L ; Soueidan

M Shukla

Ben Simon

E ; Tonchev

O ; Dimitrov

S Angelov

Title: 3D organization of the chromatin fiber

The local chromatin state plays a crucial role in all fundamental DNA--templated processes, such as transcription control, DNA replication or repair, signaling, etc. The precise 3D organization of the second level folding, the so--called 30 nm fiber, has been a matter of intense speculations and debates over the past 40 years. Two competing models have been proposed on the basis of in vitro data, the solenoid and zigzag arrangements. In the solenoid model, consecutive nucleosomes interact with each other and follow a helical trajectory with bent DNA linker. In the zigzag model, alternate nucleosomes interact with each other with straight, twisted or coiled linker DNA. During my thesis, I developed a new biochemical approach, called ICNN (Identification of the Closest Neighbor Nucleosomes), allowing a direct "visualization" of the neighboring nucleosomes within H1--dependent compacted chromatin. We showed that within H1-compacted regular nucleosomal array, N±2 nucleosomes are the nearest neighboring interaction partners of any arbitrary nucleosome N. This finding provides an unambiguous evidence for the zigzag two--start helix conformation of the 30 nm fiber.

Furthermore, this organization remains independent on the DNA linker length, demonstrating that the nucleosome repeat length (177--227 bp) does not affect the chromatin structure and therefore cannot be a reason for chromatin structural heterogeneity as suggested in the literature. Chromatin structure and dynamics might be affected by the incorporation of histone variants. Our ICNN experiments with H2A.Z arrays showed no difference of folding between H2A.Z--and H2A--containing fibers. This finding suggests that the H2A.Z--specific transcriptional regulation involves mechanisms other than chromatin folding. CENP--A is a hallmark for centromeric chromatin that is indispensable for cell division. Published crystals structure showed that CENP--A-containing nucleosomes are more "open" than conventional ones due to the shorter α--N CENP--A helix. Besides, recent results in our lab showed that H1 does not stably bind to the CENP--A nucleosomes and that no stem organization of the linker is formed (not published). Our ICNN study at the chromatin level confirmed the absence of H1--induced folding of a regular CENP--A array. Interestingly, replacement of CENP--A with the α--N H3 --CENP--A mutant restored proper H1 binding and folding of the fiber into the usual zigzag conformation, as does the conventional H3--containing fiber.

Résumé de thèse

Titre de thèse: Organisation 3D de la fibre de chromatine L'état local de la chromatine joue un rôle crucial dans tous les processus génétiques comme le contrôle de la transcription, de la réplication et de la réparation de l'ADN, de la signalisation, etc... La structure précise de l'organisation 3D du deuxième ordre de compaction de la chromatine, aussi appelé fibre de 30 nm, a été le sujet d'intenses débats durant les 40 dernières années. En se basant sur les données in vitro, deux modèles concurrents se distinguent: le solénoïde et le zigzag. Dans le modèle solénoïde, des nucléosomes consécutifs interagissent pour former une trajectoire hélicoïdale avec courbure de l'ADN de liaison. Dans le modèle zigzag, deux hélices d'empilement de nucléosomes se forment, reliées par l'ADN de liaison qui peut être droit ou tordu. Durant ma thèse, j'ai développé une nouvelle approche expérimentale biochimique, appelée ICNN (Identification of the Closest Neighbor Nucleosome), permettant de déchiffrer les interactions entre nucléosomes voisins au sein de la fibre et ainsi de définir sous quelle forme la chromatine se compacte. Nous avons démontré que dans une fibre compactée H1--dépendante, les nucléosomes N+2 sont les plus proches voisins d'un nucléosome N arbitrairement choisit. Ces résultats montrent, sans ambiguïté, l'organisation zigzag de la fibre de 30 nm. De plus, cette organisation reste indépendante de la longueur de l'ADN de liaison, démontrant ainsi que la longueur des nucléosomes (177--227 bp) n'affecte pas la structure de la chromatine et ne peut donc pas être responsable de l'hétérogénéité structurale décrite dans la littérature. La dynamique et la structure de la chromatine peuvent être affectées par l'incorporation de variants d'histones. Nos expériences utilisant des assemblages H2A.Z ne montrent aucune différence de repliement entre les fibres contenant H2A.Z et H2A. Ces données suggèrent que le mécanisme moléculaire de régulation de la transcription spécifique de H2A.Z n'est pas lié au repliement de la chromatine. CENP--A est un élément de la chromatine centromérique indispensable à la division cellulaire. La structure par diffusion des rayons--X dans des cristaux montrent que les nucléosomes contenant CENP--A sont plus ouverts que le nucléosomes conventionnels à cause de l'hélice α--N CENP--A plus courte.

Parallèlement, des résultats récents de notre laboratoire ont montré que H1 ne se lie pas d'une façon stable aux nucléosomes contenant CENP--A, ne conduisant à aucune organisation de l'ADN de liaison (données non publiées). Notre étude au niveau de la chromatine a confirmé l'absence de repliement de la fibre contenant CENP--A en présence de H1. De manière intéressante, le remplacement de CENP--A par le mutant α--N H3 --CENP--A restaure la bonne liaison de H1 et le repliement de la fibre avec une conformation habituelle en zigzag comme dans une fibre contenant H3. I want to express my gratitude to the group of people that were collaborating with me on the project of chromatin fiber structure: Dr. Stefan Dimitrov for his enthusiastic knowledgeable discussions and his influence on my thesis. Dr Manu Shukla for teaching me the chromatin biochemistry and for giving me the necessary tools to continue what he started.

I would like to thank, Dr. André Verdel, Dr. Marc Lavigne and Dr. Gael Yvert for their time and efforts to evaluate the thesis.

I would like to extend my gratitude to all the members of the Anguelov's team past and present. In particular I would like to thank my favored indians Dr. Charles Richard john Lalith (Richy baba) and Dr. Imtiaz Nisar Lone (mimi) for the worm welcome they gave me when I first joined the team and for the great fun times we had in the algeco. My special thanks to Rama, my lab drinking buddy, for all the stories we shared about our drunken nights. My thanks to Ogi, Hervé and Elsa for all their help and time during my PhD.

During my four years stay in ENS I met some wonderful people that I enjoyed spending time in ENS and in bars. Special thanks goes to Fanny for the great time we spent in ENS, specially playing badminton and drinking coffees. I can't thank her enough for the best night of my life, the night I ran 14 km at 2am on an icy pathway and the night that I fell more than 7 times. I can't thank you enough for all the corrections you made and all the "ca se dit pas!!!". I also would like to thank Lydia, for all the "pause clope" that we did, for all the sweets that we ate (without anybody knowing) and for the great time we are having together every Wednesday (I tried to make a sentence with hydratation in it and I couldn't). A special thanks goes to Paulina and Xuan Nhi, for making this last year sweet with our afternoon coffee and lunch breaks and specially for listening to 'bad luck' stories all of last year. I also would like to thank Mehrnaz, Loan and Marion one of the first people I met once I started in ENS, for the wonderful time we spend together and for hicking and skiing trips. To Mehrnaz I would like to add, don't be afraid we are going to all be here for you. Special mention goes to Juliana for all the time we spent in the "Lapin blanc" and hope I'll see you soon in Japan (if you invite me). I can end the ENS chapter without thanking, mulumulu ducon (alias maxime) and Etera for being the main consumers and providers for 'Bar Lamoush'.

I owe 'monsieur le bricoleur' (alias Mathieu) a big thank you, for being present for me when I needed help, especially for repairing my bike and my door but all also for all the fun night and yummy burger we ate together. A special thanks to caro (Dufaure de citre), for the brunches and for the 'confession intime' discussion we had and hope we continue doing. I would like also to thank all the people I met during my college days. Starting by Johanna, I would like to thank you and your family for accepting me into your family during Christmas days and for the time we spend at the riverside. I'm really proud to have as my friend. I would like also to thank Fatima, for all the time she been late to movies, restaurant and parties. I hope I'll be there to see become doctor soon.

A special thanks to Julie, for all the times you have been mean to me (specially with lorraine) and for all the times you've been nice to me and for all great summer week end we had at your parent's house. I would also like to thank Lolo for making me a chartreuse addict, for the great friend you have been during all this years and for introducing to your 'copinette group', Marie--Audrey, Laure and Caro (Jaillet). I would like also to thank Elodie, Anais and Laura for the great support they have been during the past years; I know I can count on them whenever they are needed.

A special thought to my childhood friends, to be more precise to my teenagehood friends. Elma, we've been friends for a long time, we've been through good and bad times, and I'm glad I can still count you as close friend. I would like to thank Claire, also known as claira mama, for making my visits to Lebanon fun and enjoyable for the past 10 years.

Last but not least, I would like to thank my family, who has been there for me all along, who sent me to France at 17 to have a better life. I dedicate this thesis to my father Issa, to my mother Aida, to my two brothers, Maher and Samer and finally to my older sister Liliane. A special mention goes to Maher, who put up with me during 8 long years. A special thanks also my beloved sister, I don't where will I be if you weren't there for me each second of my life. Thank you for teaching me to be the strong, independent and open--minded woman that I am today. I hope one day, I can repay 1% of what you've given me. And finally I would like to thank my mom, who had the courage to send her youngest daughter 3000 Km away allowing me to have a better life and a better education. 30 nm exist in vivo?............................................................................................................................60 3.6.2 Tables:

Lists of Abbreviations:

2D To address these questions, multitudes of techniques are employed to study the dynamics of nucleosome and chromatin structure that will provide the molecular basis for cellular processes.

Chapter 1: DNA organization in eukaryotic cells.

Chromatin overview

Stuffing the long strands of chromosomal DNA into the eukaryotic nucleus requires the DNA to compact in length approximately 10--50.000 folds. Indeed, eukaryotic cells contain ~5x10 9 DNA base pairs (bp) in a 5--7μm nucleus and the DNA molecules that code the human genome should measure ~2 meters if they were laid end to end. This vast amount of DNA is packed by the histone proteins into a hierarchical structure called the chromatin. The word "chromatin" has been derived from the Greek word "khroma" meaning colored based on its staining ability in dye. The dynamics and organization of this chromatin influences many function of the genome. Its primary functions can be divided into 4 categories, 1) to package DNA into a smaller volume to fit in the cell, 2) to reinforce the DNA to allow mitosis, 3) to prevent DNA damage and 4) to control gene expression and DNA replication.

Chromatin history

The history of chromatin began in late 19 th century when biologist W. Flemings suggested the name 'chromatin' [1] while working on nuclear division (mitosis was another word suggested by him)(1880). Flemings, influenced by H. Zacharias microscopy studies of protease--digested nuclei, wrote: " … in the view of its refractile nature, its reactions, and above all its affinity to dyes, is a substance which I have named chromatin…" [2].

During this time F. Miescher developed methods for the isolation of nuclei from pus leukocytes and described what he then called 'nuclein' as a strong phosphorus--rich acid [3]. Later on, in 1884 A. Kossel, who was working with Miescher in Hoppe--Seyler's laboratory, described the 'histone' in the acidic extract from erythrocytes nuclei [4].

The first half of the 20 th century was great for the field of genetics but showed no advances in the understanding of the chromatin structure. chromosomes and on plant chromosomes using proteases and nucleases suggesting that both the salivary gland and plant chromosome are composed of a continuous framework and a matrix that occupies a considerable volume [5]. Most of the investigators were back then agreeing that chromosomes and chromatin formed the structural basis of the genes and that the histone's primary function was carrying genetic information [6].

Major structural biology discoveries were made during the second half of the 20 th century due to the development of X--ray imaging techniques. The discovery of the protein α--helix in 1951 by L. Pauling [7] followed by the famous discovery of double helix structure of DNA published in 1953 by J. Watson & F. Crick [8], M. Wilkins [9], and R. Franklin & R. Gosling [10] were turning points in the chromatin field. Another key advance in the field was done by G. Zubay and P. Doty who were able to prepare a soluble chromatin molecule [START_REF] Tachiwana | Structures of human nucleosomes containing major histone H3 variants[END_REF]. In the 60s, J. Gall demonstrated that the backbone of the chromatid is a continuity of one single molecule of DNA [START_REF] Gerchman | Chromatin higher--order structure studied by neutron scattering and scanning transmission electron microscopy[END_REF]. The fractioning of histones by E. Johns [START_REF] Ghirlando | Hydrodynamic studies on defined heterochromatin fragments support a 30--nm fiber having six nucleosomes per turn[END_REF] and the discovery of association between histone modification and chromatin transcription by V. Allfrey [START_REF] Williams | Chromatin fibers are left--handed double helices with diameter and mass per unit length that depend on linker length[END_REF] were among the important advances made during this decade on the chromatin.

The first electron microscopy images performed on chicken erythrocyte nuclei in 1973 by A. Olins & D. Olins [START_REF] Bordas | The superstructure of chromatin and its condensation mechanism. II. Theoretical analysis of the X--ray scattering patterns and model calculations[END_REF] and C. Woodcock [START_REF] Bordas | The superstructure of chromatin and its condensation mechanism. I. Synchrotron radiation X--ray scattering results[END_REF] showed the 'beads on a string' structure. 6 months later, R. Kornberg published his chromatin model, based on nuclease assays and histone cross--linking, showing that about 200 bp of DNA formed complex with four histone pairs [START_REF] Makarov | A triple helix model for the structure of chromatin fiber[END_REF]. In 1975, this chromatin subunit received the name of 'nucleosome' [START_REF] Song | Cryo--EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units[END_REF]. The discovery of the nucleosome represented a 'quantum jump' in the understanding of the chromatin structure. During the next 30 years a race took place to uncover the structure of chromatin and the 30 nm fiber (Figure1).

DNA packaging

In order to fit into the nucleus, DNA must be packaged into a highly compacted structure known as chromatin. In the first step of this process DNA is condensed into an 11 nm fiber that represents an approximate 6--fold level of compaction [START_REF] Correll | Short nucleosome repeats impose rotational modulations on chromatin fibre folding[END_REF]. This is achieved through nucleosome assembly.

The nucleosome is the smallest structural component of chromatin, and it is produced through interactions between DNA and histone proteins. A histone octamer is formed from the histones H2A, H2B, H3 and H4, although in some cases other histone variants may also be found in the core (e.g. H2A.Z, MacroH2A, H2A.Bbd, H2A.lap1, H2AX, H3.3, CENP--A and others). Additionally, a 147 bp DNA segment then wraps itself around the histone octamer 1.75 times, thus completing the formation of a single nucleosome. As shown in figure 2, the nucleosome is part of a wider process, whereby multiple nucleosomes form in a linear fashion along the DNA molecule. This ultimately produces the 11 nm fiber, which is traditionally described, based on its appearance, as "beads on a string". Here, adjacent nucleosomes are connected via "linker DNA", which is usually bound to the H1 histone and is between 20--80 bp long. Additionally, flexible histone tails extend away from nucleosomal DNA and can interact with other nucleosomes, stabilizing more complex 3D structures [START_REF] Boulard | Histone variant nucleosomes: structure, function and implication in disease[END_REF]. Of note, nucleosomes are dynamic entities and can undergo spontaneous sliding, "splitting" or even complete dissociation. The level of compaction attained through the formation of the 11 nm nucleosome fiber is insufficient to package the whole genome into the nucleus. Instead, this fiber forms the basis for other higher--order chromatin structures that are established through additional folding and bending events. Despite the extensive knowledge already gained on the structure of the 11 nm nucleosome fiber the intermediate chromatin structures commonly known as the 30 nm fiber is a matter of debates. Studies over the past decades have emphasized the critical importance of chromatin components whose nature and spatial organization are sources of information that contribute to cellular function and identity. Changes in chromatin structure have been found to play a fundamental role in the regulation of multiple genomic processes, from gene expression to chromosome segregation and the maintenance of genome integrity and stability.

Two popular models were proposed based on in vitro data: the solenoid and the zigzag.

In each case, the 11 nm nucleosome fiber undergoes additional folding to form a 30 nm fiber [START_REF] Nekrasov | Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics[END_REF]23]. In the one--start solenoid model, bent linker DNA sequentially connects each nucleosome core, creating a structure where nucleosomes follow each other along the same helical path [23,24]. Alternatively, in the two--start zigzag model, straight linker DNA connects two opposing nucleosome cores, creating the opposing rows of nucleosomes that form so called "two--start" helix. In zigzag model, alternate nucleosomes become interacting partners [START_REF] Nekrasov | Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics[END_REF]25]. Interestingly, some studies offer a model, in which intermediate 30 nm fibers contain both the solenoid and zigzag conformations [26].

One aspect shared by most of the models for higher--order chromatin organization is the dynamic existence of decondensed loops among more compact chromatin structures. In most cases, higher--order chromatin has to be decondensed so that the transcription machinery can gain access to the genes [27,28].

Types of chromatin

Interphase chromatin can be divided into two domains, euchromatin and heterochromatin. These can be distinguished in a cytological manner by differential staining [30]. The weakly and strongly stained regions are the euchromatin and the heterochromatin, respectively (Figure 3A). These two vary on different genetic levels such as gene activity, histone modifications and nucleosome packaging [31]. They might also have a different higher--order packaging [32,33] and nuclear organization [34].

Heterochromatin

Heterochromatin was initially defined as the portion of the genome that retains deep staining with DNA specific dyes as the dividing cell returns to interphase from metaphase. Heterochromatin regions consist of repetitive DNA, including satellite sequences and middle repetitive sequences related to transposable elements and retroviruses. These regions are typically gene--poor, replicate in late S--phase and generally have a reduced frequency of meiotic recombination [35]. Heitz first introduced it in 1928, by identifying regions of mitotic chromosomes that retained a compact structure during interphase. Autoradiographic studies using tritium--labeled uridine ([ 3 H]UdR) provided convincing evidence that RNA synthesis is only possible in the diffuse state, whereas condensed chromatin is transcriptionally inactive [36]. Two reasons were established for inactive chromatin:

1. The rare coding DNA present in these regions is temporarily and reversibly inactivated.

2. The majority of this region composed of non--coding DNA is incapable of transcription.

The heterochromatin is spread everywhere in the chromosome in small regions but resides in the centromere, telomeres and the Barr body of the inactivated X chromosome [START_REF] Heard | Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome[END_REF]37].

However, heterochromatin plays an important role in gene expression during development and differentiation [38]. Many components are necessary in generating heterochromatin, such as repetitive DNA sequences, methylation of histone H3 lysine 9, heterochromatin protein 1(HP1) and RNA interference (RNAi) [39]. The post-translational modifications of the key lysine residues on histone protein H3 determine the formation of different chromatin structures. The methylation of N--terminal lysine residues of histone H3 is among the most studied histone modification. The lysines 4,9,27,36 and 79 can be modified by methytransferases and demethylases. Transcription and chromatin structure are affected by mono--or di--methylation of these residues, while epigenetic regulation is attributed to tri--methylation (H3K4me3, H3K9me3, H3K27me3, H3K36me3, H3K79me3) (Figure 3C) [40,[START_REF] Shilatifard | Chromatin modifications by methylation and ubiquitination: implications in the regulation of gene expression[END_REF]. Three different kinds of condensed chromatin have been observed within interphase nuclei: tissue--specific condensed euchromatin, facultative heterochromatin and constitutive heterochromatin.

Tissue--specific condensed euchromatin

Although almost all cells in an organism contain the same DNA, only a part of the available information within a genome is expressed in any given cell type. This selectivity depends on the inactivation of some euchromatic components of the genome in a particular cell. The condensation happens at specific differentiation states as part of a tissue--specific transcriptional control system. This condensation state is highly variable from one cell type to another within a given species. This condensation state is reversible [START_REF] Nakatsu | Activity of DNA templates during cell division and cell differentiation[END_REF], but once it is dispersed, it does not automatically adopt a transcriptionally active form as euchromatin.

Facultative heterochromatin

The facultative heterochromatin is found mainly at the developmentally--regulated loci, next to rRNA genes or at the transcriptionally active region of euchromatin [34]. It plays a key role in normal cell lineage development, cell differentiation by somatic methylation and inactivation of germline--specific genes [START_REF] Feldman | G9a--mediated irreversible epigenetic inactivation of Oct--3/4 during early embryogenesis[END_REF].

Furthermore, the facultative heterochromatin might be involved in allelic exclusion, genomic imprinting and inactivation of the X chromosome, the gene loci of immunoglobulin and T receptors α and β [START_REF] Heard | Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome[END_REF][START_REF] Skok | Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes[END_REF]. For example, the inactivation of the X chromosome in female mammals can occur because one X chromosome is packed as facultative heterochromatin and is thus silenced while the other packed as euchromatin and expressed [START_REF] Talbert | Spreading of silent chromatin: inaction at a distance[END_REF]. Proteins like Polycomb--group proteins and non--coding genes such as Xist regulate the formation of facultative heterochromatin.

Constitutive heterochromatin

These are the regions to which Heitz originally gave the name heterochromatin. They are mostly composed of non--coding and largely repetitive DNA such as clusters of satellite sequences and transposable elements of centromeres, pericentric foci and telomeres [34]. These transposable elements are highly mutagenic because they target coding genes for insertion causing chromosome breakage and genome rearrangement [START_REF] Henikoff | Heterochromatin function in complex genomes[END_REF]. This DNA is permanently non transcribable rather than simply repressed. The constitutive heterochromatin plays an essential role in keeping genomic integrity by preventing abnormal chromosome segregation, recombination and DNA replication.

Euchromatin

Euchromatin is lightly packed chromatin and highly concentrated in active gene. It is compacted during cell division and relaxed during interphase. The replication of this chromatin occurs during S--Phase and the recombination during meiosis. It is also been

shown that the condensation coincides with the lack of synthesis of mRNA during mitosis. Euchromatin is subjected to post--translational modifications (PTMs) such as histone methylation and acetylation (Figure 3B). Some of these modifications have been suggested to have a global effect (indirect) and others affect mostly the boundary between euchromatin and heterochromatin. The molecular mechanisms responsible for different gene silencing in euchromatin are still largely unknown [START_REF] Rusche | The establishment, inheritance, and function of silenced chromatin in Saccharomyces cerevisiae[END_REF].

Centromeres

Mitosis is the process by which eukaryotic cells divides to produce two daughter cells that contain the same number of chromosomes as the parent cell. The centromere plays a crucial role in mitotic segregation. The genes found along the centromere are highly divergent while the genes found in the chromosomes arms are highly conserved. The main function of the centromere is to build the kinetochore at meiosis and mitosis, which will serve as the physical connection of the chromosomes to the microtubule-based spindle [START_REF] Eichler | Repetitive conundrums of centromere structure and function[END_REF]. However, the repetitive DNA typically found in this region is required for neither the identity nor the function of the centromere. The architecture and the scale of centromeric chromatin vary between different eukaryotic species.

However, all centromeres contain the histone H3 variant CENP--A, a centromere--specific protein [START_REF] Earnshaw | Three human chromosomal autoantigens are recognized by sera from patients with anti--centromere antibodies[END_REF][START_REF] Earnshaw | Proteins of the inner and outer centromere of mitotic chromosomes[END_REF]. The centromere identity is defined by the presence of CENP--A in the chromatin [START_REF] Palmer | Purification of the centromere--specific protein CENP--A and demonstration that it is a distinctive histone[END_REF].

Even though the word "centromere" is derived from the greek centro ("central") and mere ("part"), chromosomes can be classified into four types based on the location of the centromere: metacentric, submetacentric, acrocentric and telocentric (Figure 4).

Telomeres

Telomeres are ribonucleoprotein structures that protect the end of linear chromosomes from recognition as double--stranded breaks and activation of DNA damage response.

They are G--rich and short repeat sequences that comprise the physical termini of chromosome [START_REF] Greider | Telomere length regulation[END_REF]. The number of repeats and the length of telomeres are subject to regulation and influence biological processes such as aging and cancer [START_REF] Blackburn | Telomeres and telomerase: their mechanisms of action and the effects of altering their functions[END_REF]. Telomere length is controlled by a homeostatic mechanism that involves telomerase and conventional replication machinery. Telomerase, a telomere--specific protein, is a specialized reverse transcriptase that synthesizes the G--tail using intrinsic RNA template and thus prevents premature shortening of the chromosomes [START_REF] Lingner | Reverse transcriptase motifs in the catalytic subunit of telomerase[END_REF].

Chapter 2: The nucleosome

Nucleosome overview

As mentioned in the previous chapter, the genetic information of eukaryotic cells is stored in a 2 meter--long DNA molecule. At the first level of compaction, two super-helical turns of DNA are wrapped around a protein assembly of 8 histone molecules to form the nucleosome core particle. Hundreds of thousands of nucleosomes are further organized into multiple higher levels.

Due to its packaging, the structure and the accessibility of DNA in living cells is different from that of linear, naked DNA. This is of fundamental importance for our understanding of all processes that use DNA as a substrate, such as transcription, replication, DNA repair, etc. Eukaryotic cells developed complex mechanisms to modulate and control chromatin dynamics

DNA structure

The DNA molecule has a right--handed double--stranded helical structure in which the two strands run opposite and interwined to each other. Each helix is a polymer of four basic nucleotides. The polymer backbone is composed of the alternating sugar (deoxyribose)--phosphate units, attached to which are four types of heterocyclic nitrogenous bases namely Adenine, Thymine, Guanine and Cytosine, which hold the two strands together. Adenine from one strand forms two hydrogen bonds with Thymine form the other stand, while Guanine forms three hydrogen bonds with the Cytosine from the opposite strand. The DNA double helix also has two different--sized "grooves": a major groove and a minor groove (Figure 5A). DNA double helical structure has been classified into three types namely B--DNA, A--DNA and Z--DNA. B--DNA is the most abundant form of DNA commonly found under physiological conditions in a cell. In this structure, the helix takes a turn every 3.4 nm, and the distance between two neighboring base pairs is 0.34 nm. Hence, there are about 10 pairs per turn. In a solution with higher salt concentrations or with alcohol added, the DNA structure may change to an A form, which is still right--handed, but makes a turn every 2.3 nm and there are 11 base pairs per turn. A--DNA forms are present under some biological conditions that are not yet well understood. Another DNA structure is called the Z form, because its bases seem to zigzag. Z--DNA is left--handed and also narrower than the other two types of DNA. One turn spans 4.6 nm, comprising 12 base pairs. The DNA molecule with alternating G--C

These are found in the same equal molar stoichiometry in all eukaryotic organisms [START_REF] Hayashi | Asymmetry of chromatin subunits probed with histone H1 in an H1--DNA complex[END_REF] and their sequences are highly conserved in evolution.

Sequences and domain structure of histone

The core histones are divided into three types of structural domains: the histone fold, the histone fold extensions and histone tails. α--helix, loops and coil elements are the main secondary structure in histones (Figure 6).

1. Histone fold: this central region of 70 aa (amino--acid) of the protein chain contains a 3 to 4 turn α--helix (α1), a loop of 7--8 aa, an 8--turn α--helix (α2), a loop of 6 aa and a final 2 to 3 turn α--helix (α3).

2. Histone fold extensions: It contains less uniform structural elements. It extends N--terminally from the histone--fold domain of H3 (αN) and C--terminally from the histone fold domains of H2A and H2B (αC).

Histone tails:

The N--terminal part of all core histones is made up of random--coil elements of different lengths (16aa for H2A to 44 aa for H3). In the context of a single nucleosome core, these tails are flexible and unstructured [START_REF] Correll | Short nucleosome repeats impose rotational modulations on chromatin fibre folding[END_REF]. H2A also has also a 10 aa C--terminal tail that varies between H2A variants. These regions are also subjected to many posttranslational modifications such as acetylation, methylation, phosphorylation, ubiquitination, sumoylation, biotinylation, glycosylation, parpylation and ADP--ribosylation, which will be discussed later on.

Histone octamer

The histones assemble in heterodimer pairs, H2A and H2B on one hand and H3 and H4

on the other hand. The dimers are formed through histone--specific interactions, called the handshake motif [START_REF] Arents | The nucleosomal core histone octamer at 3.1 A resolution: a tripartite protein assembly and a left--handed superhelix[END_REF]. The L1--α2--L2--α3 regions will interact in head--to--tail orientation through extensive hydrophobic contacts between helices. The short terminal helices fold and rotate over the central helix, which will cause for the terminal helices to interlink and for central helices to overlap (Figure 8). Furthermore, β--bridges are formed due to loop interaction: the C--terminal loop (L1) of H2A interacts with the N-terminal loop (L2) of H2B, while the C--terminal loop of H3 interacts with the N--terminal domain of H4. These bridges will serve as primary docking sites on the histone surface. The central and C--terminal helices (α2--α3) of the two H3 within the H3--H4 dimers interact together and thus form a four--helix pack. These interactions result in the formation of the H3--H4 tetramer. The hetero--tetramer has the shape of a twisted open horseshoe [START_REF] Xie | Structural similarity between TAFs and the heterotetrameric core of the histone octamer[END_REF] and plays a role in nucleosome positioning. In addition, it exists as a soluble complex in physiological ionic strength solutions and binds much more strongly to the DNA than the H2A--H2B dimers. To obtain a fully folded histone octamer, two units of H2A--H2B dimers bind to the opposite side of the H3--H4 tetramer.

Nucleosome core particle (NCP)

The nucleosome core particle has been determined by high resolution X--ray crystallography [START_REF] Davey | Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution[END_REF][START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF] (Figure 8B). The NCP comprises 147 DNA bp and the histone (Figure 8A). The orientation of the histone octamer favors the positioning of the arginine side chains in the minor grooves of the B--DNA which results in a left--handed superhelix. adapted respectively from [START_REF] Ransom | Chaperoning histones during DNA replication and repair[END_REF] and [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF].

The interactions between the histone octamer and the DNA are divided into three groups:

1. Charge neutralization of the acidic DNA phosphate groups 2. Hydrophobic interactions.

3. Hydrogen bonds, mainly between main--chain amide group and phosphate oxygen atoms.

The flexible histone tails represents another means of DNA--protein or protein--protein interaction. Indeed, the N--terminal of both H2B and H3 are random coil segments that pass through the DNA gyres and are reported to interact with the linker DNA [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF][START_REF] Angelov | Preferential interaction of the core histone tail domains with linker DNA[END_REF].

Nucleosome positioning

As mentioned earlier, nucleosomes are arranged into regularly spaced arrays, with the length of the linker region connecting nucleosomes. It has long been known that the nucleosomes favor some position in the genome. Thanks to high--resolution genome-wide analysis, a common pattern has been discovered: the nucleosomes are absent at almost all enhancer, promoter and terminator regions [START_REF] Yuan | Genome--scale identification of nucleosome positions in S. cerevisiae[END_REF][START_REF] Valouev | Determinants of nucleosome organization in primary human cells[END_REF]. Nucleosome positioning can vary from an almost perfect positioning, in which a nucleosome is located at a given 147 bp stretch in all DNA molecules in a substantial fraction of cell population, to no defined positioning, in which nucleosomes are located at all possible genomic positions with equal frequency across a cell population.

The histone octamer exhibits some DNA sequence specificity, but unlike DNA binding proteins that achieve specificity by direct and strong interactions between few DNA base pairs and amino acids, the specificity of nucleosome formation resolves around the ability for a given 147 bp sequence to bend around the histone octamer [START_REF] Drew | DNA bending and its relation to nucleosome positioning[END_REF]. In vitro, many DNA sequences have been tested for the ability to position nucleosomes and the highest--affinity sequence so far identified is the artificial 601 element isolated by SELEX [START_REF] Lowary | New DNA sequence rules for high affinity binding to histone octamer and sequence--directed nucleosome positioning[END_REF]. The 601 sequence has been used numerous in vitro studies because it assembles into a very stable, highly positioned nucleosome. The rDNA is another nucleosome positioning sequence is widely used in in vitro experiments [START_REF] Hansen | Conformational dynamics of the chromatin fiber in solution: determinants, mechanisms, and functions[END_REF]. Although displaying lower positioning capacity that the 601, it has the advantage to be a natural DNA sequence.

New regulators, called chromatin remodelers, are emerging as important controls of gene expression. They form a complex chromatin remodeling machinery that uses the energy of ATP hydrolysis to control the nucleosome positions. In other words, some chromatin remodeling complexes can establish a specific nucleosome positioning patterns that defines accessibility of DNA and with it the 'on' or 'off' states of nucleosome--dependent gene expression [START_REF] Cairns | The logic of chromatin architecture and remodelling at promoters[END_REF].

The linker histone

The protein known these days as the linker histone was initially described as the lysinerich nuclear protein. It was separated from the other major nuclear proteins by ion-exchange chromatography [START_REF] Johns | Studies on nuclear proteins. The binding of extra acidic proteins to deoxyribonucleoprotein during the preparation of nuclear proteins[END_REF][START_REF] Bonne | Single--strand DNA binding protein from rat liver: interactions with supercoiled DNA[END_REF]. While studying the nucleosome organization within the chromatin, the linker histone was found to be binding to the DNA between nucleosomes and helps chromatin compaction.

Sequence and domain structure of linker histone

Even though a lot of biochemical similarities were found between linker and core histones, they differ in function, architecture and evolutionary origin. Structurally, the canonical linker histones of higher eukaryotes can be separated into three domains: a short N--terminal (20 aa) a long lysine--rich C--terminal (100 aa), and a non--polar central globular domains (80 aa), while the two tails are unstructured [START_REF] Hartman | Studies on the role and mode of operation of the very--lysine-rich histone H1 in eukaryote chromatin. The three structural regions of the histone H1 molecule[END_REF]. The linker histone family is highly diverse, in that 11 stage and species--specific variants have been discovered so far. They all differ in sequence, molecular weight, biochemical/biophysical and immunochemical properties [START_REF] Cole | Microheterogeneity in H1 histones and its consequences[END_REF].

The Globular Domain of histone H1 (GH1)

The central domain belongs to the 'winged helix' family of DNA--binding proteins. The structure of the GH1 was resolved by crystallographic studies on the closely related linker histone H5 (H1 variant in avian erythrocytes) [START_REF] Ramakrishnan | Crystal structure of globular domain of histone H5 and its implications for nucleosome binding[END_REF]. Even though the GH1 has been internally located in the 30 nm fiber, a lot of controversy lies around its exact location within the nucleosome.

The GH1 adopts a mixed α/β fold, with three α--helices forming the core of the domain and β--strands (Figure 9A). α1 and α2 are separated by β--strands, followed by α3 and 2

anti--parallel β--strands and a loop are formed by the extension of these 2 β--strands. 2D

NMR experiment on GH5 and chicken histone GH1 showed a conserved 3D structure of the globular domain among linker histones [START_REF] Ramakrishnan | Crystal structure of globular domain of histone H5 and its implications for nucleosome binding[END_REF]. Even though the linker histone seems to be involved in stabilization of a higher--order of chromatin structure, it does not seem to be essential for DNA compaction.

For the past 30 years, efforts have been directed towards understanding the location of globular domain on either native or reconstituted nucleosomes. A limited digestion of the whole chromatin by microccocal nuclease (MNase) releases an intermediate particle containing a 166 bp nucleosome and the linker histone, often called chromatosome [START_REF] Simpson | Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones[END_REF].

Several models have been postulated to decipher the exact location of the globular domain on either native or reconstituted nucleosome substrates (Figure 9) [START_REF] Thomas | Histone H1: location and role[END_REF]. The first such model was proposed in 1986 and states that the GH1 binds to 10 bp of the entering and 10 bp of the exiting DNA (linker DNA) of the nucleosome in such a way that it is placed near the dyad axis in a symmetrical manner (Figure 9A). This model was validated by DNase I and •OH radical foot printing on the nucleosome dyad [START_REF] Staynov | Footprinting of linker histones H5 and H1 on the nucleosome[END_REF][START_REF] Syed | Single--base resolution mapping of H1--nucleosome interactions and 3D organization of the nucleosome[END_REF].

However, Zlatanova and coworkers challenged this model by proposing asymmetrical GH1 binding model. Asymmetrical model suggests that GH1 binds to 20 bp of linker DNA on either entering or exiting DNA (Figure 9C) [START_REF] An | Linker histone protects linker DNA on only one side of the core particle and in a sequence--dependent manner[END_REF]. A third mode called as "bridging"

model" proposes that the linker histone interacts with the dyad and with only one free DNA arm (either entering or exiting) (Figure 9B) [START_REF] Brown | Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo[END_REF]. Fluorescence recovery after photobleaching (FRAP) studies suggested the presence of only two DNA binding sites in the globular domain. One of the two binding sites fits within the major groove close to the dyad axis and the other within the minor groove on the linker DNA close to the NCP [START_REF] Brown | Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo[END_REF]. One of the reasons for the controversial models could be partly attributed to the way H1 is deposited on the reconstituted nucleosomes. The above--mentioned in vitro studies used salt dialysis to deposit H1 on the nucleosomes. However, this method might leads to improper assembly of H1 [START_REF] Shintomi | Nucleosome assembly protein--1 is a linker histone chaperone in Xenopus eggs[END_REF]. Another reason that could contribute to the controversy is the use of poorly positioned nucleosomes. Indeed, the widely used 5S

rDNA was shown to exhibit several translational positions, which in turn would interfere with the mapping of histone H1 nucleosomal DNA contacts.

The C--terminal Domain (CTD)

The C--terminal domain is very poorly conserved between different linker histone subtypes and species. The CTD is lysine--rich region with a repeat sequence S/TPXK leading to an even distribution of around 30--50 net positive charges [START_REF] Hansen | Intrinsic protein disorder, amino acid composition, and histone terminal domains[END_REF][START_REF] Subirana | Analysis of the charge distribution in the C--terminal region of histone H1 as related to its interaction with DNA[END_REF].

The CTD is involved in the folding of the nucleosome arrays into chromatin fibers [START_REF] Allan | Roles of H1 domains in determining higher order chromatin structure and H1 location[END_REF] and is required for high affinity binding to chromatin in vivo [START_REF] Hendzel | The C--terminal domain is the primary determinant of histone H1 binding to chromatin in vivo[END_REF]. The data collected on the binding affinity of histone H1 subtypes to the chromatin are somewhat contradictory.

Early work showed a difference in the binding affinity of the H1 subtypes to the nucleosome arrays and thus variability in their ability for inducing chromatin compaction [START_REF] Liao | Condensation of dinucleosomes by individual subfractions of H1 histone[END_REF]. In another study where H1 subtypes were purified from mouse liver and testis and their binding affinity tested with reconstituted mono--nucleosomes, the binding affinity was shown to be identical for all subtypes except H1b [START_REF] Talasz | In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor[END_REF]. The methods of H1 preparation could be the reason for these contradictory results. Indeed, it has been shown that the acid--extracted H1 may have altered folding ability of the GH1 and CTD domains, which is not the case of salt--extracted proteins.

However, the specific sites or residues within the H1 CTD required for chromatin binding remain undefined. It is believed that the excess of positive charges on the CTD neutralizes the negative charges on the DNA and helps with its condensation. The exact mechanism of chromatin stabilization by the CTD is still unclear. Even if it is disordered in solution, it can adopt a segmental α--helical form upon interaction with DNA or in presence of secondary structure stabilizers (such as trifluorethanol and NaClO 4 ) (Figure 10) [START_REF] Verdaguer | Helical structure of basic proteins from spermatozoa. Comparison with model peptides[END_REF]. These results indicate that CTD binding to linker DNA within chromatin can induce folding in this specific area leading to the hypothesis that the depletion of the CTD leads to the loss of H1--dependent chromatin compaction.

Functional roles of the linker histones

In vitro studies on H1--containing chromatin showed a strong inhibition of transcription [START_REF] Shimamura | Histone H1 represses transcription from minichromosomes assembled in vitro[END_REF]. Furthermore, remodeling by SWI/SNF seems to be modulated by the linker histone H1 by partially inhibiting the remodeling activity [35].

However, in vivo, the concept of H1 as a general repressor has been disproved by experiments showing almost no transcriptional differences when H1 is depleted in lower eukaryotes possessing a single H1 gene. Genetic 'knockout' experiments in higher eukaryotes have been complicated by the presence of compensatory factors such as H1 alleles or variants [START_REF] Brown | Differential effect of H1 variant overexpression on cell cycle progression and gene expression[END_REF].

Interestingly, studies showed a direct relation between the ratio of linker histone H1 and the nucleosome repeat length. For example, neuronal chromatin has a 0.45 H1 ratio and a162 bp NRL while glial chromatin has a 1.04 H1 ratio and a 201 bp NRL (reviewed in [START_REF] Woodcock | Role of linker histone in chromatin structure and function: H1 stoichiometry and nucleosome repeat length[END_REF]). Alongside other data, it is stipulated that the role of histone linker is more architectural than regulatory.

The dynamics of H1

Measuring the dynamics of a biological molecule in a living cell was a very important task. It became possible with the advances in imaging technologies and the discovery of fluorescent proteins. FRAP is one of these techniques used to study the spatial and temporal dynamics of protein binding and/or diffusion. After photobleaching by micro-irradiation, the time required for H1--GFP to achieve recovery of fluorescence is determined by the binding affinity of H1 to the nucleosome. FRAP done on H1.2 showed a recovery time of approximately 1--2 min, which is rather fast, compared to the core histone showing little or no recovery [START_REF] Th'ng | H1 family histones in the nucleus. Control of binding and localization by the C--terminal domain[END_REF][START_REF] Misteli | Dynamic binding of histone H1 to chromatin in living cells[END_REF]. However, compared to transcription factor or other chromatin--binding proteins, which have a time recovery of 25 s, it is rather slow. These FRAP experiments of H1 described it as a highly dynamic and mobile protein rather than a statically bound protein. Based on this distribution, 3 models were suggested to determine the domain responsible for the low affinity pool. The first model designates the CTD to be key for the low affinity binding by interacting first with the linker DNA. It will then allow a stable and efficient binding of the globular domain [START_REF] Brown | Mapping the interaction surface of linker histone H1(0) with the nucleosome of native chromatin in vivo[END_REF]. If the CTD fails to connect with the linker DNA, H1 binding would be compromised. The second model suggested that the GH1, through nonspecific electrostatic interactions, mediates the first contact, thus allowing the CTD to acquire a 3D structure capable of high affinity binding. To confirm this model, FRAP experiments were done on a mutated CTD. When Ser/Thr sites were mutated to Glu, Ala and Lys, most of the deviations in the curves were seen after 65--90 s (high binding phase), while the phase prior to 50 s (low affinity phase) was found unchanged [START_REF] Hendzel | The C--terminal domain is the primary determinant of histone H1 binding to chromatin in vivo[END_REF]. Nevertheless, some inconsistency in the results does not validate this second model. Indeed, the Thr152Glu mutation showed alteration in the low affinity phase as well as in the high affinity phase, which suggested that both domains might be responsible for the low affinity binding. These findings led to a third model where the GH1 and CTD of H1 make the contacts simultaneously with linker DNA through an electrostatic clamp (Figure 12).

The eviction of H1 from its stable binding site is yet another unclear process.

The linker histone per nucleosome stoichiometry

The linker histones H1 are involved in both the nucleosome structure, as mentioned earlier and in the formation of a higher--order chromatin structure. The 30 nm fiber was first described as a solenoid arrangement of a chain of nucleosomes in the presence of H1. This solenoid fiber was also detected in the absence of H1 at an increased ionic strength. However, this fiber was not as condensed and regularly oriented as the fiber in However, the generalization of 1 histone H1 per nucleosome has persisted in the years.

Besides, it is not very clear whether the quantification of the H1/H5 levels is as precise as claimed and whether many of these in vivo reports are not rather measurement artifacts.

In vivo, mouse embryonic stem (ES) cells null for three H1 genes showed dramatic chromatin changes such as increasing of nucleosome spacing, reduced compaction and a decrease in histone modification. However, this depletion showed that the expression of very little number of genes was affected. The affected genes are all normally regulated by DNA methylation [108].

The linker histone isoforms

The H1 family of linker histones is the most divergent class of histones protein. Eleven subtypes have been identified in the human genome. This family of histones can be subdivided according to different criteria. Linker histone variants differ in their timing of synthesis, rate of synthesis, turnover rates, phosphorylation status and ability to compact chromatin. Broadly, they can be classified into three groups based on their expression pattern:

1. Genes expressed during the S--phase of the cell cycle (histone H1.1 to H1.5)

2. Genes with variable modes of expression in somatic cells (histones H1.0 and H1x)

3. Genes expressed in germ cells (histones H1t, H1T2, H1LS and H1oo)

The genes of linker histones have been found to exist either clustered (H1.1 to H1.5 and H1t) or solitary (H1.0, H1x, H1T2, H1LS and H1oo) and their distribution in the genome was found highly conserved between the human, mouse and rat genomes.

Cell--cycle dependent H1

The five main histone H1 genes (H1.1 to H1.5) share the particular characteristics as the majority of histone genes. They are located in the major cluster of chromosome 6, they

Germ--cell line specific H1 histones

It should be kept in mind that the H1t gene is also located on chromosome 6 and has a 3' hairloop end but this protein is only expressed in morphologically distinct spermatocyte chromatin [124]. It has been reported that the H1t binds less tightly than the somatic H1 variant, causing the chromatin to be more sensitive to nuclease. This property of H1t keeps the chromatin in a decondensed state during meiosis which facilitates spermatogenesis events such as recombination or protein transition [125].

H1LS is a histone H1--like protein expressed only in elongating and elongated spermatids. It has several biochemical properties that are similar to those of linker histones including aggregating chromatin and binding to mononucleosomes. Since H1LS is expressed in late spermatids (that do not contains the core histones), it might use another mechanism for chromatin condensation that the linker histones. In addition, based on the expression pattern of H1LS, it was suggested that it participates in chromatin remodeling and regulates transcription during spermiogenesis [116].

Oocyte--specific linker histone H1oo is the mammalian homologue of the oocyte--specific linker histone B4 in frogs and cs--H1 in sea urchin. It is expressed in early stages, as early as germinal vesicle, metaphase II, polar bodies formation and two--cell stage embryo, but it disappears in the 4--8 cell embryonic stages. The functions of this protein are unclear but it might play a key role in control of gene expression during oogenesis and embryogenesis through perturbation of the chromatin structure [126].

Non cell cycle dependent H1

H1.0 and H1c genes are both solitary genes, located respectively on the 22 nd and 3 rd chromosomes. Their genes are intronless and they both express polyadenylated mRNAs.

The expression of this replication--independent variant of H1 is linked to cessation of DNA synthesis [127]. Although having the shortest C--terminal tail, H1.0 binds to the chromatin with a moderate affinity compared to some others H1 variants with longer C-terminal tails [START_REF] Th'ng | H1 family histones in the nucleus. Control of binding and localization by the C--terminal domain[END_REF].

However, their patterns of expression are different. Indeed, H1.0 gene is expressed in only some cell types and the protein H1.0 plays a role in terminal differentiation [128].

As for the H1x gene, it is ubiquitously expressed and the level of the protein remains stable throughout the cell cycle [129].

The histone tail and higher--order structure

Each histone has an N--terminal tail domain that extends to the exterior of the nucleosome and contacts extra--nucleosomal constituents in chromatin (Figure 13).

Together with the C--terminal tail of H2A, the count goes up to 10 tails projecting outside the nucleosome through the superhelical gyres [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]. The N--terminal tails of H2B and H3 go between the superhelical gyres whereas the N--terminal tails of H2A and H4 go under or over them. The tails present a low affinity to the nucleosome core DNA. In absence of DNA, they adopt an unstructured random coil conformation, which subjects them to a higher proteolytic cleavage compared to the histone fold domains [130]. They are highly charged positively due to the predominance of lysine and arginine residues. The histone tails main implication was reported to be regulation of transcription in vivo. However, it was noted that they play a small role in nucleosome stability and accessibility to DNA binding factors. The involvement of the tails in higher--order chromatin structure was first noticed during experiments involving proteolytic removal of the tail domain.

Indeed, early experiments on native chromatin extracted from chicken erythrocytes nuclei showed that the digestion of the tails resulted in unfolded chromatin. Once these tails were replaced by an extraneous polypeptide, the chromatin acquired back its original compaction under specific ionic strength conditions [131].

These findings were later on confirmed on native and reconstituted chromatin. Indeed, the self--association of oligonucleosomes in presence of divalent cations (Mg 2+ ) showed that the H3/H4 tail mediates the compaction directly and not through the electrostatic interaction that might occur. Furthermore, hybrid nucleosome array where either H2A/H2B dimers or H3/H4 tetramers lacked their N--terminal tail did not show any compaction even in high salt concentration. These results combined bring us to the conclusion of the equal importance of all the core histone tails [132]. On the contrary, in presence of divalent cations, nucleosomes containing only H3/H4 tails were able to oligomerize unlike nucleosomes containing only H2A/H2B tails [133]. Due to the importance of histone tails in the chromatin structure, it is also important to note the effect of the post--translational modifications that these tails encounter on the chromatin.

Indeed, as mentioned earlier, the histones tails are subjected to many modifications such as methylation, acetylation, ubiquitination, phosphorylation, etc. (Figure 13).

The histone tails--induced interactions can be divided into two categories: intra-nucleosome interactions where the tails interact with DNA/protein of their own nucleosomes and the inter--nucleosome interactions, where the tails mediate the interactions between different nucleosomes within condensed chromatin. The core histone tail domains can however participate in both kinds of interactions simultaneously. Understanding how tails interact within and between nucleosomes is essential for understanding chromatin folding and genetic regulation. Throughout the years some intra--nucleosome contacts of tail domains have been characterized. Using high--intensity UV laser crosslinking it was shown that the core histone N--termianal tails, especially H3 and H4 interact with linker DNA as well in cellular chromatin as in reconstituted nucleosomes and these interactions persists upon acetylation [START_REF] Angelov | Preferential interaction of the core histone tail domains with linker DNA[END_REF]135]. It was also shown that the C--terminal tail of histone H2A interacts with the DNA near the nucleosome dyad in the NCP and also near the edge of the nucleosome core region when linker DNA is present [136,137]. The N--terminal tail of H2A has been reported to bind to the nucleosome core DNA as well, at two positions around 40 bp from each side of the dyad [138]. In addition to H2A, the N--terminal tail of H4 presents a similar kind of binding, where the H4 tail binds to core DNA at a distance of 1.5 helical turn on both sides of the dyad [139].

Moreover, it has been demonstrated that the H3 tail binds within the nucleosome DNA when the nucleosome array is in beads--on--string conformation, but when the array undergoes salt--dependent folding and oligomerization the tails engage in inter-nucleosome interactions. Since H3 contributes to both types of interactions, crosslinking studies showed that only 20% of these interactions are inter--nucleosome whereas the 80% left are intra--nucleosome interactions. Furthermore, the exposed regions of H3 tails contribute to the inter--nucleosome interactions, while the regions near the histone fold are associated with the intra--nucleosome interactions. The same studies performed in presence of H1 showed that the histone H1 does not affect these interactions but does however stabilize the chromatin structure [140].

While the nucleosome crystal structure gave a lot of information on the intra-nucleosome contacts of the tail domains, this information was not complete. Indeed, the lack of linker DNA in the crystal of the nucleosome and the presence of a very high divalent cation concentration may affect the actual interaction within the native chromatin. Interestingly, on the first X--ray structure of the nucleosome, it was noted that the H4 tail domain of one nucleosome interacts with the surface of the H2A/H2B dimer of a nearby nucleosome [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF].

Histone modifications

The four core histones, the linker histone and their variant are all likely to be subjected to post--translational modifications (PTMs), which play a crucial role in chromatin dynamics. PTMs are particularly abundant on the N--terminal histone tails, but they also exist within the core and on the C--terminal tails. The histone code hypothesis is proposed in order to explain and understand the pattern of PTMs and their biological consequences. This hypothesis states that PTMs act through two distinct mechanisms:

• Regulating DNA accessibility by favoring inter--nucleosome contacts.

• Being used as a docking site to initiate biological processes.

The histones can be subjected to a wide range of PTMs such as acetylation, phosphorylaton, methylation and ubiquitination (Figure 14). Distinct histone amino--terminal modifications can generate synergistic or antagonistic interaction affinities for chromatin--associated proteins, which in turn dictate dynamic transitions between transcriptionally active or transcriptionally silent chromatin states.

The combinatorial nature of histone amino--terminal modifications thus reveals a "histone code" that considerably extends the information potential of the genetic code.

Hyperacetylated oligonucleosomes exhibit a reduced tendency to fold into secondary and tertiary structures compared to non--acetylated arrays [142]. For example, the substitution of Lys by Glu, which mimics the acetylation of the histone tails, inhibits the array condensation [143]. Interestingly, the H3 acetylation only disturbed DNA wrapping stability whereas H2A acetylation showed different outcomes depending on the other histone modifications. This phenomenon can be described as the acetylation-dependent histone--histone interactions [143].

Histone acetylation

All core histones can be acetylated but the majority of acetylations occur on lysines in the N--terminal of H3 and H4. It occurs during DNA replication and when genes are activated.

This process is performed by a class of enzymes called Histone Acetyl Transferases (HATs). HATs can be cytoplasmic or nuclear. The cytoplasmic HATs acetylate histones prior to nuclear organization and chromatin assembly, whereas the nuclear HATs acetylate histones in order to affect transcription or other DNA--dependent processes.

Biochemical analysis of a variety of proteins and protein complexes involved in transcription activation allowed the identification of a large number of HATs [144].

Tetrahymena p55 was the first nuclear HAT identified that provided a link between histone acetylation and transcriptional activation [145]. Recently, a connection between HATs and transcriptional elongation has been made. Indeed, coding regions need to be poorly acetylated to prevent aberrant initiation of transcription. Through a complex mechanism of methylation, a Histone DeAcetylase Complex (HDAC) is recruited and specifically deacetylates coding regions [146]. H4K16 plays many major roles, for example, maintaining boundaries between euchromatin and heterochromatin in yeast.

In Drosophila, it is also involved in the process of dosage compensation (equalization of gene expression between male X chromosome and female X chromosome). It was also determined that the H4K16 inhibits the formation of higher--order chromatin Histidine phosphorylation of histone H4 occurs on histone residues H18 and H75. A clear relationship has been established between an enhanced activity of the H4 histidine kinase and regeneration of damaged liver in porcine [155]. However, no mammalian histone H4 histidine kinase has been characterized yet.

Histone methylation

Histones H2B, H3 and H4 are the only histone modified by methylation. Lysine and arginine residues at the N--terminal tail of H3 and H4 are methylated. These modifications do not affect the charge of the protein but they increase its hydrophobicity and reduce its ability to form hydrogen bonds.

Histone methyltransferases (HMTs) are classified into two families the histone lysine methyltransferases and the histone arginine methyltransferases, based on substrates and structural motifs. Each HMT can methylate multiple substrates.

H4K20 is one methylation site that might alter the nucleosome structure because of its location in the basic region that binds to the DNA. The level of H4K20 methylation varies during the cell cycle, reaching maximum levels at G1 and mitosis and decreasing at S phase. In contrast, H4K16 acetylation reaches maximum levels at mid S phase and decreases to lower levels at G1 and mitosis phases. These observations, alongside other studies, showed that H3K20 methylation inhibits H3K16 acetylation and/or vice versa [156].

Histone H3 may be methylated at lysines 4, 9, 27, 36 and 79. It has been shown that transcribed condensed regions were highly enriched in acetylated H3 and di--methylated H3K4. On the contrary, repressed condensed regions are poor in acetylated histones and enriched in methylated H3K9. These two domains can be separated by an insulator element, which supports the histone code hypothesis that each histone modification has a distinct biological consequence [157]. This methylation is responsible for recruiting HPI to heterochromatic regions.

Histone ubiquitination

Histone H2A was the first identified protein to be modified by ubiquitin in cells [158].

Together with H2B, they are the most abundant ubiquitinated proteins in the nucleus.

Multiubiquitination of intracellular proteins is required for selective degradation by the proteasome through the ubiquitin--dependent pathway. However, monoubiquitinated histones are not degraded by the proteasome and used for intercellular signaling as for most monoubiquitinated proteins. The most common ubiquitinated form of histone is monoubiquitination, where a single ubiquitin is added to a lysine residue (Lys 119 for The precise mechanism by which ubiquitination contributes to gene regulation, DNA repair and other biological processes is still poorly understood.

The classical interpretation of the effect of epigenetic modifications, implying that a single modification is independent and has a predictable and stable outcome, has now evolved. A new model has been established based on the multitude of crosstalks between specific histone modifications. The main assay for crosstalk detection involves site--specific antibodies that specifically recognize the modification coupled to mass spectroscopy analysis, but this requires a prediction of possible interactions. However, the use of microarrays and next generation sequencing will increase the crosstalk identification.

Histone Variants

Even though histones are among the slowest evolving proteins, they have non--allelic variants that can have significant differences in primary sequence. Some of them have different biophysical characteristics that might alter the properties of the nucleosomes, while others have specific locations in the genome. In general, histone variants are present in single copies in a gene and are expressed throughout the cell cycle. Some of these variants are replacements histones that can be substituted to histones during development and differentiation. Histone H4 does not appear to have a known sequence variant but variants were reported for all three remaining conventional histones.

Histone H2A variants

The H2A is among the largest family of identified histone variants with some universal variants found in almost all organisms such as H2A.Z and H2AX (Table2). The C--terminal tail is where sequence diversification between variants is found, regarding length as well as amino acid sequence.

H2A.Z Histone Variants

Histone H2A.Z is the best--studied histone variant; it shares roughly 60% of homology with canonical H2A within the same species. H2A.Z is important to many organisms such as mouse, fly, Tetrahymena etc… Surprisingly, despite the divergence in sequence, H2A.Z nucleosome structure shows high similarity to the canonical one [163]. The significant differences are in L1 loop, which is important to interactions between the H2A--H2B dimers and the DNA within the nucleosomes. These differences of structure led to the hypothesis of a homotypic H2A.Z nucleosome, because the presence of both types of dependent nucleosome remodeling complexes such as Swr1 complex in budding yeast [167]. It has been demonstrated that the interaction between Swr1 complex and H2A.Z is essential for the deposition. Since the mechanism by which H2A.Z is targeted by these complexes need to be resolved, it would be interesting to check if these complexes will remain associated with H2A.Z when it is incorporated within the chromatin. Recently, an answer was partially given suggesting that histone chaperone ANP32E evicts H2A.Z/H2B dimer by interacting with a short region of the docking domain of H2A.Z

[168].

H2AX Histone variant

H2AX has an extended C--terminal moiety characterized by a unique SQE (Ser--Gln--Glu) motif, which is targeted for post--translational phosphorylation in response to DNA damage. Genome--wide analysis showed that H2AX is evenly distributed and represents 2 to 25% of the mammalian histone H2A pool [169]. Phosphorylated H2AX accumulates at the site of high--intensity laser--induced double strands break (DSB), generating structures called foci [169]. In response to a double strand break, an accumulation of repair proteins within the damaged foci has been noticed by fluorescent microscopy.

Two contradictory role have been assigned to H2AX on one hand that H2AX foci are essential for recruiting the DNA repair proteins [170], while on the other hand H2AX

foci could be essential for the retention of these proteins rather than for recruiting [171]. Emerging data indicate that H2AX may have other functions than repair. These data showed that H2AX is phosphorylated independently of DSB and that these modifications are under cell cycle regulation. However, the exact function of these foci is not known.

MacroH2A

MacroH2A is a vertebrate--specific variant, which has two distinct domains. The N-terminus that is similar to H2A, sharing 64% identical amino acids and a large C-terminus that has no similarity to other histones. The C--terminal domain is lysine--rich and includes a random coil. MacroH2A--containing nucleosomes show the same structural similarity to canonical nucleosomes and wrap the same amount of DNA [172].

However, due to a four amino--acid difference within the L1 loop, macroH2A--containing nucleosomes are more stable in structure and form more compact chromatin [173]. The main role attributed to microH2A in mammalian cells was transcriptional repression, based on its enrichment in the transcriptionally inactive X--chromosome (Xi) and its association to inactive alleles of imprinted genes. However, further studies showed macroH2A enrichment in inducible genes and bivalent genes in stem cells where it remains bound before and after transcription, leading to the hypothesis that macroH2A is necessary for establishing a chromatin environment for gene transcription. Many hypotheses have been proposed to explain the repression mechanism of macroH2A. On one hand it was suggested that macroH2A directly prevents the binding of transcription factors and chromatin remodeling [174], and on the other hand, it was suggested that macroH2A inhibits the p--300--dependent histone acetylation in vitro, and indirectly represses the transcription [175].

H2A.Bbd

The histone H2A

Histone H2B variants

The few documented H2B variants are capable of completely replacing the major H2B

subtypes, and they have specialized functions in chromatin compaction and transcription regulation. The sperm--specific H2Bs of sea urchins have a long N--terminal tail that is highly charged. This tail is involved in chromatin condensation, which implies that SpH2B plays an important role in the chromatin packaging in sperm [182].

Histone H3 variants

Histones

CENP--A, the centromere--specific histone

CENP--A is a centromere--associated protein that is required to build a fully functioning kinetochore. It was purified alongside the nucleosome core proteins, which indicated that it forms a complex with the core histones [187]. Homologues of CENP--A were identified from yeast to mammals, where their loss was found lethal. The homology between CENP--A and H3 is essentially found in the α--helical carboxy--terminal histone-fold domain. The N--terminal tail of CENP--A is highly variable between species and it is mostly required to recruit kinetochore proteins to the centromere [188]. Furthermore, in mammalians cells, CENP--A is overexpressed throughout the cell cycle where it is deposited in non--centromeric regions. These mislocalized CENP--A will recruit CENP--C and other kinetochore components which will drive the alignment and segregation of chromosomes.

The CENP--A--H4 tetrasome has been found to be more rigid that the H3--H4 tetrasome,

Histone chaperones

The term histone chaperone was first used to describe the role of nucleoplasmin in the prevention of histone DNA aggregation during nucleosome assembly. However, they are more generally used to define a group of proteins that bind to histones and regulate nucleosome assembly [START_REF] Ransom | Chaperoning histones during DNA replication and repair[END_REF]. In general, histone chaperone can be classified as either H3--H4 or H2A--H2B chaperones based on their preferential histone binding. However, most of the histone chaperones bind to both H3--H4 tetramer and H2A--H2B dimer. In addition to the canonical histones, variant histones have probably unique histone chaperones that recognize and deposit them specifically. Each histone chaperon can participate in four distinct steps of the nucleosome assembly.

1. Histone protein importers. For example, Nap1 transports the newly synthesized histone from the cytoplasm to the nucleus [192].

2. Maintaining histone supply. For example, NASP acts as a histone reservoir and regulates histone supply under stress conditions [193].

3. Regulating histone--modifying enzymes. For example, ASF1 serves as a bridge between histone sand the PTM enzymes [194].

4. Depositing histones during nucleosome assembly [195].

Table 3 summarizes the major histone chaperones and their role in nucleosome assembly.

Histone chaperone Target Function

Anti

Regulation of macroH2A.1 incorporation during DNA damage

Table 3: Histone chaperones and their functions during nucleosome assembly. Table inspired from [196]

Nap1 shares sequence homology with a large class of histone chaperones, including Nap1--like proteins (NAPL) in human cells and Vps75 in yeast. In vitro studies show that Nap1 has the same affinity for H3--H4 tetramers and H2A--H2B dimers but in vivo Nap1 preferentially binds to H2A--H2B. These contradictory findings suggest that there is a regulatory mechanism for the interaction between Nap1 and H2A--H2B dimers [192].

Like all histone chaperones, Nap1 intervenes at various steps of H2A--H2B deposition and exchange. It starts by mediating the interaction between the importin Kap114 and H2A--H2B, which allows the dimers to move from the cytoplasm to the nucleus. Then, alongside ACF (ATP--dependent Chromatin remodeling Factor), they help the deposition of H3--H4 and H2A--H2B onto the DNA for the nucleosome formation. Finally, Nap1 disrupts non--productive histones--DNA interactions [197]. Nap1 was also identified as a chaperone of linker histone B4 in Xenopus laevis [198].

The crystal structure of yeast Nap1 shows a α--helix fold that is mainly responsible for dimerization and a β--sheet, which is similar to other histone chaperone proteins. yNap1 exists as stable dimers and self--associated oligomers in solution. No crystal data on higher eukaryotic Nap1 have been resolved yet [199].

In addition to nucleosome assembly, Nap1 is implicated in transcriptional regulation and cell cycle regulation. In S. cerevisiae, the expression of 10% of all genes is affected by the depletion of nap1 gene, which indicates transcriptional regulation. yNap1 was found to interact with B type cyclin (clb2), kinase Gin4 and NBP (Nap1 binding protein), which suggests that Nap1 participates in the control of mitotic events [200].

Chromatin remodelers

Chromatin remodeling is an enzyme--assisted and ATP--dependent histone or nucleosome mobilization, which influences local chromatin structure to facilitate or prevent protein accessibility, which is required to initiate DNA--templated reactions.

These enzymes are called chromatin remodelers and they play an important role in maintaining the promoters either in permissive state or in non--permissive state [START_REF] Cairns | The logic of chromatin architecture and remodelling at promoters[END_REF].

Accordingly, remodelers have been shown to modulate transcription, replication and DNA repair. There are four families of chromatin remodelers, SWI/SNF (switching defective/sucrose non--fermenting), ISWI (imitation Switch), CHD (Chromodomain Helicase DNA binding) and INO80 (Inistol requiring 80). All four share some properties

Chromatin fiber models

Based on the experimental evidence of the past 30 years, several models for the folding of nucleosomes into the chromatin fiber have been proposed. They can be divided into two main classes based on the number of helical starts, the one--start helix and the two-start helix. The solenoid model, the best example for one--start helix, was first proposed by Finch and Klug in 1976 [23] whereas the two--start model, resulting in a zigzag arrangement, was first proposed by Worcel in 1981 [207] and improved by Woodcock and colleagues in 1984 [START_REF] Nekrasov | Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics[END_REF]. Twisting and coiling of the nucleosome stacks can produce different forms of zigzag models.

The solenoid model

The solenoid model is a single start helix with a pitch of 11 nm and a mass of 6 nucleosomes per 11 nm resulting in roughly 6 nucleosomes/turn. The nucleosomes have their faces tilted around 20° from the fiber axis. Although the DNA linker length might change, the diameter of the helix stays 30 nm. The nucleosomes are arranged with the dyad pointing into the center of the fiber, which means both the linker DNA and linker histone H1 are also located at the center (Figure 16A) [24] The super coiled linker model is another one start helix where the linker follows a coiled path similar to the DNA wrapped around the octamer. In this case, the dyad position is dependent on the actual linker length (Figure 16 B) [START_REF] Nekrasov | Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics[END_REF]. The diameter as well as the mass per unit is dependent on the linker length, for example the minimum pitch of one nucleosome is 25 nm, which corresponds to 12.5 nm spacing between nucleosomes. Until now, different models of crossed linker models have been suggested based on the number of helical gyres n from the helical repeat (e.g n = 1, 2, 3 or 5) (Figure 17A) [209].

The twisted ribbon model is another zigzag--based model, which suggests that the fiber is formed by coiling of a ribbon--like structure into a hollow cylinder. In this model, the helical pitch increases and the mass per unit decreases with increasing linker length.

However the fiber diameter is independent of the linker length and stays constant. Since the center is hollow, the linker DNA and the histone H1 occupy the same perimeter as the nucleosome cores (Figure 17B) [START_REF] Nekrasov | Histone H2A.Z inheritance during the cell cycle and its impact on promoter organization and dynamics[END_REF].

the main reason for this multitude of models, and the debate on 30 nm structures was becoming more and more important.

In silico chromatin models

3D modeling of the chromatin was first introduced in the 80s where the models suggested were compatible with EM observation of isolated fiber and measurement of the linking DNA [207]. The first geometrical model suggested that the geometry of native chromatin fiber extracted from nuclei can be described using two angles. The first angle (α) is the angle between the entry and exit linker of a single nucleosome and the second angle (β) is the angle between consecutive nucleosomes. These two angles are affected by the variable DNA length, which leads to an irregular fiber (Figure 19A) [211]. This theoretical model was used for further investigation of the chromatin fiber in order to achieve a better understanding of the folding, however, in vivo experiments showed no evidence that the DNA linker is straight. More in silico models were suggested throughout the years using sophisticated 3D analysis such as Monte--Carlo simulation or the Discrete Surface Optimization (DiSCO). These models were suited to fit a fiber compaction into a 30 nm fiber but none allowed the fiber to reach compaction as the one seen for metaphasic chromosomes. A simple explanation to these results might be that not all the known parameters of chromatin fiber were taken into consideration. For example, the integration of the electrostatic charges of a nucleosome to DiSCO analysis proved to be more useful for chromatin dynamics than chromatin structure (Figure 19B). The most reasonable explanation is that the first DiSCO model did not consider the effect that H1 might have on linker DNA [212]. The elucidation of the histone tail contribution to the chromatin structure and their involvement in nucleosome-nucleosome interactions was a starting point to new features to account in chromatin models. Integrating these results in a new mesoscopic theoretical model, suggested that the chromatin folds into an irregular zigzag with a dominant interaction between nucleosome N and N+4 [213].

Electron microscopy and X--ray crystallography

It is important to note that at the beginning most of the studies were done on purified chromatin or DNA from nuclei but the production of the "601 sequence" by Jonathan Widom group allowed much more extended experiments for determining the 30 nm fiber structure [START_REF] Lowary | New DNA sequence rules for high affinity binding to histone octamer and sequence--directed nucleosome positioning[END_REF]. A SELEX experiment was carried out to isolate and characterize the highest nucleosome affinity members of a large set of DNA fragments. The affinity of 5x10 2 different chemically synthetic random DNA molecules of 220 bp was tested for the histone octamer. The highest affinity 147bp sequence selected out of the random DNA molecules showed by far stronger nucleosome positioning properties than natural sequences already known. Since then, the 601 sequences have been extensively used because of their strongest nucleosome positioning properties, enabling reconstitution of a single nucleosome over a long DNA stretch containing a single copy of this high--affinity

Atomic force microscopy

Atomic force microscopy is a technique that can directly image single molecules in solution. It provides a powerful tool for obtaining insights into the basics of biological materials. In both, objects are bound (immobilized) on a surface. In EM, analysis is made by electron beam (microscopy), while in AFM analysis is made by a vibrating nano--tip.

EM has in principle a higher resolution, while AFM is more flexible and easier to use.

The first AFM images coupled with geometrical modeling showed the chromatin fiber as having an irregular organization without adding salt or H1 but once salt was added, the chromatin tended to organize in zigzag form (Figure 21) [217]. The irregular organization of the chromatin beads--on--chain was challenged when EM studies coupled with computer modeling suggested that at low salt the chromatin is organized into a zigzag model with loose entry/exit angles. This angle decreased once an increasing concentration of salt was added. In summary, the 30 nm fiber zigzag organization is predisposed by the organization of the unfolded chromatin [104].

The accessibility of sites within folded and unfolded chromatin was studied by combining biochemical analysis to AFM. The data revealed that folded nucleosome arrays display a less important accessibility to the sites, but the array remains intrinsically dynamic allowing for sites to be slightly accessible [218].

Optical tweezers

For a further understanding of the chromatin structure in presence or absence of salt, the inter--nucleosome interactions were measured using optical tweezers followed by computer modeling. Optical tweezers is a single--molecule technique that can exert forces up to 100 pN on particles ranging from nanometers to micrometers. Focusing laser creates the optical trap; in this case the laser beam traps a polystyrene bead with one chromatin fiber stuck to it. Stretching was applied with a glass micropipette at different salt concentrations. The stretching forces should act on the entry/exit angle of the DNA, which is fixed by the interactions with the linker histone H1. The stretch and release experiment revealed that at physiological ionic strength the fibers present a dynamic structure that interconvert between open and closed states suggesting an irregular zigzag (Figure 22A) [219].

Single--molecule force spectroscopy (SMFS) is a unique new way to study the interactions involved in chromatin folding. This technique does not expose the chromatin fiber to staining, drying, extreme buffer conditions or surfaces. Using optical tweezers, this method provides a quantitative measure of the force needed for stretching the chromatin and predicting the inter--nucleosome interactions that modulate it structure. Surprisingly, it was reported that the linker histone H1 does not affect the stiffness of the fiber but only stabilizes it. Interestingly, these results are in accordance with a solenoid, one--start helix model (Figure 22B) [220].

Whatever the real structure of the chromatin within the cell nuclei id, the in vitro structure of the 30 nm fiber remains elusive and controversial, mainly due to the lack of direct data because of insufficient resolution requiring extensive mathematical treatment, modeling and fitting.

The nucleosome repeat length effect on the chromatin structure

Chromatin fibers of various species and tissues are characterized by different Nucleosome Repeat Length (NRL) of linker DNA. For example, single cellular organisms have short NRLs (160--189 bp), whereas mature cells usually have longer NRLs (190--220bp). Recent studies showed a strong positively--correlated linear relationship between the number of H1/nucleosome and the NRL, with increasing the NRLs showing a higher H1/nucleosome ratio [221]. Furthermore, EM imaging combined with sedimentation coefficient measurement suggests that the folding of the chromatin is both NRL--and linker histone--dependent. Indeed, it has been shown that only 197 bp NRLs can fold into 30 nm fiber whereas 167 bp have a limited compaction resulting in a thinner fiber (diameter of 21 nm only) with a two--start helix (zigzag) organization [222]. In addition, it was suggested that the medium--sized NRLs (177--207 bp) have a constant diameter of 33 nm and that longer linkers (217--237 bp) form a 44 nm--diameter fiber, while both fold into interdigitated one--start helices (solenoid) [215].

These same EM data were analyzed independently by another group while taking in consideration the linker histone H1. The analysis showed that NRLs ranging from 155 to 211 bp reveal a compacted fiber with possible one--, two--and three--start structures [223].

Computational modeling studies using an already established mesoscale model suggest that short to medium NRLs (173 to 209 bp) with linker histone condense into irregular zigzag structures. However, for longer NRLs, the chromatin folds into solenoid structures [224]. Models taking into consideration the basics physics of chromatin such as electrostatics, DNA and nucleosome mechanics, histone tail flexibility, hydrogen bonding fluctuations, showed that the linker histone H1 has very little effect on the short NRLs but an important one on medium NRLs [224]. Moreover, Monte Carlo simulations of oligonucleosome model showed that non--uniform NRL fiber containing one short linker forms a bent ladder rather than a compact 30 nm fiber. But for chromatin fibers with medium and long NRLs, the preferred organization would be a regular zigzag conformation that has been described in uniform fibers. However, long NRLs are described to compact into irregular fiber, with linker DNA having a bent or interdigitated conformation. These findings led to the conclusion that the polymorphism of the chromatin structure is triggered by the NRL variations.

The nucleosome acidic patch effect on higher--order structure

The nucleosome acidic patch is negatively charged region formed by a cluster of eight acidic residues: Glu56, Glu61, Glu64, Glu90, Glu91, Glu92 of H2A and Glu102, Glu110 of H2B. The crystal structure of the nucleosome first presented this cluster as a surface with uneven charge distribution [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]. This cluster forms a curved and asymmetrically charged surface called the acidic patch. As shown in Figure 18, the acidic patch has the form of a narrow pocket where the bottom is formed by the α2--helix and the C--terminal extension of H2A and the edges are formed by the α1--and αC--helices of H2B (Figure 23).

Moreover, the presence of the H2A residues Y50, V54 and Y57 at the bottom of the pocket makes this pocket hydrophobic due to the non--polar character of these residues.

The mechanistic details of the H4 tail--acidic patch interaction remain unresolved but proven to be necessary to the chromatin folding. However, molecular dynamics simulation of the H4 tail acidic patch showed that residues V21 of H4 and E64 of H2A are close in space when chromatin is folded. This model also suggested that residues R3, K16, R19 and K20 of the histone H4 face the acidic patch groove and form non--covalent interactions with the acidic patch residues [225]. Even though the acidic patch plays a role in chromatin folding, its absence was not shown to inhibit the compaction of the 30 nm fiber. The acidic patch is also found to bind to non--histone proteins, which creates a competition between these proteins and the H4 tail domain, thereby triggering remodeling of the higher--order structure of the chromatin [226].

Role of histone variants in chromatin structure

The biological roles of histone variants have been largely discussed and many questions still need to be answered. The structural organization of chromatin containing histone variants is one of these remaining questions. X--ray structures, cryo--EM and AFM images of nucleosomes containing histone variants such as H2A.Z, H2A.Bbd or CENP--A helped to shed some light on this issue. However, much less is known about the 30 nm fiber containing these histone variants.

Role of H2A histone variant H2A.Z

Intra--nucleosomal interactions

The H2A.Z nucleosome crystal structure [164] showed an extended acidic patch compared to the H2A nucleosome, and Asn and Lys residues were replaced by Asp and Ser respectively. The main question remains about the involvement of this extended acidic patch in the folding of the chromatin.

In order to provide an answer a mutated H2A containing the additional acidic patch residues was expressed and purified. Sedimentation studies compared H2A.Z and the mutated H2A to H2A arrays and the results showed that the H2A.Z and mutated H2A arrays showed similar folding patterns and were considered more compact compared to H2A arrays under the same divalent cation concentration, confirming that due to their extended acidic patch, H2A.Z arrays promote the formation of the 30 nm. [227,228]

Inter--nucleosomal interactions

Increasing the concentrationsof divalent cations above the intramolecular folding range induces a cooperative and reversible oligomerization. During this oligomerization, the arrays self--associate into large nucleoprotein complexes resembling a chromosomal fiber during interphase. In order to study the H2A.Z implication in this self--association, sedimentation experiments were carried out on H2A.Z--containing nucleosome arrays.

The results showed that with 2 mM MgCl 2 the H2A.Z fiber presented a much slower sedimentation than the H2A array [228]. Moreover, microfuge--based assay provides further evidence that H2A.Z inhibits oligomerization and requires a much greater amount of divalent cation in order to achieve it [227]. Briefly, H2A.Z array seem to promote the 30 nm fiber formation but at the same time inhibits the oligomerization of this array.

Role of H2A histone variants: H2A.Bbd

H2A.Bbd has different characteristics from H2A, for example the absence of a C--terminal tail, no lysine residues in its N--terminal tail and the lack of residues that contribute to the acidic patch in the nucleosome core particle in mammals [178]. The H2A.Bbd nucleosome was reported to have a more relaxed conformation than the H2A nucleosome [178]. Furthermore, the H2A.Bbd nucleosome displays lower stability in vitro and the dimer H2A.Bbd--H2B is rapidly exchanged in vivo [229] [230]. All these indications of an unfolded chromatin, together with the H2A.Bbd enrichment in euchromatin regions of the genome strongly suggests that it might play a role in up-regulation of chromatin transcription [177,179].

Role of H3 histone variant: CENP--A

For the past 20 years, the structural and mechanical features of the CENP--A nucleosomes have been widely studied. Even though a recent crystal structure of the CENP--A--containing nucleosome helped uncover some of its important features, the controversy about CENP--A chromatin structure and dynamics is still lingering.

The variant histone CENP--A was first described to be present in nucleosome--like structures following micrococcal nuclease digestions and that it co--purified with nucleosome particles [187]. CENP--A has been a marker for centromere location because of its dynamic nucleosomes, its histone stoichiometry and the nature of the wrapping of the DNA, from left to right (while in canonical nucleosome it is from right to left). Even though the central role of CENP--A arrays has been identified, the inter--nucleosomal interactions occurring are still completely unexplored.

The histone stoichiometry within the CENP--A--containing nucleosome remains the subject of many speculations. AFM studies on an ex vivo CENP--A nucleosome arrays showed a size reduced by half relative to canonical H3 nucleosomes. These results led to believe that the CENP--A nucleosomes are tetrameric (containing one copy of each histone). Moreover, EM, like AFM, revealed a small particle separated by long linkers that resisted ionic condensation [231]. However, these results were controversial, because mammalian centromeric nucleosomes are described as octameric, whereas budding yeast nucleosome can go through two forms, i.e. octameric or hexameric complexes. These different models are due to the various eukaryotic species used to isolate the CENP--A nucleosomes.

The crystal structure of the CENP--A nucleosome gave a larger idea of the intra-nucleosome interaction, describing the CENP--A nucleosome to have loose terminal DNA contacts, resulting in DNA wrapping [190]. The loose terminal DNA contact was also confirmed by micrococcal nuclease assay, where CENP--A nucleosome was shown to cover 20 bp less than the canonical nucleosome.

To further illustrate the CENP--A nucleosome controversy, it was recently published by Miell et al. [232] that the CENP--A nucleosome does indeed have a reduction of height in octameric nucleosomes. CENP--A and H3 were purified from two distant organisms, human and S. pombe, reconstituted into an octameric nucleosome and crosslinked before conducting AFM experiments [232]. These results were indeed surprising, because they contradicted other AFM studies done on octameric CENP--A nucleosomes 3.6 Beyond the 30 nm fiber

Does the 30 nm exist in vivo?

The controversy regarding the structure of 30 nm fibers has been substantiated by few reports because of inability to observe the 30 nm fiber in in vivo cryo--EM vitreous sections visualization (CEMOVIS). This was first done 20 years ago on mammalian mitotic cells and only an aggregation of 11 nm nucleosome fiber was observed [234].

However, it was argued that the cryo--EM images had been corrupted by the CTF (contrast transfer function), but once CTF were corrected, the spectrum still showed no evidence of 30 nm peaks. Furthermore, 3D structure of Xenopus chromosomes assembled in vitro using electron tomography detected no fiber--like structures [235]. It is important to note that the original concept of the 30 nm fiber started with conventional transmission or scanning EM showing fibers of 30 nm diameters in isolated chromatin, nuclei and mitotic chromosomes [236]. However, if the 30 nm fiber can be visualized by EM only, this might be explained by environment changes triggered by sample preparation. In addition, it has been noted that nuclei of specific cell types such as starfish spermatozoids of chicken erythrocytes are almost full of 30 nm fiber.

These observations have led to the hypothesis that in vivo the chromatin does not fold into a 30 nm fiber. Therefore, much more experiments are needed to exclude artifacts and to eventually confirm these hypotheses.

Tertiary structure

As for proteins the concepts of primary, secondary, tertiary and quaternary structures can be applied to chromatin structure hierarchies (Table 4).

Even though no definitive structure has been appointed to the secondary structure of the chromatin, there is no doubt about the higher--order organization, which is obvious in the metaphase chromosome. In order to study the tertiary level of chromatin structure, experiments were performed using optical microscopy, which is limited to a ~300 nm resolution. The images revealed that the tertiary structure in natural chromosome is beaded. The number of adjacent beads can go from one to six beads with diameters going from 0.4 to 0.8 μm. Similar results were obtained with tandem DNA. Moreover, these beaded structures can be adapted to chromonema models and radial--loop/scaffold models (Figure 25). The chromonema models propose a fiber that yields different fiber thicknesses. It starts with a thinner fiber that yields into a thicker fiber that appears as beads under the light of the microscope. This structure is stabilized by fiber--fiber interactions. The thickness of beads depends on the thickness of the underlying fiber. For example, if the underlying fiber measures around 30 nm, the beads would appear to be 500 nm in diameter. In contrast, if the underlying fiber is thicker (around 100 nm fiber), the beads would only be twice as big, around 200 nm. On the other side, it has been described that chromatin fibers form loops attached to a protein scaffold. Under the light of the microscope, the cluster of loops would form a bead with a diameter around 0.3 μm (which is close to the average diameter of the beads) [237].

The metaphase chromosome

The metaphase chromosome is part of the chromatin higher--order structure, where DNA is compacted 10,000 to 20,000 fold. To this day, there is no accepted model because of the several features that must be accounted for. One of these features for example is the diameter of the chromosome arm, which is different from one species to another. This chromosome has been described as DNA loops surrounding a core structure called scaffold, which is at the base of the chromosome architecture. Studies of some of the component isolated from the scaffolds revealed a family of structural maintenance of chromosomes (SMC) proteins. Also, two proteins complexes called

Cohesin and Condensing play a role in sister chromatin adhesion and chromosome condensation [238]. Experiments showing the extension of the chromosome revealed that chromosomes exhibit a remarkable degree of elasticity and repeatedly return to their original state after stretching [239]. In contrast, these results do not apply to hierarchical models of chromosome structure, which predicts that stretching the chromosome would lead to unfolding and reduction of the chromosome diameter. The metaphase chromosomes appear to be more complicated than they look like, hence understanding their composition, formation, maintenance and architecture remains to be achieved.

II. Objectives

The compaction of genomic DNA into chromatin is involved in the regulation of key biological processes such as transcription, replication and DNA repair. Nucleosomes, which form the repeating units of chromatin, vary in their histone composition.

Linear arrays of nucleosomes are folded and compacted into 3D assemblies of higher-order structures. Secondary chromatin structures are defined as the structures emerging from the folding of an individual array (strings--on--beads) to produce a particular fiber, the 30 nm fiber. Given the fundamental role of the 30 nm fiber structures in regulating all DNA--and chromosome--dependent processes, its precise molecular organization and 3D arrangement have been extensively debated. However, progress has been slow and result interpretation controversial due to the limitation of current microscopy approaches and to the complexity of the question. Initially, structural studies on fibers assembled on natural DNA sequences were hindered by variation of the length of the linker DNA. However, significant advances have come from the construction of regularly spaced tandem DNA repeats for precise nucleosome positioning [START_REF] Lowary | New DNA sequence rules for high affinity binding to histone octamer and sequence--directed nucleosome positioning[END_REF]. Under physiological salt conditions, and or/presence of H1 an array of nucleosomes folds into a secondary chromatin structure with the shape of a 30 nm fiber.

Despite decades of effort, the structure of the 30 nm fiber has not been resolved yet due to its highly compacted nature, which prevents the path of the DNA from being visualized by electron microscopy. Two models have been proposed on the basis of in vitro data, the solenoid and zigzag arrangements, which were both supported by different studies. Crosslinking studies and the crystallization of a tetranucleosome array have provided strong evidence that the 30 nm fiber adopts a zigzag form. However, the short repeat length and the absence of linker histone H1 made these results questionable [25,214]. On the contrary, cryo--EM on long regular arrays with constant repeat length and in the presence of linker histone H1 concluded to a multiple--start interdigitated solenoid model [248]. Finally, a recent study using cryo--EM suggested that there is not one uniform type of organization but rather heterogeneity of nucleosome interactions. In another study, the fiber showed a predominantly two--start organization that was interspersed with bent DNA typical of 'one--start' solenoids [26].

Of note, the nucleosome repeat lengths have been reported to affect the chromatin folding as well. Indeed, some results suggest that short to medium NRLs (173--209 bp) favored a zigzag structure, whereas longer NRLs (218 bp and above) favored solenoid structures [224].

Chromatin, at all levels of organization, is not static but very dynamic. This dynamicity and plasticity is crucial to ensure proper functioning of the cell. Modification of chromatin structure is prime step in regulation of all the DNA--templated processes like transcription, replication, repair and recombination. These processes require quick changes in the chromatin organization and structure. The dynamic control of genome accessibility is governed by contributions from DNA sequence, ATP--dependent chromatin remodeling, post--translational modification of histones and histone variants incorporation [249]. However, controversial results were reported on the folding of the chromatin and its organization upon replacement of conventional histones and histones variants.

The first aim of this work was to develop a novel robust in vitro biochemical approach, we termed ICNN (Identification of Closest Neighbor Nucleosomes), which allows the direct and unambiguous determination of the 3D nucleosome arrangement within the 30 nm chromatin fiber. The method is based on direct identification of the closest interacting neighbors of each nucleosome, by using disulfide crosslinking between nucleosomes within a compacted chromatin, and affinity pull--dawn and qPCR.

To clarify the chromatin folding structure, the ICNN approach is applied in 3 complementary work axes:

1) The characterization of the 30 nm fiber organization (zigzag or solenoid) in presence of H1 on 12--mer nucleosome arrays with different NRLs to investigate the contribution of H1 and of the NRL length on the resulting chromatin structure.

2) The identification of the role played by the histone variant H2A.Z (H2A variant) on the chromatin folding structure.

3) The analysis of the CENP--A (H3 variant)--containing chromatin folding behavior in presence of linker histone H1, and more precisely the specific role of the αN domain of CENP--A in the process of folding.

Introduction

Chromatin exhibits repeating structure. The nucleosomes, the fundamental units of chromatin, are connected with linker DNA with different length (varying between ∼10 and ∼80 bp) and form the 10 nm chromatin filament [1]. Upon binding of the linker histone H1 the chromatin filament is folded into the 30 nm chromatin fiber [2--8].

Although, the structures of the conventional [9] and histone variant nucleosomal core particles [10,[START_REF] Tachiwana | Structures of human nucleosomes containing major histone H3 variants[END_REF] were solved at very high resolution, the 3D organization of the chromatin fiber is still poorly understood. The available data suggests that the structure of the fiber, which contains ~6 nucleosomes/11 nm [2,[START_REF] Gerchman | Chromatin higher--order structure studied by neutron scattering and scanning transmission electron microscopy[END_REF][START_REF] Ghirlando | Hydrodynamic studies on defined heterochromatin fragments support a 30--nm fiber having six nucleosomes per turn[END_REF]] could be described either as one start helix with consecutive arrangement of nucleosomes and bent linker (the solenoid model, [2] and Figure 1a) or as two start structure with non--consecutive arrangement of nucleosomes and straight linker ([6, 14--18] and Figure1b). The two-start structure was described, in turn, either as a helical ribbon arrangement of nucleosomes or as two intertwined helical stacks of nucleosomes. Recently a polymorphic structure of the chromatin fiber based on biochemistry experiments and EM imaging was proposed [START_REF] Grigoryev | Evidence for heteromorphic chromatin fibers from analysis of nucleosome interactions[END_REF]. In addition, the folding of the fiber might depend on the nucleosome repeat length [START_REF] Correll | Short nucleosome repeats impose rotational modulations on chromatin fibre folding[END_REF].

Each model for a regular 3D arrangement of the nucleosomes within the chromatin fiber supposes well--defined neighboring nucleosomes. For example, in the case of the solenoid model the neighbors of the N nucleosome are N±1 nucleosomes (Figure 1a). In contrast, in the two--start structure model the N nucleosome has for neighbors the N±2 nucleosomes (see Figure 1b). Therefore, unambiguous determination of the spatial arrangement of the nucleosomes in the compact chromatin fiber will help to discriminate between the different models and will allow shedding light on the chromatin condensation process.

Histone variants are key epigenetic players, which are usually coded by two distinct genes. Each cell is expressing histone variants. The current view is that the incorporation of histone variants confers novel structural and functional properties of the nucleosome [START_REF] Boulard | Histone variant nucleosomes: structure, function and implication in disease[END_REF]. H2A.Z is a universal histone variant present from yeast to men, which is implicated in several vital cell processes, including transcription, repair and mitosis [22--26]. Several studies were focused on both the structure of the H2A.Z nucleosome and the H2A.Z chromatin fiber [27--32]. The H2A.Z chromatin organization was, however, investigated with mainly low resolution physical--chemistry methods and, in addition, the available data show some controversy [29--33].

Description of the novel Identification of Closest Neighbor Nucleosomes (ICNN) approach

In this work we are describing a novel robust approach, termed ICNN (Identification of Closest Neighbor Nucleosomes), which allows the determination of the 3D nucleosome arrangement within the compact fiber with very high precision (Figure 1c-h). The method is based on the ability to crosslink nucleosomes containing both single amino-acid cysteine substituted both histone H4 (H4--V21C) and H2A (H2A--E64C) within compact chromatin [34]. ICNN uses strongly positioned 601 DNA nucleosomal arrays reconstituted with the histone octamer consisting of H4--V21C and H2A--E64C mutated histones and both wild type H3 and H2B. Linker histone H1 is deposited in In vivo--like manner by using its chaperone Nap1. Each individual 601 repeat within the array contains 2 sets of 2 distinct mutated nucleotides (each individual set of the 2 mutated nucleotides is in proximity to the respective nucleosomal end, Figure 1e). The use of specific primers, which 3'--end is "finishing" at these sets of mutated nucleotides, allows the specific amplification of the repeat of interest, and thus, the "visualization" of the nucleosome of interest. The repeats are separated by a blunt ScaI restriction site. In addition, each individual repeat contains an inserted cleavage site for a distinct restriction enzyme (Figure 1e). The presence of these last distinct restriction cleavage sites allows the specific replacement of a selected repeat with the same repeat, but containing biotin, a procedure necessary for the usage of ICNN (see below).

The ICNN approach consists of the following steps (see schematics in

Analysis of the 3D nucleosome arrangement within compact chromatin with varying repeat lengths

We have initially applied the ICNN approach for analyzing the neighbors of nucleosome #5 within compact chromatin, reconstituted by using 12 tandem 197 bp 601 repeats (see schematics of Figure 1). We have first characterized the efficiency of reconstitution (Figures 2a,b). In the absence of H1, the reconstituted arrays migrate as an up--shifted band compared to the band of free DNA (Figure 2a, lanes 2 and 3). Addition of Nap1

does not affect the migration of the reconstituted chromatin (Figure 2a, lane 3), suggesting, as expected, no Nap1 association with chromatin. The addition of the Nap1--H1 complex results, however, in increase of the migration rate of the arrays (Figure 2, We next condensed the arrays by using H1--Nap1 complewe in 50 mM monovalent ions buffer and carried out the bisulfide crosslinking. The efficiency of crosslinking was tested by SDS PAGE. Two additional bands, corresponding to H4--H4 and H2A--H4 adducts, were observed in the H1 bound arrays (Figure 3a, lane 2), a result in agreement with [34]. The composition of these bands was further confirmed by mass-spectrometry analysis (Figure S2). No such bands were, however, observed in the arrays, incubated with Nap1 only (Figure 3a, lane 1). We attribute this absence of nucleosome crosslinking in the arrays without H1 to reflect the very weak compaction of the arrays under these salt conditions [34]. The efficiency of nucleosome crosslinking within the H1--bound arrays was further revealed upon digestion with the restriction enzyme AvaI--ScaI (Figure 3b). Extensive digestion of the H1--folded crosslinked arrays results in the generation of several species, showing slower migration rates compared to this of the mono--nucleosomes. Note that under our conditions of crosslinking up to 3--4 additional bands with lower electrophoretic mobility were detected (Figure 3b, left panel, lane3). Treatment with 100 mM DTT leads to complete disappearance of these slower migrating bands and increasing the intensity of the mono--nucleosome band (Figure 3b, left panel, lane 4). We attributed these bands, in agreement with the available data [34], to reflect the presence of crosslinked monomer species (containing up to 3--4 monomers), which integrity is maintained thanks to the disulfide histone-histone crosslinking. Accordingly, DTT treatment leads to de--crosslinking and the presence of only monomeric particles in the treated sample. This is further supported by the electrophoretic analysis of the DNA isolated from the non--treated and DTT--treated AvaI--ScaI crosslinked arrays (Figure 3b, right panel). Indeed, in both cases only monomeric repeat 601 DNA was observed. This demonstrates that the digestion is achieved to completion and that the detected slower migrating bands in the nucleoprotein gel originated from the crosslinked monomer species.

We have next carried out a streptavidin chromatin pull--down. After isolation of DNA from the pull--down samples, we have used qPCR to identify the neighboring nucleosomes, which are crosslinked with nucleosome #5 within the compact array. By using specific set of primers, we have amplified (in 12 separate reactions) and thus measured the amount of DNA corresponding to each nucleosome (repeat) in the sample and normalized it relative to the amount of amplified DNA, corresponding to nucleosome #5. As seen (Figure 3c), the signals for nucleosome # 1, 3, 7, 9, and 11 compared to this of nucleosome #5, are 8%, 22% 42%, 24% and 13 %, respectively. No signals were detected for the any one of the "even" nucleosomes in the array. In addition, in the DTT treated samples, only a signal for nucleosome #5 was observed,

showing that the qPCR amplified DNA reflects the crosslinking of neighboring nucleosomes via the disulfide histone H4--H4 and H2A--H4 bridges (Figure 3c).

Accordingly, in the absence of histone H1, again only a signal for nucleosome #5 is detected (Figure 3c). All this indicates that within the 197 bp compact fiber, nucleosome #5 is in contact with both nucleosomes #3 and #7. As for the signal for nucleosome 1, 9 and 11, we attributed it to the pull--down of crosslinked "trimeric" and "tetrameric" particles, containing different combinations of crosslinked "odd" nucleosomes. Thus, the 197 bp chromatin fiber should exhibit a "N±2 " 3D nucleosome arrangement (see Figure 1b). To further confirm this, we have carried out the same type of experiments but with biotin--labeled either nucleosome #4, or #8 or #9 (Figure 4b). The obtained results are in complete agreement with this for nucleosome #5. We conclude that within the 197 bp compact chromatin the nucleosomes exhibit a N±2 type of non--consecutive arrangement, fully compatible with the recent cryo--EM data [START_REF] Song | Cryo--EM study of the chromatin fiber reveals a double helix twisted by tetranucleosomal units[END_REF]. To test if the same type of 3D organization is also common for fibers with different repeat length, we have performed the same experiments, but by using compact nucleosome arrays with 177 and 227 bp repeat lengths. These arrays show an electrophoretic behavior similar to this of the 197 bp ones (Figure S3, S4). In contrast to the arrays without histone H1, the histone H1--bound both 177 bp and 227bp arrays exhibits efficient nucleosome--nucleosome crosslinking (Figures S3,S4), a result in agreement with the data for the 197 bp arrays. The qPCR data, summarized in (Figure 4a,c), clearly show a N±2 spatial nucleosome arrangement for both samples. Therefore, this type of arrangement is general and does not depend on the length of the linker DNA.

Of note, the efficiency of crosslinking of a given nucleosome to its neighbor, located towards the center of the fiber, is ∼1.5--2--fold higher compared to this of nucleosome located to the end of the fiber (Figures 3d, 4a-c). For example, the relative crosslinking for nucleosome #8 to nucleosome #6 is ∼40%, while this for nucleosome #10 is ∼ 20%.

We attribute this to reflect the higher dynamics ("breathing") of the end located nucleosomes. Furthermore, the qPCR data without H1 (summarized in figure S7) shows clearly no chromatin compaction, in accordance with electrophoresis results.

Our results are in agreement with the structure in figure 4d, as well as a more refined version presented in figure 4e. However, our results do not confirm the irregular space between nucleosomes. This model resulted from fitting of cryo--EM images with the crystal structure of tetramer without H1 [37]. This irregular spacing might be an artifact of the fitting process. We suggest a two start model similar to figure 4e but with regular alignment of the tetranucleosome.

It is important to note that the ICNN approach showed reproducible results. Two distinct experiments were performed on both arrays 12x197 #9 and 12x177 #4 presented same type of nucleosomal arrangements (respectively Figure S5a, b andS5c, d). The H2A.Z chromatin fiber exhibits N±2 nucleosome spatial organization H2A.Z is an essential histone variant and its genome wide localization and structure of the H2A.Z containing chromatin is believed, as noted above, to be of crucial importance for the regulation of transcription as well as other important events in the cell (recently reviewed in [38]). With this in mind, we next studied, by using ICNN, the 3D arrangement of compact 197 bp H2A.Z nucleosome arrays with nucleosome #8 being biotin labeled. The reconstituted under our conditions chromatin was of excellent quality (Figure S6). The crosslinking at 50 mM NaCl was as efficient as for conventional H2A 197 bp nucleosomal arrays (Figure 3 and Figure S6). As expected, either the absence of H1 or the treatment with DTT abolished completely the crosslinking in the H2A.Z compact chromatin (Figure S6). The data for the 3D arrangement of nucleosome #8 in H2A.Z arrays are identical to these for the nucleosome arrangement in conventional H2A compact arrays, i.e. H2A.Z chromatin exhibits N±2 3D nucleosome arrangement. We conclude that the presence of H2A.Z does not affect the 3D arrangement of nucleosomes in compact chromatin. This suggests that the implication of H2A.Z in numerous vital for the cell phenomena, should not be achieved via the H2A.Z fiber structure, but instead by its mere presence at specific genomic locations. In this context, the role of H2A.Z chaperones in selective deposition/removal of the H2A.Z [39] would be of crucial importance, since this allows modulating at a "wish" the accessibility of the DNA binding factors cognate sequences buried in the H2A.Z nucleosomes.

In summary, we have described the application of the ICNN approach for identification of the In vitro 3D nucleosome organization in the compact both conventional and H2A.Z nucleosomal arrays. However, ICNN could have a broader application in chromatin studies, including analysis of the alterations in chromatin fiber conformations and their spreading along the fiber upon H1 loss, binding of transcription factors or chromatin remodeling machines. Of particular interest would be to study the conformational changes along the fiber upon active transcription and replication.

The ICNN approach uses tandem arrays, which contain a biotinylated repeat inserted at specific site (see Materials and Methods). The insertion of the biotinylated repeat is performed by its ligation with the remaining two DNA fragments of the tandem repeat.

Then arrays are reconstituted by salt dialysis and Nap1 is used to deposit H1.

Importantly, a further development of the approach could be envisaged, which could use already reconstituted chromatin templates and the ligation could be carried out at the level of these templates. This would allow the insertion of one or several nucleosomes at a selected site of the arrays. This nucleosome could contain epigenetic modifications such as acetylation or methylation and thus, the local effect on the structure of the fiber due to these histone modifications could be analyzed by the ICNN approach. Some recent, mainly cryo--Electron Microscopy data, defied the longtime accepted existence of genome--wide ordered 30 nm chromatin fiber In vivo (for recent review, see [40]. The potential of application of ICNN for In vivo studies would shed light on this question. CENP-A centromeric chromatin exibits non-canonical 30 nm fiber structure Soueidan,L; Dimitrov,S; Angelov,D.

Background

All the conventional histones, except H4, have histone variants. Histone variants are non--allelic forms of the conventional histones (1). The incorporation of histone variants confers novel structural and functional properties to the nucleosomes (3). A spate of recent studies have implicated the incorporation of histone variants into the nucleosome in the regulation of transcription, repair, senescence, cell division, meiosis, epigenomics events, etc (4).

Histone variant CENP-A

CENP--A replaces histone H3 at the pericentromeric chromatin. It is a key player of centromere organization and contributes to centromere identity. It has a crucial role in mitosis, as it is required for the proper recruitment of kinetochore components which drive the alignment and segregation of chromosomes (5). CENP--A comprises a well-conserved histone--fold domain and a highly divergent amino--terminal tail. Very recently the crystal structure of the CENP--A nucleosome was solved (2). Intriguingly, in contrast to the canonical nucleosome (where 147 bp of DNA are wrapped around the histone octamer), only the central 121 bp were visible (Figure 1). The thirteen base pairs from both ends of the DNA are invisible in the crystal structure, and the αN helix of CENP--A is shorter than that of H3, which is known to be important for the orientation of the DNA ends in the canonical H3 nucleosome (6).

Onto centromeric CENP--A chromatin is assembled the so--called constitutive centromere associated network (CCAN) of 16 proteins (termed generally as CENPs) distributed in several functional groups as follows: CENP--C, CENP--H/CENP--I/CENP--K/, CENP--L/CENP--M/CENP--N, CENP--O/CENP--P/CENP--Q/CENP--R/CENP--U( 50), CENP--T/CENP--W, and CENP--S/CENP--X (7). Importantly, CENP--C and CENP--T function to direct kinetochore formation (8).

3D nucleosome arrangement in the CENP-A chromatin

Very recently, in our laboratory it was shown that H1 is indeed unable to properly bind to the CENP--A nucleosome (Imtiaz Lone, personal communication). This suggests that the CENP--A chromatin would not be able to condense in a structure of the "N±2" type, characteristic for the conventional 30 nm fiber. To test this we have used the ICNN approach described in the attached paper. Briefly, we have reconstituted 12x197 bp nucleosome arrays by using nucleosome #8 (N8) with inserted biotin and histone octamer, consisting of H4--V21C and H2A--E64C mutated histones and both wild type H3 and H2B (see Materials and Methods section of the attached manuscript for details).

Linker histone H1 was deposited in In vivo--like manner by using its chaperone NAP1.

Digestion with either AvaI/ScaI (AvaI/scaI unique cleavage site for 601 DNA is in the linker DNA) or HhaI (HhaI has an unique cleavage site in the nucleosomal DNA) shows the full nucleosome occupancy of the arrays. Of note, upon addition of the NAP1--H1 complex, the band corresponding to the 12x197 bp CENP--A nucleosome arrays, is up-shifted compared to these of both conventional H3 nucleosomal arrays with and without H1 (Figure 2a). Note that the conventional H1--bound arrays exhibits higher mobility relative to the mobility of arrays without H1. This suggests that histone H1 is not properly associated with the CENP--A arrays. If this, as indicated also by other data in the lab is true, we may expect a weaker and distinct condensation of the arrays and no crosslinking of the CENP--A nucleosomes. The crosslinking was tested first by SDS electrophoresis (Figure 2c). As seen, in contrast to conventional arrays, no H4--H4 or H2A--H4 crosslinking was observed in the H1 bound samples. This was further supported by the ScaI--AvaI digestion of both conventional and CENP--A nucleosomal arrays (Figure 2b). The appearance of oligomers with higher molecular masses, reflecting the crosslinked monomeric units, was detected in the ScaI digested conventional samples. Of note, treatment with DTT led to disappearance of the oligomers. No such bands were, however, detected in both non--DTT treated and treated digested with AvaI/ScaI CENP--A arrays (incubated with either NAP1--H1 or with H1 only). This reveals that the 3D organization of the nucleosomes in the CENP--A arrays should be distinct from the N±2 organization of the conventional chromatin fiber, which is further demonstrated by the qPCR results presented on Figure 3. "Rigidifying" the DNA ends of the CENP-A-A nucleosome allows the assembly of fiber with 3D "N±2"-type arrangement of nucleosomes.

The flexibility of the DNA ends of CENP--A nucleosome reflects the peculiar structure of CENP--A amino--terminal region within the nucleosome (2). Indeed, the αN helix of CENP--A is one helical turn shorter than this of conventional H3 and the preceding region, in contrast to this of H3, is completely disordered ((2), see also Figure 1). However, both the length of αN helix and the loop segment preceding the αN helix (this loop interacts directly with the DNA ends in H3 nucleosome ( 6)) are required for maintaining the DNA orientation at the entrance and exit of H3 nucleosomes. The "defects" in the organization of the amino--terminal region of CENP--A appear, thus, to be responsible for inherent flexibility of the DNA ends of the CENP--A nucleosome (2) and the inability of histone H1

to bind to it. This would, in turn, determine the inability of the CENP--A nucleosome arrays to adopt 3D "N±2" --type nucleosome arrangement (see above). tandem repeats of 601 DNA sequence in presence of linker histone H1 showed a zigzag structure with straight linker DNA. Moreover, the structure described the chromatin as a left--handed twist of repeating tetranucleosome structural units [216] (Figure 28, right panel).

The crystal structure of the nucleosome core revealed that the surface of the nucleosome has an uneven distribution of charges [START_REF] Davey | Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a resolution[END_REF][START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF]. The most striking feature is a cluster of 7 acidic AA contributed from histone H2A, later referred to as acidic patch. It was reported that the N--terminal tail of histone H4 (residues 16--25) originating from adjacent nucleosome interacts with histone H2A through the acidic patch leading to chromatin compaction [288]. Crosslinking studies demonstrated that disulfide bridges can be generated between the N--terminal tail of histone H4 and the H2A acidic patch in condensed chromatin only [25]. In this work, we developed a new approach called the ICNN (identification of the closest neighbor nucleosome) allowing unambiguous determination the 3D arrangement of nucleosomes within a linker histone H1--dependent compacted fiber. The ability of disulfide cross--linking between neighboring nucleosomes due to interactions between the acidic patch of H2A and the H4 tail is the main feature of this approach. 601 DNA nucleosomal arrays were reconstituted with histone octamers containing mutated H2A (H2A--E64C) and H4 (H4--V21C). Linker histone H1 was deposited using histone chaperone NAP--1 in order to induce compaction.

The ICNN approach was first used to investigate the 3D structure a 12x197 bp nucleosome array containing biotin at the nucleosome N5. After chromatin compaction in presence of linker histone H1 and crosslinking of adjacent nucleosome, the fiber was cleaved to monomers and the biotinylated nucleosome N5 was pulled down by streptavidin Chromatin Affinity Precipitation and the DNA analysed by qPCR. The results showed signal for nucleosomes N # 1, 3, 7, 9 and 11 with respectively 8%, 22%, 42%, 24% and 13% of crosslinking. However, once treated with DTT these signal disappeared proving that the disulfide bonds between neighboring nucleosomes are responsible for the signal detected. Nucleosome N5 interacts with its N±2 neighbors, which corresponds to a zigzag structure.

It is clear that the linker histone H1 binds to the nucleosome core particle and promotes compaction of chromatin array into 30 nm fiber. The linker histone has 2 structural domains, the globular domain (GH) and the C--terminal domain. The binding mechanism of the linker histone H1 has been debated for years, as has its effect on the 30 nm structure. To investigate the direct effect of the linker histone H1 on the structure of the 30 nm fiber, we conducted a similar set of experiments where we substituted the H1-dependent compaction by Mg 2+ --dependent compaction. Indeed, the analysis of the 12x197 bp nucleosome array containing N5--biotin nucleosomes in presence or absence of 1mM divalent cation Mg 2+ by the ICNN approach showed a similar behavior of the compacted chromatin (Figure 29). Of note, in presence of Mg 2+ the nucleosome N5 interacts with nucleosomes # 1, 3, 7, 9 and 11 with respectively 6%, 15%, 35%, 20% and 10% of crosslinking. As expected, without Mg 2+ the nucleosome arrays show no cross-linking. Furthermore, the effect of the position of the pulled down nucleosome on the 30 nm structure was evaluated by applying the ICNN approach. Three additional 12x197 bp arrays were produced with biotin--labeled either nucleosomes N4, N8 and N9. This demonstrates that in a uniform chromatin, no structural polymorphism is present. The data for all 3 constructs confirmed that the nucleosome N interacts with the nucleosomes N±2 independently of the position of N. These results led us to conclude that in presence of H1 the 12x197 bp nucleosome array folds into a uniform zigzag conformation. Of note, our data did not confirm the two--start with tetranucleosomal units repeat models suggested after cryo--EM analysis [216]. Indeed, if the chromatin was in a tetranucleosomal unit repeat conformation, the interactions between nucleosomes N5 and N7 should be stronger than the interactions between N4 and N6, because the former falls within the same tetranucleosome, while the latter happens within the limits of two tetranucleosomes. However, we did not notice a significant difference of the percentage of crosslinking between N4--labeled arrays and N5--labeled arrays and their respective interacting N±2 partners.

A long--time persisting paradigm is that nucleosome repeat length variation might play a role in both regulating fiber compaction and selective DNA exposure. NRLs are not uniform within a single fiber, but how this variation affects fiber compaction is still unclear. Indeed, it was suggested that short 167 bp NRLs have a limited compaction, which results in a thin fiber [289], and an interdigitated one--start helix for medium To investigate this dilemma, we completed our study on NRLs effect by applying the ICNN approach to two additional fibers with different NRLs, 177 and 227 bp, that correspond respectively to the range of short and long NRLs.

As seen, both fibers exhibit the same N±2 (zigzag) conformation as the 197 bp fiber upon H1 compaction. We concluded that the NRL does not affect the 3D organization of the 30 nm fiber, which remains a two--start helix for all repeat lengths tested. Even and CENP--A (H3 variant). Of note, information on the 3D structure of NCP organization and on the compaction levels of histone variants--containing arrays is scarce.

The ICNN approach can be used as a powerful biochemical technique that describes the degree of compaction of a given chromatin. With that in mind, we decided to investigate the 3D organization of chromatin fibers containing either H2A.Z or CENP--A. H2A.Z was largely studied for its implication in many important cellular processes such as transcription, DNA repair and mitosis. The crystal structure solved by Luger and colleagues [164] described the nucleosome core containing H2A.Z to be similar to the canonical with the exception of the extended acidic patch, which might interfere with chromatin folding. However, contradictory data comparing H2A.Z-- and H2A--containing arrays described the H2A.Z arrays to be more compact than H2A--containing arrays in one case and less compact in the other.

We used the ICNN technique on reconstituted 12x197 bp arrays with biotin--labeled nucleosome N8 with H1--dependent compaction in the same conditions as canonical arrays. The data showed similar behavior for the H2A.Z containing--array displaying a zigzag organization where nucleosome N interacts with N±2. We did not notice any significant difference leading to believe that the extended acidic patch helps the chromatin to be more or less compact. The signals reported for nucleosomes N8 (# 2, 4, 6, 10, 12) were 8%, 15%, 40%, 24%, 8% of crosslinking respectively for H2A.Z chromatin and 6%, 15%, 42%, 20%, 7% of crosslinking for H2A chromatin. We then concluded that the H2A.Z chromatin displays a zigzag compaction with similar intensity as the H2A chromatin.

In most structural studies concerning array condensation, the chosen DNA template, histone origins and production systems, and the presence of linker histone H1 play a crucial role in determining the structural organization. For example, a biochemical analysis of the folding using 5S DNA template and divalent cations for inducing compaction, shows an accentuated folding into higher--order structure [228]. In our case, the 601 DNA template and linker histone H1--dependent folding were used to perform the experiments, resulting in equal folding levels of H2A.Z--and H2A--containing arrays.

Finally, we focused on the structural changes occasioned by the presence of CENP--A in a nucleosome array. The centromere--specific CENP--A is a H3 histone variant, which is important for recruitment of the kinetochore necessary to the alignment and segregation of the chromosomes. CENP--A proteins are conserved and essential components of the centromere, and thus are the best candidates for epigenetically marking the location of the centromere. In 2011, the crystal structure of NCP containing CENP--A revealed that CENP--A octamers only occupy 121 bp of nucleosomal DNA with the remaining ±13 bp of DNA invisible within the structure. This major structural difference was mainly attributed to a shorter α--N CENP--A helix [190].

In our lab, early studies on CENP--A nucleosome using •OH footprinting and EMSA methods showed that the linker histone H1 do not bind specifically to the CENP--A nucleosome (data not shown). To investigate the specific role played by the "defective" α--N CENP--A helix in linker H1 binding and linker histone H1--dependent folding, a mutated CENP--A (αN H3 --CENP--A) protein containing the α--N H3 helix and the preceding loop region of H3 was purified. The one base pair resolution •OH footprinting was used to evaluate the H1 binding to αN H3 --CENP--A--containing nucleosome. The data showed a clear recovery of H1 binding onto the nucleosome (not shown). We thus hypothesized that the αN H3 helix and the loop 1 are responsible for rigidifying the end of the nucleosomal DNA, which allows binding of the linker histone H1 as seen with canonical nucleosome. These results suggested that in presence of linker histone H1, CENP--A nucleosomal arrays will not fold into higher--order structures. However, H1--dependent folding might be recovered within replacing CENP--A with mutated αN H3 --CENP--A.

To investigate these hypotheses, we first used the ICNN approach on reconstituted 12x197 bp arrays with CENP--A octamer containing the H4V21C and H2AE64C mutated histones, allowing intra--fiber crosslinking. The biotin--labeled arrays showed no compaction in presence of linker histone H1. The absence of folding of the CENP--A chromatin is in agreement with our early observations, in which the linker histone H1

does not bind to the CENP--A nucleosome. We speculated that the absence of binding of the linker histone H1 to the CENP--A nucleosomal array leads to an unfolded chromatin.

Indeed, the recovery of linker histone binding ability in the mutated αN H3 --CENP--A nucleosome led, as expected, to a proper H1 binding and folding of the αN H3 --CENP--A array. As seen, the N±2 interactions between nucleosomes were detected similarly to the conventional chromatin. These combined results confirm that the αN helix "defect" of CENP--A is responsible to the absence of H1--induced folding of CENP--A, since H1 Nowadays, and in near future, special attention will be focused on the following problems:

--The data presented in this thesis point out to a clear edge--dependent asymmetry, meaning that we noticed a more efficient N±2 crosslinking from the longer side of the array. This asymmetry occurs for fibers with different linker lengths and different biotin--labeled nucleosomes. We tentatively attributed it to the finite length of our arrays and the more dynamic conformation at the edges. To directly address the origin of these observations, we will extend the length of our N1--N12 601 arrays by adding at each end one flanking 6x601 repeat. The ICNN detection technique will only "visualize" the nucleosomes N1--N12 located at the central part of the 24--mer array. Since the chromatin is a never--ending array in vivo, the physiological relevance of this question is obvious.

--The 3D organization of chromatin containing heterogeneous NRLs. We will create heterogeneous arrays containing a single or multiple nucleosomes with different NRLs and by means of the ICNN technique we will address the fiber organization. The resulting structural disordering, sometimes called polymorphic structure, has only been discussed by mesoscale modeling but has never been assessed by direct experimental approaches, which make these experiments relevant in addition to being closer to the in vivo conditions.

-- Altering linker length is not the only possibility to create heterogeneity. Incorporation of a single CENP--A nucleosome within a conventional array will also most probably create local structural disturbances. We are wondering whether and how the lack of H1 binding on the CENP--A nucleosome might affect its binding to neighboring conventional nucleosomes and what structural perturbations might be induced. Will the disturbance be localized to the CENP--A nucleosome position or will it affect the whole fiber? These questions will be addressed by a combination of our ICNN approach and cryo--EM imaging. The evidence of mixed H3/CENP--A nucleosomes at centromeres in vivo makes this investigation physiologically relevant.

--A combination of our biochemical ICNN method with X--ray diffusion in crystals and cryo--EM imaging appears to be the best choice to gain more structural information on the chromatin fiber. Efforts in this direction and the necessary collaborations have been already undertaken.

--The most compelling question is the in vivo organization of the chromatin fiber. It would be a remarkable achievement to extend the ICNN approach in vivo, in order to deepen our understanding of the local chromatin organization in its physiological environment. Besides, such an approach would provide direct experimental answers at the molecular level to many speculations on the role of the chromatin structure in genetic and epigenetic regulation. Efforts in this direction are already undertaken in our lab. Ligation was performed for 1h at 25°C By DNA T4 ligase. The agarose gel pattern displayed a ladder--like migration. Each band corresponding to number of repeats (2,3,4, etc.) of the 601 fragments ligated together. The resulting DNA band migrating at around 2400 bp was eluted from the gel and purified with the PCR clean--up kit (Promega).

The 601x12X fragment was then cloned into a pGEMT--easy plasmid (promega). 100 ng of vector and 300 ng of 601x12X repeats were mixed together with a DNA T4 ligase for 2 h at 25°C, transformed into DH--5α E.coli bacteria, plated on ampicillin--containing agar plates, and incubated overnight at 37°C. Single colonies were picked and incubated overnight in LB medium. Plasmids were extracted by miniprep and the presence of multimers was verified by an EcoRI digestion that releases the entire array. 3 different linker length were chosen for the 12 repeat arrays: 30,[START_REF] Eichler | Repetitive conundrums of centromere structure and function[END_REF]and 80 bp (respectively 177,197,and 227 bp NRLs).

1.1.2 N1--N12 array design.

Designing experiment.

Based on the 601 Widom sequence of the 147 bp corresponding to core DNA, 12 clones named N1 to N12 were designed, with 3 unique mutations on each end of the core DNA (illustrated in figure 30). These mutations will allow specific amplification of the repeat of interest (produced by MWG (Eurofins) and delivered in the pMAT vector). Each DNA repeat is separated by a blunt ScaI cleavage site. Each individual repeat has an inserted distinct restriction enzyme (other than ScaI). This distinct restriction site allows the specific replacement of a selected repeat with a biotin--labeled repeat. Table 1: PCR primers used for inserting a biotin label. X : biotin position.

Assembling the N1--N12 array

PCR using biotin--labeled primers were performed in the same conditions described earlier.

Chapter 2: Protein production

Xenopus core histones

The histone mutants H4V21C, H3C111A, H2AE64C and the wild--type H2B were produced using a modified version of the Luger procedure. 200 ng of plasmid (for each histone) were used to transform BL21 (DE3) pLYsS bacteria. Bacteria was induced with 0.2 mM IPTG (isopropyl--beta--D--thiogalactopyranoside) at 37°C for 3--4 hours at 200 rpm. Each liter of bacteria was pelleted at 5000g for 20 min at room temperature. 20 ml of wash buffer containing 50 mM Tris--HCl pH 7.4, 100 mM NaCl, 1mM Na--EDTA pH8, 1mM benzamidine and 10 mM ß--mercaptoethanol was added per liter of pelleted bacteria. Once the pellet were resuspended, the mixture was sonicated at 70% amplitude for 20 min and then pelleted for 30 min at maximum speed at 4°C. The supernatant was discarded and the pellet resuspended with wash buffer supplemented with 1% Triton X--100.

The mixture was pelleted again for 30 min at maximum speed at 4°C. This step was repeated twice and then 3 additional times without Triton X--100. After careful washing, the pellet was resuspended in 10 ml guanidinium buffer containing 7 M guanidinium--HCl, 20 mM Tris--HCl pH 7.4 and 10 mM DTT, incubated for 1 h at 4°C with gentle rotation (5--7 rpm) and then centrifuged for 30 min at maximum speed at room temperature. The supernatant was recovered gently and kept aside at room temperature. This procedure was repeated once. The two supernatants recovered were pooled and dialyzed overnight against SAU100 buffer (7 M Urea, 20 mM sodium acetate pH 5.2, 100 mM NaCl, 5mM β--mercaptoethanol and 1 mM Na--EDTA). The next day, samples were spun at 8000 g for 5 min before loading the supernatant onto an SP sepharose cation exchange column (GE Healthcare). The elution was done by SAU buffer with increasing NaCl concentration, ranging from 100 mM to 1500 mM. After checking on 18 % SDS--PAGE gel, the fractions containing the purified protein (500 to 700 mM NaCl) were mixed together. Before loading the purified protein to the ion exchange column Resource S 6 ml (GE Healthcare), the NaCl concentration was reduced to around 70--100mM by buffer exchange. The elution of the histone was done with a linear gradient ranging from 0.1 to 1 M NaCl in SAU buffer. After checking purity on SDS gel, these pure fractions were mixed and stored at --80°C.

Variant histones

CENP--A C75A, αN H3 CENP--A C75A and H2A.Z E64C were all sub--cloned into pET28 plasmid, which allows the production of 6x His--tagged histones (Tanaka Y et al 2004).

BL21 (DE3) pLYsS we amplified in the same conditions as for conventional core histone.

Chromatin reconstitution

For 20 µg reconstituted chromatin, 20 µg of the 601 wt arrays and 1 µg of biotinilated arrays were mixed with 4 µg of chicken carrier DNA (20 % of total amount of DNA), ~20

µg octamer, 2 M NaCl, 10 mM Tris pH7.4, 1 mM EDTA and 100 mM DTT. The mix was transferred into dialysis tubing and the reconstitution was done by dialysis against a slowly decreasing salt buffer. The NaCl concentration starts at 2 M and decreases slowly up to 500 mM NaCl. Indeed, with the help of a peristaltic pump, low salt buffer is added to the high salt buffer beaker at the rate of 1.5 ml/min for 18 h. Once finished, the dialysis bags were transferred to a 300 mM NaCl buffer and left for buffer exchange for 2 h, which was followed by a final dialysis in 10 mM NaCl buffer overnight. All NaCl buffers for reconstitution include 10 mM Tris pH 7.4, 0.25 mM EDTA, 10 mM β--mercaptoethanol and the desired amounts of NaCl.

Chromatin check by restriction enzymes

300 ng of chromatin were mixed with 0.5 mM MgCl 2 and 10 units of AvaI or HhaI and incubated at 37°C for 2 h. AvaI restriction site is present in the linker DNA and HhaI restriction site is present close to the dyad site in the core histone DNA. In case of undersaturation, the histone octamer is not sufficient to wrap the entire DNA amount, which gives access for HhaI digestion. In case of oversaturation, the excess of histone binds non--specifically to the linker DNA blocking the access for AvaI. Exact nucleosome saturation is achieved (i.e. exact equilibrium between the DNA and the octamer) when the array is fully digested to mononucleosomes by Ava I, while no any digestion is observed with HhaI.

Chromatin crosslinking and affinity precipitation

25 µg of nucleosomal array was first treated with 100 mM DTT in order to break the pre--existing disulfide bonds on ice for 2hrs.

Chromatin compaction by H1 deposition

Histone H1 mixed with histone chaperone Nap1 at 1:2 molar ratio was incubated for 30 min at 30°C in the following buffer: 20 mM Tris-HCl pH 7.5, 0.5 mM EDTA, 100 mM NaCl, 1mM DTT, 10% glycerol, 0.1mM PMSF, added to the chromatin (at a concentration of 40 ng/µl ) at a 1.25: 1 ratio and incubated for 1h at 30°C in a total volume of 100 µl .

Crosslinking with glutathion

Chromatin was first dialyzed against crosslinking buffer (10 mM Tris pH 9, 50 mM NaCl, 250 µM EDTA) for 3 h at room temperature using the cellusepT2 dialysis tubing 10 mm width. The purpose of this dialysis is to eliminate the DTT present in the chromatin mixture, whereas its presence will block the crosslinking. After dialysis, the chromatin (30 ng/µl) was adjusted to 25 µM of oxidized glutathione, 50 mM NaCl, 10 mM Tris pH 9

and was incubated at 37°C overnight for histone--histone crosslinking.

Digestion and DTT treatment

Before ScaI--AvaI digestion, the crosslinked chromatin was incubated with 2.

Affinity precipitation

To each tube containing 2 µg of chromatin (--/+DTT), 1.5 ng of IC nucleosomes (estimated amount of the N1--N12 present in each tube) and 1 µl of 1% NP40 were added. 5% of the total volume was taken for input. In this case, 5 µl was kept at 4°C for later use. The rest was mixed to blocked streptavidin magnetic beads. µg/ml BSA, 0.01% NP40 and 10 ng/µl erythrocyte chicken nucleosome. The beads were then subjected to proteinase K treatment in order to recover the attached DNA. Indeed, 200 µl of STOP buffer were added to the beads alongside 1 µl of proteinase K (934 U/ml -- Fermentas) and incubated at 55°C for 2 h in an hybridization oven for its capacity to provide a rotation of the tubes and prevent the beads from settling down. Alongside the tubes containing the beads, the input tubes were subjected to the same treatment (stop buffer and proteinase K for 2 hours at 55°C). The supernatant was collected and DNA extracted by phenol/chloroform and precipitated by ethanol.

DNA quantification and qPCR.

After DNA precipitation, The total DNA was quantified using the Quant--it Picogreen dsDNA assay kit (Invitrogen). The concentration of each sample (usually between 1.5 and 2 ng/µl) was determined with the help of a standard calibration curve. The DNA was diluted up to 16.6 pg/µl with ultrapure Millipore water and 3 µl (50 pg) were added to each well of qPCR 196--well plates (Eurogentec), together with 1x Sybr Green Mix (Fast start Universal SYBR Green (Roche)) and 10 pmol/µl detection primers (Figure30)

and completed to 15 µl with ultrapure water. The amplification was done on a

StepOnePlus real--time PCR system (Applied Biosystem).

3. 5 . 2

 52 Role of H2A histone variants: H2A.Bbd .......................................................................................................57 3.5.3 Role of H3 histone variant: CENP--A ............................................................................................................

91 IV 1 Chapter 2 : 2 . 1 Chapter 3 :

 9112213 Figures:

Figure 1 : 5 Figure 2 : 7 Figure 3 : 9 Figure 4 : 11 Figure 5 : 13 Figure 6 : 14 Figure 7 : 15 Figure 8 : 16 Figure 9 : 24 Figure 13 : 29 Figure 14 : 31 Figure 15 : 41 Figure 16 : 46 Figure 17 : 47 Figure 18 : 48 Figure 19 : 49 Figure 20 : 56 Figure 24 : 59 Figure 25 : 62 Figure 26 : 63 Figure 28 : 74 Figure 29 :

 15273941151361471581692413291431154116461747184819492056245925622663287429 Figure 1: Historical diagram representing major discoveries of chromatin studies.............5 Figure 2: Organization of eukaryotic chromatin fibers......................................................................7 Figure 3: Euchromatin and heterochromatin properties..................................................................9 Figure 4: Chromosome classification. ..................................................................................................... 11 Figure 5: Structure of the DNA................................................................................................................... 13 Figure 6: Schematic representation of all the core histones......................................................... 14 Figure 7: Histone octamer assembly ....................................................................................................... 15 Figure 8: The organizationof the nucleosome core particle ......................................................... 16 Figure 9: Three major models showing the binding of globular domain to nucleosome. 20 Figure 10: The H1 CTD is disordered in the absence of interacting partners....................... 21 Figure 11: A typical FRAP curve for H1 binding to chromatin showing multiple populations ............................................................................................................................................... 23 Figure 12: alternative models for the reversible association of histone H1 with the nucleosome............................................................................................................................................... 24 Figure 13: Schematic drawing of a nucleosome with the four canonical histones (H2A, H2B, H3 and H4) and the linker histone H1 ............................................................................... 29 Figure 14: Histone post--translational modifications ....................................................................... 31 Figure 15: Structure of the DNA entrance and exit of the human CENP--A nucleosome... 41 Figure 16: Models for the solenoid 30 nm chromatin fiber .......................................................... 46 Figure 17: Models for ZigZag 30nm chromatin fiber. ...................................................................... 47 Figure 18: The 30 nm fiber structure 1.................................................................................................. 48 Figure 19: The 30 nm fiber structure 2.................................................................................................. 49 Figure 20: The 30 nm fiber structure 3.................................................................................................. 51 Figure 21: The 30 nm fiber structure 4.................................................................................................. 52 Figure 22: The 30 nm fiber structure 5.................................................................................................. 54 Figure 23 : A Close--up View of the H4 tail domain-acidic patch interactions observed in Crystal. ........................................................................................................................................................ 56

  Photobleaching GD: Globular Domain GH1: Globular Domain H1 H1: Histone H1 H2A: Histone H2A H2A.Bbd: Histone: H2A Barr body--deficient H2B: Histone H2B H3: Histone H3 H4: Histone H4 HATs: Histone Acetyltransferases

  During this time we witnessed the rediscovery of the Mendel principle in 1900 by H. de Vries, the development of the gene theory and the principle of linkage in 1910 by T.H. Morgan. In 1928, F. Griffith identified the 'transforming principle' by infecting mice with 2 strains of pneumococcus (Streptococcus pneumoniae) and proving the transfer of the genetic information. Later on, in 1944 O. Avery, C. MacLeod and M. McCarty proved that DNA was the molecule responsible for bacterial transformation. The discovery of polytene chromosomes in Drosophila and of gene localization in 1933 by E. Heitz and H. Bauer, T. Painter and in 1935 by C. Bridges provided experimental material for exploring the chromatin structure. Inspired by this work, D. Mazia conducted in 1941 a series of analyses on the Drosophila salivary gland polytene

Figure 1 :

 1 Figure 1: Historical diagram representing major discoveries of chromatin studies. Image adapted from [19].

Figure 2 :

 2 Figure 2: Organization of eukaryotic chromatin fibers. The lowest level of DNA packaging is the nucleosome, in which two superhelical turns of DNA are wound around the outside of a histone octamer. Nucleosomes are connected to one another by short stretches of linker DNA. At the next level of organization, the string of microsomes ('beads on a string') is folded into a fiber about 30 nm in diameter, and these fibers are then further folded into higher-order structures. At levels of structure beyond the nucleosome details of folding are still uncertain. Figure adapted from [29]

Figure 3 :

 3 Figure 3: euchromatin and heterochromatin properties. (A) The distribution of euchromatin (E) and heterochromatin (H) in a normal thymus lymphocyte. (B-C) Characteristics of euchromatic and heterochromatic regions. Images adapted from [42].

Figure 4 :

 4 Figure 4: Chromosome classification. On the basis of the location of the centromere, chromosomes are classified into four types: metacentric, submetacentric, acrocentric, and telocentric. Figure adapted from [54].

Figure 6 :

 6 Figure 6: Schematic representation of all the core histones. The boxes indicate the helices of the histone fold domain and histone fold extensions. Figure is inspired from [61]

Figure 7 :

 7 Figure 7: Histone octamer assembly. (A) A histone octamer is the eight protein complex found at the center of a nucleosome core particle. It consists of two copies of each of the four core histone proteins (H2A, H2B, H3 and H4). The octamer assembles when a tetramer, containing two copies of both H3 and H4, complexes with two H2A/H2B dimers. Figure adapted from [63]. (B) SDS electrophoresis gels of the histone octamer and all histone cores.

octamer and forms 1 .

 1 67 turns of a superhelix. The H3--H4 tetramers occupy the central 60 bp region whereas each of the H2A--H2B dimer binds to the 30bp adjacent to the central region. The remaining 13 bp on each end of the DNA are held by the H3 αN helix.

Figure 8 :

 8 Figure 8: The organizationof the nucleosome core particle (A) Unraveling of the nucleosomal DNA to indicate which regions of the 147 bp of DNA are organized by which histone proteins. The orange dotted line indicates the H3/H4 tetramerization interface. The N-terminal alpha helix of H3 interacts with a different DNA gyre from that with which the remainder of the H3/H4 dimer interacts. (B) The nucleosome core particle: The views are down the DNA superhelix axis for the left particle and perpendicular to it for the right particle. Ribbon traces for the 146-bp DNA phosphodiester backbones (brown and turquoise) and eight histone protein main chains (blue: H3; green: H4; yellow: H2A; red: H2B. (Protein Data Bank (PDB): 1KX5). Figures

  The term "nucleosome positioning" is used to indicate where nucleosomes are located with respect to the genomic DNA sequence. The sequence--based mapping approaches identify the positions of individual nucleosomes in a single cell at a specific time, which might overlook the dynamic process of nucleosome positioning. Nucleosome occupancy has a critical biological role because nucleosomes inhibit the access of non--histone protein to the DNA. Transcription factors have different modes of bindings depending on their ability to be inhibited by nucleosomes. For example, a class of transcription factors such as FoxA and GATA are bound to their target sequence located at the nucleosome occupied site. These classes of factors are supposed to recruit nucleosome remodelers, thus opening up the local chromatin and allowing the binding of other transcription factor that otherwise would be inhibited by the nucleosome[START_REF] Zaret | Pioneer transcription factors: establishing competence for gene expression[END_REF]. Several factors can influence nucleosome positioning in vivo, including DNA sequence preferences of the nucleosomes themselves, DNA methylation, histone variants, PTMs, chromatin remodelers, DNA--binding proteins and higher--order structure.

Figure 9 :

 9 Figure 9: Three major models showing the binding of globular domain to nucleosome. (A) Symmetrical model. (B) Bridging model. (C) Asymmetrical model. In grey, Nucleosomal DNA, in bleu, histone octamer and in red, linker histone H1.

Figure 10 :

 10 Figure 10: The H1 CTD is disordered in the absence of interacting partners. Upon interaction with DNA or other targets in chromatin, the domain adopts a secondary structure. Figures adapted from[START_REF] Caterino | Structure of the H1 C--terminal domain and function in chromatin condensation[END_REF]

  Moreover, interrogations were issued concerning the domain responsible for the low binding affinity. Is it the globular domain or the C--terminal domain? In order to answer this question, a binding kinetics of histone H1 was performed using the FRAP experiments and mathematical modeling. As shown in figure 11, three different pools of histone H1 exist: a rapidly diffusing pool, a low--bound pool and finally a strong--bound pool [100].

Figure 11 :

 11 Figure 11: A typical FRAP curve for H1 binding to chromatin showing multiple populations. Figure adapted from [101]

Figure 12 :

 12 Figure 12: Different models for the reversible association of histone H1 with the nucleosome. In model I, the C-terminal domain (CTD) associates nonspecifically through electrostatic interactions with the linker DNA. In model II, the globular domain initiates a low-affinity binding interaction between the linker DNA and histone H1. In model III, both the globular domain and the CTD associate with the linker DNA to form an electrostatic clamp. Figure adapted from [102]

  presence of H1[103]. At low ionic strength, the loss of H1 leads to a decondensation of the chromatin to a more open and randomly folded beads--on--a--string form[104]. Based on the changing chromatin conformation in presence of mono-- or divalent ions and on the polyelectrolyte theory of Manning, it was concluded that the mechanism of compaction is primarily electrostatic, with H1 playing a key role[105]. These results were confirmed in vivo, when the loss of H1 in Tetrahymena led to an increase of the nuclear volume, consistent with expectations based on the disruption of the nuclear electrostatic balance[106]. The presence of several H1 subtypes complicates the studies of H1 stoichiometry. A thorough quantification of H1 stoichiometry indicated a 1 H1 per nucleosome stoichiometry in lymphocyte and glial nuclei and 0.8 H1 per nucleosome in liver nuclei[107]. Moreover, in erythrocyte nuclei, which contain the 2 subtypes H1 and H5, it was shown of 0.9 H1 and 0.4 H5 per nucleosome that added up to a ratio of1.3:1.

  lack introns, they have short 5' and 3' non coding sequences and their mRNA ends with a hairpin loop involved in the regulation of their expression[120]. The gene transcription of H1.1 is restricted to the proliferation phase, where the cells undergo successive divisions. Furthermore, H1.1 has the highest turnover among the different variants, with a half--life of five days. Reported studies showed that the level of H1.1 decreases from 5% to 0.5% during the post--natal development of cortical neurons in rat brain and is replaced by the H1.4 variant [121].H1.2 exhibits the highest levels of expression compared to the other somatic variants. It has been shown that H1.2 interacts with a group of protein (YB1 and PURα) and forms a that regulates p53--mediated trans--activation [122]. The H1.2 complex represses p53 via a direct H1.2/p53 interaction causing a blockage of the p300-mediated acetylation of chromatin. In addition to its nuclear role, H1.2 translocates to the cytoplasm in response to apoptotic stimuli such as DNA damage. Moreover, reducing or depleting H1.2 increases cellular resistance to apoptosis[123]. H1.2, H1.3 and H1.4 are present in non--dividing and quiescent cells of almost all tissues. H1.3 and H1.4 seem to be missing in active chromatin. H1.5 is highly expressed in the heterochromatin regions and at the nuclear periphery and unlike H1.2, H1.3 and H1.4, it has a very low level in quiescent cells.

Figure 13 :

 13 Figure 13: Schematic drawing of a nucleosome with the four canonical histones (H2A, H2B, H3 and H4) and the linker histone H1. The covalent PTMs [methylation (Me), acetylation (Ac), ubiquitination (Ub), and phosphorylation (Ph)] are highlighted on the N-and C-terminal tails of each histone. Figure adapted from [134].

Figure 14 :

 14 Figure 14: Histone post-translational modifications. All histones are subject to post-translational modifications, which mainly occur on histone tails. The four post-translational modifications are depicted in this figure: acetylation (blue), methylation (red), phosphorylation (yellow) and ubiquitination (green). The numbers in gray under each amino acid represent their position in the protein sequence. Figure adapted from [141].

  [147]. The single acetylation of the Lys 16 seems to have the most important effect on chromatin folding compared to other modifications. The nucleosome--nucleosome interactions decrease once Lys 16 is acetylated, which contributes to the unfolding of the compacted chromatin. A sedimentation experiment was performed on 12x177 bp nucleosome arrays containing wild--type (WT) H4 and K16Ac H4 octamers. Arrays containing WT H4 in presence of 1 mM MgCl 2 formed compact fiber, sedimenting at 54S whereas the arrays containing acetylated H4 were unable to compact, thus sedimenting at 44S. Moreover, in vivo studies consisting in separating chromatin fractions via Micrococcal nuclease (MNase) digestion into MgCl 2--soluble and --insoluble component showed that H4K16Ac is present in higher amount in soluble fraction, corresponding to decondensed chromatin, suggesting its involvement in chromatin compaction[147]. The exact mechanism by which this acetylation alters the chromatin folding is still unknown. The loss of the lysine positive charge due to acetylation can plays a role but it reduces by 10% only the positive charges in the tail domain of histone H4. Histone acetylation is involved in yet another DNA--dependent process; DNA damage repair. The HAT protein TIP60 activates the DNA repair damage pathway by acetylation and activation of the ATP kinase [148]. Moreover, H3K56 acetylation is required for inactivation of the DNA damage checkpoint after DNA repair is complete, thus allowing cells to re--enter the cell cycle [149]. 2.8.2 Histone phosphorylation Histone phosphorylation has important functions in mitosis and gene regulation. Abnormal histone phosphorylation has been observed in many cancers but it remains unclear whether histone phosphorylation is the cause or the effect. The four core histone, histone variants and H1 are phosphorylated on either the N--terminal or C-terminal sides. It has been demonstrated that H1 and H3 are phosphorylated at different times during cell cycle whereas H2A and H4 are phosphorylated at stable rate throughout the cell cycle [150]. The cell--cycle dependent phosphorylation of H1 and H3 hits its highest level during the M--phase and its lowest level during the G1--phase. In vivo studies showed that the phosphorylation of linker histone H1 tails influence polynucleosome folding. Indeed, phosphorylation of N--or C--terminal ends of linker histone H1 leads to the unfolding of the chromatin which in turn regulates the access of transcription factors [151]. H3 phosphorylation increases dramatically during mitosis while H3 is dephosphorylated upon anaphase entry. H3S10 phosphorylation has been proved to weaken the association of the H3 tail to the DNA, which might promote DNA binding of other factors [152]. The centromere--specific histone H3--like variant (CENP--A) is phosphorylated by aurora B during prophase and dephosphorylated by protein phosphatase 1γ 1(PP1γ1) during anaphase. Absence of this phosphorylation is reported to delay the terminal stages of cytokinesis in HeLa cells [153]. H2A histone variants are also phosphorylated, for example H2AX is rapidly phosphorylated at Ser139 in response to double--strand break. Phosphorylated H2AX appears to decondense the chromatin, recruit factors involved in DNA repair and favor the assembly of DNA repair complexes [154].

  H2A and Lys 123 (yeast) or Lys 120 (vertebrate) for H2B). In addition, H3 and H4 are poly--ubiquitinated by ubiquitin ligase complex after UVC irradiation, but the biological function is still unclear[159]. H2A(ub) is reported to be associated to gene silencing and H2A ubiquitin ligases were found in transcription repressor complexes. Furthermore, H2A deubiquitin ligase is required for gene activation, which provides another evidence of the gene silencing role of H2A(ub)[160]. On the other hand, H2B (ub) is implicated in gene activation through various mechanisms, including promoting other histone modifications and elongation by DNA polymerase II. For example, over-expression of H2B ubiquitin ligase in mammalian cells leads to an increase in H2B(ub), which in turn leads to an increase in methylation of H3K4 and H3K79 and thus to over-expression of the HOX gene[161].

  nucleosome would disturb the particle. However, in vivo and in vitro experiments showed the presence of a heterotypic H2A.Z nucleosome in S.cerevisiae, fly and human. The docking domain of the C--terminal moiety displays a sequence difference, suggesting a possible alteration of the interaction with the linker histone H1.Finally, an increased acidic patch was observed in H2A.Z nucleosome suggesting a difference in internucleosomal interactions[164]. Different methodologies used to understand the properties of the H2A.Z--containing nucleosome might be the reason of contradictory results over the stability of this nucleosome. The biological function of H2A.Z has been extensively studied revealing a role in the regulation of transcription, DNA repair, heterochromatin formation, chromosome segregation and mitosis. Although it is not essential to yeast cells, it has been reported that the absence of H2A.Z is lethal to some species such as Tetrahymena thermophila, Xenopus laevis, Drosophila melanogaster and mice[165]. Genome--wide studies showed a non--random pattern of H2A.Z distribution[166]. Indeed, in euchromatin, H2A.Z presence peaked at the 5' end of many genes, enhancers and insulators. Furthermore, in budding yeast H2A.Z nucleosomes are present in flanking regions of transcription start sites. In yeast cells, the presence of H2A.Z at gene promoters is inversely correlated to transcription levels, and by contrast in Drosophila and human cells, the presence of H2A.Z is positively correlated to transcription[166]. Non--acetylated H2A.Z is found in the heterochromatin regions, distributed all over hundreds of kilobases. The deposition of H2A.Z is catalyzed by ATP--

  H3 have five variants: H3.1, H3.2, H3.3, CENP--A and 3.1t. H3.3 and CENP--A are the two major histone H3 variants. The distribution of the H3 variants among eukaryotes is varied, for example S. cerevisiae only contains an H3 variant that resembles to H3.3 and Cse4 (CENP--A). H3.1 may play a role in silencing and gene activation once it is dimethylated at lysine 9 and acetylated at lysine 4. H3.2 is also implicated in gene silencing through di-- and trimethylation of lysine 27. H3.1 and H3.2 are canonical H3 proteins, highly expressed in S--phase and incorporated into the chromatin during DNA replication. The histone H3.3 differs from H3.1 by 5 amino acids, is expressed throughout the cell cycle and is incorporated into the chromatin during DNA replication or independently [183]. H3.3 is linked to active transcription by modifications such as the tri--methylation of lysine 4. Despite little effect on the mononucleosome structure, recent findings demonstrated that H3.3 incorporation leads to an open chromatin that promotes transcription [184]. Moreover, H3.3 co--localizes with H2A.Z at the promoters of active genes. The 5 amino acid difference between canonical H3s and H3.3 might play a direct role in nucleosome dynamics and PTM pattern. For example, the presence of serine instead of alanine at position 31 leads to a probable phosphorylation site during mitosis. Although H3.3 is present at lower levels in dividing cells, the levels increase significantly during terminal differentiation and contribute to more than 50% of the total amounts of H3 in the cell [185]. H3.3 has a different mechanism of deposition than the canonical H3s. H3.1 is deposited by histone chaperone CAF1, whereas the H3.3 is deposited by histone chaperone HIRA [186].

  and the fact that CENP--A--containing nucleosomes are more prone to unwrapping might allow easier removal from non--centromeric chromatin[189]. CENP--A nucleosome is structurally distinct from the canonical nucleosome which might expose the CENP--A nucleosome on surface of the centromere. The crystal structure of the CENP--A nucleosome showed that the histone octamer wraps the DNA in a left--handed orientation and covers only 121 bp vs 147 bp for canonical nucleosome. As mentioned before the αN helix is important for the orientation of the DNA in the canonical H3 nucleosome, which might explain the missing 13 bp on each end of the DNA in the crystal structure of the CENP--A nucleosome (Figure15)[190]. The crystal structure also revealed that CENP--A contains 2 extra positively charged amino acid residues (Arg and Gly) residues in the loop 1, whose depletion demonstrated that they are important for a stable CENP--A retention at the centromere but not for targeting CENP--A to the centromere. All these differences between CENP--A and H3 nucleosomes might play a crucial role in the centromeric chromatin architecture[190]. The only PTM of CENP--A known to date in human cells is the phosphorylation of the Ser 7 in the N--terminal tail, the absence of which results in a delay in cytokinesis[191].

Figure 15 :

 15 Figure 15: Structure of the DNA entrance and exit of the human CENP-A nucleosome (A) Secondary structure of CENP-A in the nucleosome. The sequences of human CENP-A and H3 are aligned with the secondary structure. (B) Close-up views of the αN helices and the DNA edge regions of the CENP-A (left panel) and H3 (right panel) nucleosomes. The dashed lines correspond to the DNA region that is not visible in the crystal structure. H3 is represented in orange and CENP-A is represented in magenta. Figure adapted from [190]

Figure 16 :

 16 Figure 16: Models for the solenoid 30 nm chromatin fiber. (A) The solenoid model presented by Finch and Klug. (B) The coiled linker model, continuously bent linker model. Figure adapted from [208]

Figure 18 :

 18 Figure 18: The 30 nm fiber structure 1. (A) Rat thymus chromatin, positively stained. (B) schematic diagram showing the folding of the nucleofilament into a solenoid. (C) Helical ribbon of stacked nucleosomes, nucleosomes are drawn as flat cylinders to show the symmetrical inter-nucleosome contacts. (D) A view of the twisted-ribbon model, the model has a helical repeat of 18 nucleosomes, pitch of 32 nm, dameter of 30 nm and a central hole of 8,5 nm diameter. Figures adapted from [15], [23], [207] and [209] respectively.

Figure 19 :

 19 Figure 19: The 30 nm fiber structure 2. (A) An irregular symmetric chromatin generated with fixed linker length (46bp). (B) Upper panel shows the nucleosome-folding motif obtained for a condensed dinucleosome. The middle panel shows the 48 nucleosome system constructed from repeating the nucleosome motif and lower panel shows the same chromatin with integration of electrostatic charges. Figures adapted from [211] and [212] respectively.

Figure 21 :

 21 Figure 21: The 30 nm fiber structure 4. (A) Simulated AFM images of chromatin after partial flattening with random linker length between 60 and 64 bp. (B) Model used for the chromatin simulation in A. Figure adapted from [217]

Figure 22 :

 22 Figure 22: The 30 nm fiber structure 5. (A) Fit of the release of the force-extension curve of a chromatin fiber at low ionic strength by using an extensible worm-like chain model. Schematic drawing of the chromatin qualitatively represents the continuous deformation of the chromatin fiber as it is subjected to increasing tension at low ionic strengths. (B) Schematic representations of chromatin fiber subjected to different forces.

Figure

  Figure adapted from [219] and [220], respectively.

Figure 23 :

 23 Figure 23: Close-up View of the H4 tail domain-acidic patch interactions observed in crystal. The H4 tail is shown in lime green. Histones H2A are in grey. Acidic patch residues are in shaded pink. Figure adapted from [226]

Figure 25

 25 Figure 25 Chromonema and radial-loop/protein scaffold models. (a) Chromonema models propose a hierarchically folded chromatin fiber that yields a series fiber of different thicknesses. (b) Local disruption of fiber-fiber interactions would lead to the unraveling of the fibers. (c) Radial-loop models propose that the chromatin fiber forms loops attached to a protein scaffold. (d) The structure shown in panel c could decondense into two beads by local detachment of a loop from the scaffold. Figure adapted from [237].

  genomic behavior in cell populations, thus enabling DNA topology studies at a higher resolution. These techniques rely on the remarkably simple idea that digestion and re-ligation of fixed chromatin in cells, followed by quantification of the ligation junctions, allows the determination of the DNA contact frequencies. In the original study that describing the 3C techniques, the average 3D conformation of yeast chromosome III was determined, showing that it forms a contorted ring[246]. The method was then adapted for mammalian systems and used to prove the existence of chromatin loops in vivo between regulatory DNA elements and their target genes[247]. Other 3C--based methods have seen the light since then including 4C (Chromosome Conformation Capture--on--Chip), 5C (Chromosome Conformation Capture Carbon Copy), HiC and ChIA--PET. However, the 3C--derived methods fail to detect cell--to--cell variation and cannot assess the dynamics in the system. Integration of the 3C--based methods with high resolution live--cell imaging will lead to a better understanding of the dynamics underlying chromosomal interactions and their cell--to--cell variation.

  Figure 1c-h): (i) induction of disulfide crosslinks (H4--H4 and H2A--H4) between the neighboring nucleosomes in the compact arrays in solutions containing 1:1 ratio of reduced and oxidized glutathione as described [34]; (ii) Digestion the chromatin samples to completion with the ScaI restriction enzyme; (iii) Checking the completion of cleavage at the level of DNA, isolated from the cleavage reaction products; [34]. Note that upon complete cleavage only individual 601 repeats DNA and mono--nucleosomes have to be observed; (v) Demonstration of the efficiency of crosslinking; (vi) Native streptavidin chromatin pull down and, (vii) Quantitative PCR of the DNA isolated from the pull down samples.

Figure 1 :

 1 Figure 1: Schematic presentation of the Identification of Closest Neighbor Nucleosomes (ICNN) approach. (a) The one-start helix (solenoid) model with N±1 nucleosome arrangement (b) The N±2 "zigzag" model of two intertwined helical stacks with linkers crossing the fiber (c) Schematics of the 12 repeats of 601 DNA used for nucleosomal array reconstitution; the repeats are separated by a common blunt ScaI site (vertical green discontinuous line) and contains in close vicinity the cleavage site (indicated in orange as I_) for specific for this repeat « overhang » restriction enzyme (see also panel 2 in (d). (d) Linear alignment of the 601 nucleosomal array, containing 12 (from N1 to N12) distinct nucleosomes. (e) Upper part, schematics for an individual nucleosomal 601 repeat used for streptavidin pull down; the end of the repeats are defined by the blunt end (in green) ScaI site and it contains an unique site for an « overhang » end restriction enzyme(orange dotted line); two nucleotides (orange stars) in vicinity of each the nucleosome DNA (indicate as discontinuous oval) are mutated, which allows their unique "visualization" by QPCR, incorporated biotin

lane 4 )

 4 , which reflects the Nap1 assisted deposition and binding of H1 to the arrays. The bisulfide crosslinking does not affect the mobility of the samples indicating no changes in their structure upon crosslinking (Figure S1, lane2-5). Digestion of chromatin with AvaI--ScaI, a restriction enzyme having a single cleavage site within the linker region of each nucleosomal repeat, generates only mono--nucleosome particles, with no free DNA detected (Figure 2b, lane 3). Although, chromatin DNA was inaccessible for digestion with the restriction enzyme HhaI, which has a single cleavage site within in the interior of each individual nucleosome of the arrays (Figure 2b, lane 4). In contrast, incubation of free DNA tandem repeats with either one of the enzymes leads to their complete cleavage resulting in the presence of monomeric repeat DNA only (Figure 2b, lanes 5, 6). Taken as a whole, the above data demonstrate the high quality of our reconstituted 197 bp arrays.

Figure 2 :

 2 Figure 2: Characterization of the reconstituted chromatin used for ICCN analysis. (a) Agarose gel image of reconstituted 12x197 bp nucleosome arrays without (lane 3) and with H1 (lane 4). The histone chaperone Nap1 was used for the deposition of H1. Lane 3, only N added to the reconstituted chromatin. (b) Agarose gel image of 12x197 bp nucleosome arrays digested with either AvaI-ScaI (lane 3; AvaI-ScaI unique cleavage sites for 601 DNA is in the linker of the carrier and the biotinylated DNA substrates respectively) or HhaI (lane 4), having an unique cleavage site in the nucleosomal DNA. Lanes 5,6, digested free DNA tandem arrays with either AvaI-ScaI (lane 5) or with HhaI (lane 6) (c) Agarose gel of isolated DNA of 12x197 nucleosome arrays digested by AvaI-ScaI (lane 2) and HhaI (lane 4) after phenol chloroform. Lane 4, 5 digested free DNA tandem arrays with AvaI-ScaI (lane 4) and HhaI (lane 5).

Figure 3 :

 3 Figure 3: 3D organization of nucleosomes in reconstituted 12x197 bp nucleosomal arrays. (a) SDS PAGE image of internucleosomal disulfide crosslinking of histone H4-H4 and H2A-H4 in compacted nucleosomal arrays in the absence (lanes 1, 2) and presence of histone H1 (lane 3). Lane 2, only Nap1 was added to the nucleosome arrays. (b) Left panel, treatment with DTT results in complete reversal of the disulfide crosslinking. The crosslinked arrays were digested to completion with AvaI and then run on agarose gel before (lane 3) or after treatment with 100 mM DTT (lane 4). Right panel, DNA isolated from the samples run on lanes 1-4, respectively. (c) Right panel, N±2 3D nucleosome arrangement in histone H1 bound 12x197 bp nucleosomal arrays. The measured probability of crosslinking between the different nucleosomes is presented. Nucleosome #5 (N5) contained inserted biotin. The amount of streptavidin pull down N5 DNA was taken as 100% and the amount of the co-precipitated DNA from the other nucleosomes was normalized to it; Left panel, same as upper panel, but for 12x197 bp nucleosomal arrays without H1.

Figure 4 :

 4 Figure 4: The N±2 3D nucleosomal arrangement in condensed arrays is independent of DNA linker length. (a) Measured probability of crosslinking of nucleosomes within the condensed 177 bp repeats array. Biotin was inserted in either nucleosome #4 (N4, left), or #8 (N8, middle) or #9 (N9, right), respectively. (b, c) Same as (a) but for condensed arrays with repeat length of 197 (b) and 227 bp (c), respectively. d) Zigzag model proposed by [35] e) zigzag model proposed by [36].

Figure S2 :

 S2 Figure S2: Mass spectroscopy analysis of the crosslinked bands. (a) Schematic of the SDS gel used for the mass spectroscopy analysis. (b) Mass stectroscpy results showing that 'bande 1' contains histone H2A and histone H4, whereas 'bande 2' contains only histone H4 confirming that these band corresponds to H2A-H4 and H4-H4 crosslinks. These results are consistent with crosslinking results published in [37].

Figure S3 :

 S3 Figure S3: Agarose gel characterization of the reconstituted 12x177 bp nucleosome arrays by restriction enzymes treatment. Free DNA and nucleosome array without (a) and with H1 (b). . The histone chaperone Nap1 was used for the deposition of H1. (c) The crosslinked arrays were digested to completion with AvaI/ScaI before (lane 1, 3) or after treatment with 100 mM DTT (lane 2, 4) Treatment with DTT results in complete reversal of the disulfide crosslinking in 12x177 bp arrays (compare lanes 3 and 4).

Figure S4 :

 S4 Figure S4: Efficient disulfide nucleosome crosslinking in compact 12x227 bp nucleosome arrays. Characterization of the reconstituted 12x227bp nucleosome arrays by restriction enzymes cleavage and agarose gels (a), (b), (c) and by SDS PAGE (d). The histone chaperone Nap1 was used for the deposition of H1. (b) Nucleosome arrays without (lanes 2, 4), with H1 (lane 3), and with only Nap1 added (lane 4). (c) Treatment with DTT results in complete reversal of the disulfide crosslinking in 12x227 bp arrays. The crosslinked arrays were digested to completion with AvaI/ScaI and then run on agarose gel before (lane 1, 4) or after treatment with 100 mM DTT (lane 2, 3). (d) SDS PAGE image of internucleosomal disulfide crosslinking of histone H4-H4 and H2A-H4 in compacted nucleosomal arrays in the absence (lanes 1) and presence of histone H1 (lane 2). Lane 1, only Nap1 was added to the nucleosome arrays. Nap1 were removed by Ni-NTA beads but it did not affect the crosslinked chromatin (Figure S2, Lane 6, 7).

Figure

  Figure S5: QPCR analysis for two fully independent experiment with arrays 12x197 #9 (a, b) and 12x177 #4 (c, d) demonstrating the high reproducibility of the results.

Figure S6 :

 S6 Figure S6: 3D organization of nucleosomes in reconstituted 12x197 bp nucleosomal arrays containing H2A.Z instead of H2A. (a) Treatment with DTT results in complete reversal of the disulfide crosslinking. The crosslinked arrays were digested to completion with AvaI/ScaI and then run on agarose gel before (lane 1, 3) or after treatment with 100 mM DTT (lane 2, 4). (b) SDS PAGE image of internucleosomal disulfide crosslinking of histone H4-H4 and H2A.Z-H4 in compacted nucleosomal arrays in the absence (lanes 1, 2) and presence of histone H1 (lane 3). Lane 2, only Nap1 was added to the nucleosome arrays. (c) qPCR analysis clearly showing a similar N±2 3D arrangement as conventional H2A.

Figure S7 :

 S7 Figure S7: qPCR analysis of streptavidin pull down DNA isolated from AvaI-ScaI-digested 177 bp (a), 197 (b) and 227 bp nucleosome arrays (c) with biotin labeled nucleosomes #4, #8 and #9 (from left to right) without H1. Note the absence of crosslinking for all arrays demonstration the essential role of H1 in chromatin folding at low monovalent and absence of divalent ions.

Figure 2 :

 2 Figure 2: Characterization of the reconstituted CENP-A 12x197 bp nucleosomal arrays used for ICCN analysis. (a) Agarose gel of reconstituted 12x197 bp CENP-A nucleosome arrays without (lane 3) and with H1 (lane 4). The histone chaperone NAP1 was used for the deposition of H1. For the sample in lane 3, only NAP1 added to the reconstituted chromatin; lane 1, free DNA 12x197 bp tandem arrays (b) Arrays with (lane1, 2) or without H1 (lane3, 4) were treated with 100 mM DTT. The crosslinked arrays were digested to completion with ScaI and then run on agarose gel before (lane1, 3) or after (lane2, 4) treatment with 100 mM DTT. Note the absence of crosslinking (no lower migrating bands are present). (c) SDS PAGE analysis of internucleosomal disulfide crosslinking in CENP-A 12x197 bp nucleosomal arrays.

Figure 3 :

 3 Figure 3: Measured probability of crosslinking of nucleosomes within the condensed 197 bp repeats arrays containing CENP-A octamer or conventional one. Biotin was inserted in either nucleosome 8. To note, the absence of the N+2 3D structure in CENP-A array in presence or absence of H1 (a,b) in comparision with conventional chromatin (c,d).

Figure 4 : 4 .

 44 Figure 4: Characterization of the reconstituted αNH3 CENP-A 12x197 bp nucleosomal arrays used for ICCN analysis. (a) Agarose gel of reconstituted 12x197 bp CENP-A nucleosome arrays without (lane 3) and with H1 (lane 4) before crtosslinking. (b) Same as (a) but after crosslinking. (c) Arrays with (lane1, 2) or without H1 (lane3, 4) were treated with 100 mM DTT. The crosslinked arrays were digested to completion with ScaI and then run on agarose gel before (lane1, 3) or after (lane2, 4) treatment with 100 mM DTT.(d) SDS PAGE analysis of internucleosomal disulfide crosslinking in αNH3 CENP-A 12x197 bp nucleosomal arrays in the absence (lanes 1, 2, 3) and presence of histone H1 (lane 4). Lane 3, only Nap1 was added to the 12x197 bp nucleosome arrays. The samples were run on 18% SDS-PAGE. Note the H4-H4 crosslinks in lane 4.

Figure 5 :

 5 Figure 5: Measured probability of crosslinking of nucleosomes within the condensed 197 bp repeats arrays containing αNH3 CENP-A octamer. Biotin was inserted in either nucleosome 8. To note, the N+2 3D structure in αNH3 CENP-A array in presence of H1.

Figure 27 :

 27 Figure 27: Models of the 30 nm fiber. Orthogonal views perpendicular to the 30 nm fiber axis (top) and down the axis (bottom) of the two-start zigzag model (left), one-start solenoid model (center) and two-start tetranucleosome-unit model (right). In the two-start model, each sequential pair of nucleosomes across the fiber is colored similarly. For the one-start model, all nucleosomes in the same turn of the solenoid are colored similarly. In the two-start tetranucleosome model, each tetranucleosome repeating unit is colored similarly

Figure 28 :

 28 Figure 28: Measured probability of crosslinking of nucleosomes within the condensed 197 bp repeats arrays containing cannonical octamer. Biotin was inserted in nucleosome 8. Left panel, the chromatin display no folding in absence of divalent cation Mg 2+ . Right panel, the N+2 3D structure of the folded array in presence of divalent cation Mg 2+

(

  177--207 bp) and long (217--237 bp) NRLs but with different diameters of 33 and 44 nm respectively [215]. However, chromatin modeling presented a zigzag structure of small and medium NRLs and a solenoid structure for longer NRLs [224].

  though these results go against some data claiming different 3D conformations depending on the NRL, it does not give a definitive answer to the main question we are facing. Recent Monte--Carlo simulation data of coarse--grained oligonucleosome models described the chromatin as a polymorphic form that might include solenoid, zigzag, and irregular zigzag within the same non--uniform fiber[290]. However, this simulation takes in consideration data confirming that the small and long NRLs fold into zigzag and solenoid organization respectively, which makes their data bias and their polymophism theory unfounded. Moreover, our biochemical approach does not allow us to decipher the DNA linker position (crosslinked, helical ribbon) within the zigzag model. Electron microscopy or X-ray scattering in crystal studies are needed for further comprehension of the 3D organization of the 30 nm fiber.The efficiency and reproducibility of the ICNN techniques allowed us to extend our scope of research to new horizons. In recent years, countless structural studies have been done on histone variants revealing some variant--specific roles in the stability and exposure of the NCP[291]. Much like PTMs, histone variants can engender changes in the interactions with nucleosomal DNA and solvent accessible nucleosome surface. The most studied histone variants in the past few years are H2A.Z, H2A.Bbd (H2A variants)

  cannot stably bind to the nucleosome to induce the linker DNA stem necessary for compaction. However, a large number of questions surrounding the 3D organization remain unanswered. Even though the ICNN technique allowed an unambiguous recognition of the two--start model of the 30 nm fiber, it did not uncover linker DNA arrangement and linker histone H1 binding site. Advancement in cryo--EM and X--ray crystallography in the future will aid the visualization of the 30 nm fiber and the characterization of nucleosome binding enzymes. Despite the extraordinary progress in the past years, it is clear that we only uncovered a small part of the role of nucleosome in coordinating chromatin--templated processes. Additional work will create a new and This thesis described the development of a new biochemical technique, called the ICNN, which was specifically designed for obtaining structural information. The ICNN approach allowed us by a relatively simple crosslinking experiment to recognize the nucleosome positions within a compacted fiber. Our in vitro provide direct unambiguous evidence that uniform 601--arrays, containing properly bound linker histoneH1, fold into a two--start zigzag conformation with a regular nucleosome spacing, thus closing a 40 years old debates and controversy. Interestingly, the regular two--start conformation is invariant in respect to the nucleosome linker length variation within 177 and 227 bp NRL. This finding is important with respect to existing speculations on chromatin structural polymorphisms and its origin. In addition, we established with certitude the implication of histone variants in the organization of the chromatin structure. While H2A.Z fiber did not exhibit any particular changes in the folding conformation compared to H2A fibers, CENP--A fiber displayed a high level of folding inhibition, This finding is in accordance with the observation that linker histone H1 does not stably binds to CENP--A mononucleosomes. This "open" conformation of CENP--A chromatin is likely to be a means for allowing the recruitment of CENPs proteins and the formation of the consecutive centromere associated network. functional role in cellular regulation.

  DNA sequence (strong positioning DNA sequence) was used as a matrix to produce the 601x12X carrier arrays. AvaI restriction site was introduced at the end of the linker DNA by PCR. Placing the AvaI at different linker lengths allowed the production of arrays with different NRLs. 601x12X with AvaI restriction sites on both sides of the linker allowed the construction of the 12 repeat arrays by ligation.

Figure 29 :

 29 Figure 29: Schematic representation of the restriction enzyme sites in the N1-N12 repeats.

Chapter 3 :

 3 Chromatin reconstitution and chromatin check3.1 Histone octamerThe histone cores were all mixed in an equimolar ratio, and dialyzed overnight in HFB buffer (2M NaCl, 10 mM Tris pH7.4, 1 mM EDTA pH 8 and 10 mM β--mercaptoethanol).H3C111A, 12 µl H2AE65C, 15 µl of H2B and 46 µl of H4V21C were mixed with 50 µl of 8 M urea and 20 µl of 1 M DTT to have a total volume of 160 µl. After dialysis, 40 µl of NaCl saturated glycerol was added to the mixture, adding up the volume to 200 µl, which makes the octamer concentration 0.5 µg/µl.

  5 mM iodoacetamide at room temperature in the dark for 1 h. Later on, the mixture was dialyzed against NT buffer containing 50 mM NaCl, 10mM Tris pH 7.4 for 5 h at 4°C. After dialysis, 5µl of highly concentrated NapI (9.2 µg/µl) was added in order to remove the linker Histone H1 from the chromatin. Chromatin was digested with 250 units of AvaI, 100 units of ScaI and 25 units of XbaI (methylation site) overnight at 37°C in 200 µl of buffer containing 10 mM Tris pH7.4, 50 mM NaCl, 1.25mM MgCl 2 . The next day, each tube was separated in two 100 µl each. In one of them, 10 µl of 1M DTT was added and in the other one 10 µl of NT buffer(50 mM NaCl, 10 mM Tris pH7.4). Reactions were incubated at room temperature for 2 h. The digestion and efficiency of DTT treatment was checked on 1% agarose gel with 0.5X TBE before proceeding to affinity precipitation.

3. 4 . 4 . 2

 442 Blocking streptavidine beads5µl Dynabeads M--280 Streptavidin (Life technology) were added to 1.5 ml siliconized Eppendorf tubes and mixed with 200 µl of blocking buffer containing 10 mM Tris pH 7.4, 1 mg/ml BSA, 50 mM NaCl, 0.01% NP40. Magnetic beads were shook at 23 rpm to prevent them from settling, which would limit the blocking process. Beads were separated from supernatant on a Magnetic Particle concentrator (Dynabeads MCP --Life Technologies). The blocking step was repeated twice for 20 minute before proceeding to AP. Once the beads were ready to use, chromatin was added for binding overnight at 4°C under 20 rpm shaking. Supernatant was discarded, and beads were washed 3 times for 30 min with a buffer containing 10 mM Tris pH7.4, 50 mM NaCl, 100 µM EDTA pH 8, 100

Table 1 :

 1 The H1 histone family in mammals. ...................................................................................... 26

Table 2 :

 2 Histone variants and their functions..................................................................................... 36

Table 3 :

 3 Histone chaperones and their functions during nucleosome assembly. ............... 42

Table 4 :

 4 Level of chromatin structure and their possible global and local interactions. . 61

Table 1 : The H1 histone family in mammals. Table inspired from [119]

 1

Table 2 : Histone variants and their functions. Table inspired from [162]

 2

  Bbdhas only 48% identity with conventional histone H2A in humans. H2A.Bbd lacks both the C--terminal tail and the end of docking domain that characterizes a typical H2A family histone. Chromatin fractioning showed that H2A.Bbd is present in nucleosomes with histone H4 acetylated on the Lys--12. Photobleaching studies showed a faster recovery of H2A.Bbd than H2A in the nucleus, which indicates a higher mobility. Furthermore, stability studies on H2A.Bbd nucleosomes led to the conclusion that the nucleosome is more open and less stable than H2A nucleosomes[177]. H2A.Bbd--containing nucleosomes protect only ~120bp of DNA resulting in open nucleosomes that facilitate transcription[178, 179, 180]. Besides, H2A.Bbd display a testis specificity and its low abundance in somatic cells has made it extremely difficult to find the function for this protein[181].

.Bbd (Barr body deficient) was first identified from a search of human ESTs (expressed sequence tag) with distant homology to H2A

[176]

. Indeed, H2A.

Table 4 : Level of chromatin structure and their possible global and local interactions.

 4

Acknowledgements

I am deeply indebted to Dr. Dimitri Anguelov for giving me the opportunity to work on this challenging project and for providing the excellent environment that was necessary to complete my PhD and successfully use the wide range of techniques.

as well as the CENP--A nucleosome structure published a couple of years earlier. To solve this matter, another team repeated the same AFM experiment with human CENP--A, yeast CENP--A and canonical H3 that they obtained from four independent laboratories including the one used by Miell et al. As expected, no changes in height or diameter were noticed with both human and yeast CENP--A compared to H3. However, Miell et al. replied by collecting all AFM data that have been published on CENP--A/H3 difference of height (Figure 24). They stated that inconsistencies for AFM measurements collected from different groups are not unusual and might be due to imaging conditions such as the force and frequencies of the AFM tip, the adsorption of salt on the surface, humidity among other factors. The limitations of 2D imaging techniques and the indirect approaches have been the source of most contradictory informations collected about the chromatin structure and its dynamics.

Manuscript 1:

The novel ICCN approach reveals a 3D non--consecutive, independent of both H2A.Z and nucleosome repeat length, arrangement of nucleosomes in compact chromatin Supplementary Results: