiation during oogenesis. In somatic cells, the Pol II general transcription factor (GTF), TFIID, is the first to bind to gene promoters to nucleate pre-initiation complex (PIC) formation together with TFIIA, -IIB, -IIE, -IIF, and -IIH. In metazoans, TFIID is composed of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs). During oocyte growth TBP is replaced by a vertebrate specific TBP-type protein, TBP2 (also called TRF3 or TBPL2) and Tbp2 -/- females are sterile. To understand whether and how TBP2 is controlling transcription initiation during oogenesis, we carried out RNA-seq analyses from wild-type and Tbp2 -/-oocytes from primary and secondary follicles. These analyses show a main decrease in the expression of the most abundant genes as well as specific down-regulation of the expression of the MaLR (mammalian apparent LTR retrotransposons)-type endogenous retroviral elements. To identify the nature of the complex associated with TBP2 in the oocytes, we carried out immunoprecipitation followed by mass spectrometry. We demonstrate that, in the oocytes, TBP2 associates with TFIIA, but does not assemble into a TFIID-type complex. Altogether, our data show that a specific TBP2-TFIIA-containing transcription machinery, different from canonical TFIID, drives transcription in mouse oocytes.

TATA box, MTE and XCPE1 elements (Fig. 4A, 4C and4E). Thus, it seems that the TBP2regulated genes do not dramatically differ at the sequence level from canonical TFIID-regulated genes, but are more enriched in TATA boxes, MTE and XCPE1 elements.

Expression of mouse class III MaLR endogenous retroviral (ERV) elements are down regulated in Tbp2 -/-primary and secondary follicle oocytes During the course of the above mRNA analyses we realized that expression from many mouse repetitive elements are significantly down regulated (Fig. 5A). Mammalian repetitive elements belong to the following major classes of repeat elements: long terminal repeats (LTRs), long interspersed nucleotide elements (LINEs), short interspersed nuclear elements (SINEs) class I retrotransposons and class II DNA transposons and are transcribed by Pol II (reviewed in [START_REF] Smit | Interspersed repeats and other mementos of transposable elements in mammalian genomes[END_REF]). When the relative abundance of transcripts initiated from these repetitive elements was analysed in wild-type P7 and P14 oocytes, the expression of the LTR retrotransposon class [also called endogenous retroviruses (ERVs)], was found to be the most abundant at both P7 and P14 (Fig. 5B). As the mouse LTRs (ERVs) are further divided in ERV classes I, II and III as well as into solo LTRs and gypsy LTRs [START_REF] Hubley | The Dfam database of repetitive DNA families[END_REF], we analysed which of these classes is affected in the P7 and the P14 Tbp2 -/-mutant oocytes (Fig. 5C and Supplementary Fig. 2A). Our analyses indicated that expression from the class III ERVs was the most down regulated (Fig. 5D and Supplementary Fig. 2B). ERVs belonging to the mouse class III family contain ERVL and MaLR apparent LTR retrotransposons (reviewed in [START_REF] Thompson | Long Terminal Repeats: From Parasitic Elements to Building Blocks of the Transcriptional Regulatory Repertoire[END_REF]). In our WT oocytes RNA-seq analysis MaLR family of nonautonomous retrotransposons were found to be highly expressed, in agreement with previous observation [START_REF] Peaston | Retrotransposons regulate host genes in mouse oocytes and preimplantation embryos[END_REF] and importantly the most down regulated in both P7 and P14 Tbp2 -/-mutant oocytes (Fig. 5D and Supplementary Fig. 2B). There is more than 100 distinct MaLR LTRs [START_REF] Hubley | The Dfam database of repetitive DNA families[END_REF] and among them, three members MT-Int, MTA-Mm, and Proteins were identified by database searching using SequestHT (Thermo Fisher Scientific) with Proteome Discoverer 1.4 software (Thermo Fisher Scientific) a combined Mus musculus database (Swissprot, release 2015_11, 16730 entries) where 5 interesting proteins sequences (TrEMBL entries) were added. Precursor and fragment mass tolerances were set at 7 ppm and 0.5 Da respectively, and up to 2 missed cleavages were allowed. Oxidation (M) was set as variable modification, and Carbamidomethylation © as fixed modification. Peptides were filtered with a false discovery rate (FDR) at 5 %, rank 1 and proteins were identified with 1 unique peptide.

Normalized spectral abundance factor (NSAF) [START_REF] Zybailov | Statistical analysis of membrane proteome expression changes in Saccharomyces cerevisiae[END_REF] normalized to the bait (NSAFbait) were obtained as followed (PSM*; peptide spectrum match, SAF; spectral abundance factor, x; protein of interest) using a R script available on request (R software, version 3.5.1):

All the figures were generated using R software version 3.5.1.

RNA-seq analyses

PolyA+ RNA seq libraries were prepared using the SMART-Seq v4 UltraLow Input RNA kit (Clonetch) followed by the Nextera XT DNA library Prep kit (Illumina) according to the manufacturer recommendations from 3 replicates for each conditions (wild-type P7 (SNVT9, SNVT10, SNVT11), P7 mutant (SNVT13, SNVT14, SNVT15), wild-type P14: SNVT1, SNVT2, SNVT4), P14 mutant (SNVT5, SNVT6, SNVT7)) and sequenced 50 pb single end on

Competing interests

The authors declare that they have no competing interests.

Supplementary Table 1: Gene ontology analyses of the common down-regulated genes in

Tbp2 -/-mutant oocytes at P7 and P14.

Supplementary Table 2:

Expression of mRNA decay-associated genes in Tbp2 -/-mutant oocytes at P7 and P14.

Supplementary Table 3:

Comparison between the 3 anti-TBP2 antibodies used for proteomic analyses of the TBP2-IP from NIH3T3-II10 cells.

Supplementary Table 4:

Oocyte-specific RNA-seq data at P7 and P14.

Supplementary Table 5:

List of the antibodies used.

Results

Unpublished data

Conclusions

During my thesis, I have explored the role of TBP2 in controlling transcription initiation during oocyte growth, and have characterized the oocyte-specific TBP2-TFIIA-containing transcription machinery.

We have shown that Tbp2 loss of function results in the main decrease in the expression of the most abundantly expressed genes as well as a specific down-regulation of the expression of the MaLR retroviral elements by performing RNA-seq analyses of wild-type and Tbp2 -/-oocytes from both primary and secondary follicles.

Using an optimized immunoprecipitation method coupled to mass spectrometry and mouse molecular genetics, we have shown that in the growing oocytes, TBP2 does not assemble into a TFIID-type complex but associates with TFIIA to form a specific TBP2-TFIIA-containing transcription machinery.

By studying TBP2 occupancy in the II10 cells that ectopically over-express TBP2, we have shown that TBP2 binds to gene promoters genome-widely and the average binding profile shifts a bit downstream of the TSS, suggesting that TBP2 might be involved in a different TSS usage.

Additionally, we have applied TBP2-Dam-ID-seq, TBP2 uliCUT&RUN and carried out nascent transcripts analyses using SLAM-seq coupled with mouse TU-tagging approach, those ongoing studies will give us more insights about the role of TBP2 during oocyte growth.

Altogether, these results show that a specific TBP2-TFIIA-containing transcription machinery, different from canonical TFIID, drives transcription in mouse growing oocytes. Ravens et al. Epigenetics & Chromatin (2015) 8:45 DOI 10.1186/s13072-015-0039-z Abbreviations Ac: acetyl; bHLH-LZ: basic helix-loop-helix leucine zipper; bp: base pairs; ChIP: chromatin immunoprecipitation; ChIP-seq: chromatin immunoprecipitation coupled to sequencing; DHS: DNase Hypersensitive Site; GEO: gene expression omnibus; H: histone; HAT: histone acetyltransferase; KAT: lysine acetyltransferase; K: lysine; kb: kilobase; kD: kilo Dalton; m: mouse; me: Methyl; mD: mega Dalton; mESCs: mouse embryonic stem cells; Mof: male absent on the first; MSL: male-specific lethal; mRNA: messenger RNA; NSL: non-specific lethal; Pol II: RNA polymerase II; qPCR: quantitative polymerase chain reaction; shRNA: small-hairin RNA; siRNA: small interfering RNA; Tip60: tat-interacting protein 60 kDa; TSS: transcription start site; UTR: untranslated-region; y: yeast; WB: western Blot.

Appendix

Authors' contributions SR, CY and MS performed experiments. SR and TY analysed data. SR and LT designed the study, analysed data and wrote the manuscript. All authors read and approved the final manuscript.

Author details

Competing interests

The authors declare that they have no competing interests.

Additional files

GSM723018), H3K27ac (GSM594578) and H3K4me1 (GSM594577) using enhancer mid points [START_REF] Whyte | Master transcription factors and mediator establish super-enhancers at key cell identity genes[END_REF] as reference coordinates. Two main clusters were defined: one with significant Tip60 binding (cluster 1), and a second where Tip60 cannot be detected (cluster 2). Densities are represented in region of ∓ 2 kb around enhancer mid points. The representation is as in Fig. 1a. (B and C) Tip60 enrichment around enhancer mid points of cluster 1 (B) and cluster 2 (C). Clusters were defined in panel A. S1. Summary of all ChIP-seq datasets used representing the number of unique reads and GEO accession numbers.

Additional file 3: Table

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