Saadi Khochbin
email: saadi.khochbin@univ-grenoble-alpes.fr

Acyl-carrier protein ACSL Acyl-CoA ligase long chain specific

Keywords: Histone Posttranslational Modification, Mitochondrial Metabolism, FASTKD1, Acute Lymphoblastic Leukemia, β-oxidation, BRD4

Based on this work, we propose that cell metabolism, through modulating the histone acetyl/acyl ratio, controls a cellular reservoir of BRD4 (and probably many bromodomaincontaining proteins). High acetyl/acyl ratio favors the constitution of a reservoir of nonfunctional chromatin-bound BRD4. Low acetyl/acyl ratio, in contrast, increases BRD4 dynamics and makes it available for recruitment on the sites of action.

Our work not only added a new piece of evidence regarding the concept of metabolismdriven epigenetic modifications, but also emphasized on a collective and combinatorial actions of the relatively low abundant individual acylations.

ACKNOWLEDGEMENT

I would like to thank Uwe Schlattner, Eric Gilson, Zdenko Herceg, and Geneviève Almouzni to be members of the jury and evaluate my work.

I thank my supervisor, Saadi Khochbin for his wonderful ideas and extensive suggestions throughout the project. As a beginner, I have been exposed to too many expert thoughts, and have received too much patience from him. I would like to thank my co-supervisor, Sophie Rousseaux for her suggestions and her tremendous help in bioinformatics. I would like to thank my joint supervisor, Jianqing Mi, who not only supported the scientific project, but also attached me to the clinical knowledge. I would like to thank my PhD committee members, Carlo Petosa and Sophie Park, who evaluated my work each year and gave advice.

I would like to thank all the colleagues in SK team. I thank Domenico Iuso, who expressed numerous basic concepts in biology and taught plenty of general ideas about research. I thank Tao Wang, who is a walking encyclopedia and always gave answers. I thank Sophie Barral, Sandrine Benitski-Curtet, Florent Chuffart and Ekaterina Flin who helped me with the experiments and the data analysis. I thank Anne-Laure Vitte, Fayçal Boussouar, Thierry Buchou, Michel Pabion, Alexandra Vargas, Clovis Chabert, Ariadni Liakopoulou, Mélanie Petrier, and the previous colleagues, Naghmeh Hoghoughi, Azadeh Hajmirza to talk, to discuss, and to help. I would like to thank every colleague in IAB, I thank Sara El Kennani, Xue Yang, Tao Jia, Alex Casanova, Francesco Ielasi, Valentina Lukinovic, Oliver Destaing, Sabeen Ikram, Nicolas Reynoird etc. I remember every single piece of help they provided, and every single piece of joy they brought about in the institute. I would like to thank every working stuff in IAB, Anne-Sophie Ribba, Mylène Pezet, Jacques Mazzega, Dalenda Benmedjahed, Hélène Medjkane, Amélie Fauconnet etc. who ensure the platforms and the whole institute to run properly. I thank UGA working stuffs, the CSV secretary Magali Pourtier and a lot of other employees to deal with the students' affairs.

I would like to thank all the collaborators for their contributions. I thank Lisa Bargier and Denis I would like to thank my colleagues in Shanghai, Jin Wang, Duohui Jing, Lijun Peng, Mengping Xi, Caicike Bayin, Sisi Wang, Wenqian Xu, Wanyan Ouyang, Shanshan Guo, Gaoxian Song who make the lab function and bring joys to the lab. I thank every working stuff in Shanghai Institute of Hematology who serves the institute and provides convenience for students.

Finally, I thank my family, my parents and my siblings who support and care me all the way here. I thank my boyfriend who gives love, patience, comforts and keeps me company throughout the ups and downs in my life. I thank my precious friends Mei Kang, Hongling Diao and Xinran Wang who always stand by my side.

Thank you all.

ABSTRACT

Chromatin-based events, prominently gene transcription are the basis of cellular characteristics in normal tissues and in cancer context. Histone posttranslational modifications are parts of specific regulatory circuits controlling chromatin-based biological processes. A myriad of specific types of histone modifications, including site-specific histone acetylation and methylation, etc. have been characterized with respect to their roles in gene transcriptional regulation. A critical family of chromatin regulators bridges histone modifications and gene transcriptional output is the readers. The specificity of readers recognizing histone modifications depends not only on the modifications, but also on their combinations.

Additionally, it should be noted that histone modifications are dynamic and this process is impacted by a variety of factors, including cellular metabolites.

Previously the team identified a gene FASTKD1 whose expression is associated with poor prognosis in acute lymphoblastic leukemia (ALL). During my research, we uncovered that this gene is a negative regulator of general mitochondrial activity, and more specifically controls the mitochondrial respiration. Using gene knockout cell models, we further characterized the link between mitochondrial activity with histone modifications, and highlighted the importance of fatty acid metabolism, especially β-oxidation, in mediating histone modifications. The association of mitochondrial activity-β-oxidation and histone acylations was also confirmed in patients' primary blasts.

BET family proteins are specific readers of histone acetylation and mediate transcription regulation. Previous studies uncovered that the first bromodomain of a BET protein, Brdt, recognizes diacetylated histone marks (H4K5acK8ac) but not H4K5 butyrylated histones.

We noticed that FASTKD1-mediated mitochondrial activity prominently impacts non-acetyl acylations but not acetylations. Using our gene knockout cell model, we could demonstrate that the relative level of the acetyl and acyl marks tunes the bound state of BRD4 with chromatin. We showed that an increased ratio of acyl/acetyl disfavors BRD4-chromatin interaction, resulting in a loose and dynamic bound state, while a decreased ratio favors the binding and leads to a tight interaction. The functional output of this dynamic interaction is to re-distribute the BRD4 across the genome. More specifically, dynamic BRD4-chromatin interaction caused by high acyl/acetyl ratio makes BRD4 more available to be recruited on gene transcriptional start sites (TSS) and mediate the stimulated expression of a subset of genes mediated by BRD4. Gene functional analysis revealed that high acyl patients or high acyl/acetyl cells displayed increased expression of genes associated with ribosome synthesis, cell cycle and decreased expression of genes associated with stemness.

RESUME

Les événements contrôlés par la chromatine, notamment la transcription des gènes, sont à la base des caractéristiques cellulaires dans les tissus normaux ainsi que dans le contexte du cancer.

Les modifications post-traductionnelles des histones font partie de circuits de régulations spécifiques contrôlés par la chromatine. De nombreuses modifications d'histones, notamment l'acétylation et la méthylation des histones, ont été caractérisées et leur rôle dans la régulation de la transcription a été étudié. Les facteurs régulateurs de la chromatine capables de lire ces modifications, établissent un pont entre les modifications des histones et la transcription des gènes. La spécificité de ces facteurs peut également dépendre des combinaisons de modifications d'histones. Finalement, il convient de noter que les modifications d'histones sont dynamiques et qu'elles sont influencées par divers paramètres, notamment les métabolites cellulaires.

Mon laboratoire d'accueil a précédemment identifié un gène nommé FASTKD1 dont l'expression est associée à un mauvais pronostic dans la leucémie aiguë lymphoblastique (LAL).

Au cours de mes recherches, nous avons découvert que la protéine codée par ce gène est un régulateur négatif de l'activité mitochondriale globale, et qu'elle contrôle plus spécifiquement la respiration mitochondriale. En utilisant des modèles de cellules ou le gene codant pour cette protéine est inactivé, nous avons caractérisé le lien entre l'activité mitochondriale et les modifications des histones. En particulier, nous avons mis en évidence l'importance du métabolisme des acides gras, notamment la β-oxidation, dans l'établissement des modifications d'histones par l'acylation. Cette relation entre l'activité mitochondriale, la β-oxidation et l'acylation des histones a également été confirmée dans des blastes primaires de patients atteints de LAL.

Les protéines de la famille BET reconnaissent spécifiquement l'acétylation des histones et sont impliquées dans la régulation de la transcription. Des études précédentes de mon laboratoire ont révélé que le premier bromodomaine de l'une des protéines de la famille BET, Brdt, reconnaît les marques d'histones diacétylées (H4K5acK8ac) mais pas les histones H4K5

butyrylées. A partir de ces données, mon laboratoire avait fait l'hypothèse que le rapport acetyl/acyl au niveau des histones devrait impacter la liaison des facteur de la famille BET (Brdt, BRD4, etc..) avec la chromatine. Mon travail de thèse a permis de vérifier cette hypothèse en déployant d'autres modèles cellulaires.

En effet, nous avons aussi remarqué que l'activité mitochondriale contrôlée par FASTKD1 a un impact important sur les acylations (non-acétyl) mais pas sur les acétylations. En utilisant notre modèle de cellules ou le gène codant pour ce facteur est inactivé, nous avons pu démontrer que le niveau relatif des marques acétyl et acyl au niveau des histones contrôle l'état de liaison de BRD4 avec la chromatine. Nous avons montré qu'une augmentation du rapport acyl/acétyl diminue l'interaction BRD4-chromatine, ce qui entraîne un état de liaison lâche et dynamique, tandis qu'une diminution de ce rapport favorise une interaction forte. Le résultat fonctionnel de cette interaction dynamique est une redistribution de BRD4 à travers le génome. Plus précisément, l'interaction dynamique entre BRD4 et la chromatine causée par un rapport acyl/acétyl élevé rend la protéine BRD4 plus disponible pour être recrutée sur les sites d'initiation de transcription des gènes (TSS). Ainsi BRD4 contrôle l'expression d'un sousensemble de gènes. L'analyse fonctionnelle de ces gènes a révélé aussi bien chez les patients que dans nos cellules modèles, qu'un rapport acyl/acétyl élevé était associé à une expression accrue de gènes associés à la synthèse des ribosomes et au cycle cellulaire et à une expression réduite des gènes associés à l'état souche.

Sur la base de ces travaux, nous proposons que le métabolisme cellulaire, en modulant le rapport acetyl/acyl des histones, contrôle un réservoir cellulaire de BRD4 (et probablement de nombreuses autres protéines contenant un bromodomaine). Un rapport acétyl/acyl élevé favorise la constitution d'un réservoir de BRD4 non-fonctionnel lié à la chromatine. Un rapport acétyl/acyl faible, au contraire, augmente la dynamique de BRD4 et la rend disponible pour le recrutement sur les sites d'action.

Notre travail non seulement ajoute une nouvelle preuve de l'impact du métabolisme sur les modifications épigénétiques qui sous-tendent la biologie de la tumeur, mais également met l'accent sur l'action collective des acylations individuelles qui, lorsqu'on les considère chacune de manière isolée, sont relativement peu abondantes.

Mots-clés:

Modifications post-traductionnelles des histones, Métabolisme mitochondrial, FASTKD1, Leucémie aiguë lymphoblastique, β-oxidation, BRD4 Heterochromatin comprises some structural regions of chromosomes, such as centromeres and telomeres (so-called constitutive heterochromatin). It also corresponds to a cell-type specific and developmentally-controlled transcriptionally repressed regions, known as facultative heterochromatin.

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Chromatin structure undergoes temporal alterations dependent on cell cycle, most impressively it condenses during mitosis and decondenses in interphase. Later studies revealed that even in interphase, euchromatin is organized into specific areas, known as chromosome territories at nuclear scale (Figure 1). Recently, with the development of technologies characterizing 3D architecture of chromatin (e.g., 3C, Hi-C etc.), researchers discovered that at the megabase scale, chromosomes are segregated in two distinct compartments, designated as compartment A and B 1,2 . Type A compartment is enriched in active chromatin, where high transcriptional activity, DNase I hypersensitivity and active histone marks are found. This compartment is likely to be in the central region of nucleus as well as in regions close to the nuclear pores. In contrast, Type B compartment is known to have inactive chromatin associated with repressive histone marks, silent genes, and late replication timing. This compartment is located close to the nuclear periphery and is associated with the nuclear lamina 3,4 . The stability of these regions tunes the accessibility of the genomic regions to transcriptional machineries, thereby regulates cell specific gene transcription programs [START_REF] Dixon | Chromatin architecture reorganization during stem cell differentiation[END_REF] . The additional micro-hierarchical structures composing chromosome compartments are the so-called topologically associating domains (TADs). TADs are partitioned regions with relatively defined boundaries. In TADs chromatin contacts preferentially occur within the same domain. The contacts within the same continuous chromatin region or between distant separated chromatin regions are facilitated by loop structures. Chromatin loops are kilobases structure whose formation is assisted by chromatin architectural proteins, such as CCCTC-binding factor (CTCF) (once known as an insulator protein), cohesions and mediators. These loop structures allow the contacts between distal elements, such as promoters-enhancers, or certain DNA elements with transcriptional factors and cofactors, thereby playing an important role in gene transcription [START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF][START_REF] Pombo | Three-dimensional genome architecture: players and mechanisms[END_REF] . Indeed, TADs are viewed as units of gene transcription [START_REF] Ramírez | High-resolution TADs reveal DNA sequences underlying genome organization in flies[END_REF][START_REF] Jerkovic | Higher-Order Chromosomal Structures Mediate Genome Function[END_REF] .

Figure 1. Hierarchical organization of chromatin.

Genome is non-randomly placed in cell nucleus. Nucleosome fibers can form loops facilitated by CTCF and the cohesin complex. Chromatin loops are ordered in TADs or sub-TADs. TADs domains are located within different types of chromatin compartments: type A active chromatin and type B inactive chromatin. At the nuclear scale, each chromosome is arranged in a relatively specific place, designated as chromosome territories.

Chromatin packaging

In human cells, 2 meters of DNA must be tightly packaged into chromatin in order to fit into a micron-sized nucleus. Nucleosome is the basic unit of chromatin compaction. Each nucleosome is composed of 145-147 base pairs (bp) of DNA wrapped approximately 1.7 times around the histones core in a left-handed manner. Linker histone H1 interacts with both the dyad and entry/exit of DNA strand to further compact DNA. Adjacent nucleosomes are linked by DNA to form nucleosome arrays (so-called "beads on a string" structure observed under electron microscope). The next level of compaction is called "30nm fiber". Two models applying "30nm fiber" have been proposed: 1) the one-start solenoid where nucleosomes are arranged in a single helical stack, with the linker DNA slightly bent; 2) the two-start zig-zag structure where the neighboring nucleosomes extend back and forth, and are connected by a relatively straight linker. Though controversial, evidence indicated that both modes might exist in the real situation, the adoption of which depends on the length of linker DNA. A short linker DNA contributes to the formation of solenoid, while nucleosomes with long linkers tend to form zigzag structure. As aforementioned, the chromatin fiber could be spatially ordered in the nucleus with the assistance of other chromatin bound proteins. During the mitotic cell division, these noodle-like chromatin fibers could further be folded into a highly condensed state (Figure 2). Chromatin is highly packaged structure. Nucleosome is the basic unit of chromatin packaging. When linked, nucleosomes form the "beads on a string" structure under EM. Nucleosomes then form 30nm fiber chromatin, which can be further folded to highly condensed chromosome during metaphase. From Wikipedia.

Of note, recent studies revealed that the well-organized 30 nm fiber might merely reflect the artificial situation in vitro with low nucleosome concentration and low salt conditions. In vivo however, given 1) the physiological salt condition, 2) nucleosomes crowding [START_REF] Chereji | Ubiquitous nucleosome crowding in the yeast genome[END_REF] , and 3) taking into account that nucleosomes are irregularly positioned, etc., nucleosomes arrays are not likely to fold into 30nm fiber but rather are organized in clutches and stay in an amorphous form (Figure 1) [START_REF] Nozaki | Dynamic Organization of Chromatin Domains Revealed by Super-Resolution Live-Cell Imaging[END_REF][START_REF] Ricci | Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo[END_REF][START_REF] Baldi | Beads on a string-nucleosome array arrangements and folding of the chromatin fiber[END_REF] .

Nucleosome core particle structure

Structure of nucleosome core particle (NCP) has been determined with X-ray crystallography with near-atomic resolution (Figure 3) [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF][START_REF] Davey | Solvent Mediated Interactions in the Structure of the Nucleosome Core Particle at 1.9Å Resolution[END_REF] . Two copies of each H3, H4, H2A, and H2B form the disc-like histones core. Each core histone shares similar organization encompassing flexible Nand C-terminal tails and the histone fold, which consists of three α helices linked by two loops (α1-L1-α2-L2-α3) (Figure 3A). For each nucleosome, a segment of ~121bp DNA is organized around the histone fold, leaving ~13bp DNA at each entry/exit end. Interactions between core histones and DNA are mainly mediated by the conserved domains of core histones, where 7

unique DNA binding sites are harbored in every half of nucleosome particle, corresponding to the places where the DNA major groove faces the histone octamer. At the entry/exit of nucleosome, DNA is bound by the N-terminal α helices of histone H3. Within the histone core, two H2A-H2B heterodimers interact with the symmetric (H3-H4)2 heterotetramer through H2B-H4 four-helix bundle and H2A docking domain, the latter of which functions to lock H3-H4 by forming a β-sheet with C-terminus of H4, and can stabilize histone octamer by guiding H3 αN, which is interacting with DNA.

Core histones have long flexible tails containing alkaline lysine and arginine that can protrude NCP. For example, tails of H2B and H3 pass through DNA gyres and extend out of NCP randomly. Further, the N-terminal tail of H4 can contact the adjacent nucleosome via the acidic patch formed by H2A-H2B. Such interaction is crucial for the folding of nucleosome arrays and the high-order chromatin packaging. In the case of H2A, the C-terminus could serve as a docking domain that interacts with H3-H4, while the N terminal tail binds to the minor groove of DNA superhelix. Of note, these alkaline histone tails can be subject to a variety of covalent modifications. Apart from interacting with H4, the H2A-H2B acidic patch can act as a platform for many protein-nucleosomes complex interactions as well. respectively. Graphic from [START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF] (A) and from PDB:1KX5 17 (B).

Nucleosomes positioning

Around 75-90% of genomic DNA is wrapped in nucleosomes in eukaryotes. As introduced above, in order to be wrapped into nucleosomes, DNA must be bent around a histone octamer.

Since DNA sequences impact histone octamer binding stability, nucleosomes might be

A B
positioned across the genome depending on the genomic sequences. Indeed, Segal and colleagues demonstrated that dinucleotides facilitate the bending direction of DNA, where GC and AA, TT or AT are placed alternatively every 10 bp following DNA major and minor groove (Figure 4). Such model predicted around ~50% of nucleosomes positioning in yeast and chicken genome, indicative of a determinant role for the genomic sequence in directing nucleosome positioning [START_REF] Segal | A genomic code for nucleosome positioning[END_REF] . DNA is wrapped around a histone octamer following a 10bp-periodic sequences of GC/AA, TT or TA dinucleotides.

From [START_REF] Onufriev | The nucleosome: from structure to function through physics[END_REF] .

During the last decade, the heterogeneity of nucleosome positioning (referring to the relative position of nucleosomes with respect to certain genome DNA sequences) has been uncovered using high-throughput sequencing-based approaches. Up to now, the basic features of the nucleosome positioning landscape can be roughly summarized as 1) Silent genes harbor regularly-spaced, but poorly-positioned nucleosome arrays. 2) At active promoter and enhancer regions, nucleosomes are irregularly spaced. 3) At the 5' end of active genes immediately upstream and downstream of TSS, nucleosomes are strongly positioned and phased. A nucleosome-free region (NFR) at active gene TSS, separating the -1 and +1 nucleosomes, is observed (Figure 5) [START_REF] Baldi | Beads on a string-nucleosome array arrangements and folding of the chromatin fiber[END_REF] . The nucleosome positioning, spacing, and phasing are controlled by the internal property of genomic sequences and the action of chromatin remodelers. Since nucleosomes are the fundamental barriers for DNA accessibility, alterations of nucleosome arrays and the resultant chromatin re-organization might impact tremendously chromatin functions. Downstream of TSS of expressed genes, nucleosomes tend to be precisely phased, with strong positioning but less regular spacing (left panels). In contrast, in silent domains, nucleosome arrays are highly regular but poorly positioned to genomic DNA (right panels). From [START_REF] Baldi | Beads on a string-nucleosome array arrangements and folding of the chromatin fiber[END_REF] .

Gene transcription is the first highway to biological properties

Since 1950s, the discovery of DNA structure has paved the way towards understanding life.

Generations of researchers built their research on this discovery and established the basics of the modern biology. What we are learning in middle schools is the central dogma of molecular biology, describing the flow of genetic information passing from DNA to RNA and then to proteins. Here I will give a brief introduction on the first level of genetic information expression in this central dogma, which is gene transcription in eukaryotes.

Types of RNA polymerases

Transcription is catalyzed by RNA polymerases, which produce RNAs reverse complementary to DNA template. There are three types of RNA polymerases (Pol) in eukaryotes, RNA Pol I, II, and III, with each responsible for transcribing distinct types of RNAs. RNA Pol I produces large ribosomal RNA, RNA Pol II synthesizes messenger RNA and many non-coding RNAs, while RNA Pol III transcribes transfer RNA, small ribosomal RNAs and some other small RNAs.

Promoters and enhancers

Two key DNA elements involved in transcription are promoters and enhancers. Promoter is a region of DNA where RNA polymerases assemble to initiate transcription. Promoter regions comprise of core promoter, proximal promoter, and distal promoter. Of them, core promoters refer to the minimal DNA region that direct the precise initiation of transcription. Core promoters contain several sequence motifs, including TATA box (5'-TATAAA-3') (~ -30bp), initiator (± 3bp), and downstream core promoter element (DPE) (~ +30) etc., and are featured by a nucleosome-depleted region (NDR) on the TSS (transcription start site) in the case of active promoters [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] .

In metazoan, core promoters can be divided into three types according to their activation status, the motifs, and the local nucleosomes architecture. The first type is known as "disperse" and "broad" promoters mainly found in housekeeping genes. These promoters are featured by constitutively activated status, dispersed distribution of transcription initiation, and a typical NDR, and often overlap CpG islands. Second type is "sharp" "focused" promoter, which possesses sharp initiation, unprecise nucleosome positions, and often contain TATA box and initiator elements. These promoters are found in cell-type-specific genes that function during differentiation. The third type is "poised" promoter, which are covered by bivalent histone histone marks (both active H3K4me3 and repressive H3K27me3). These promoters are found in embryonic stem cells, where they control developmental transcription factors and ensure the correct expression of development-associated genes in certain tissue type [START_REF] Haberle | Eukaryotic core promoters and the functional basis of transcription initiation[END_REF] .

Enhancers are DNA sequences that can regulate the activity of their target promoters by recruiting transcription factors and cofactors. In metazoan, enhancers are relatively large elements that can be up to several hundred base pairs and are often placed distant from (e.g., kb) the target promoters. Currently, active enhancers are well-predicted by the coverage of H3K4me1 and H3K27ac and in the case of active enhancers, by the co-localization of p300/CBP [START_REF] Raisner | Enhancer Activity Requires CBP/P300 Bromodomain-Dependent Histone H3K27 Acetylation[END_REF] . Similar to promoters, active enhancers harbor NDRs. At both edges of NDR, enhancers DNA can be transcribed by RNA Pol II divergently to produce unstable enhancer RNAs (eRNAs) [START_REF] Andersson | Determinants of enhancer and promoter activities of regulatory elements[END_REF] . The existence of eRNAs reflects the active status of enhancers, although their actual functions are yet to be determined. It is noteworthy that specific subsets of large enhancers, known as locus control regions (also known as super-enhancers, SEs) were identified and became famous for their roles in controlling cell type-specific gene expression

or key oncogenic program. SEs are distinguished by the co-occurrence of transcription factors binding, Mediator and Pol II co-activators [START_REF] Smith | Enhancer biology and enhanceropathies[END_REF] .

In order to regulate distal promoters, enhancers should first physically contact distal promoters, which has been suggested to be enabled by the chromatin loop structure, where the interacting proteins, including Mediator complexes and cohesions, facilitate the enhancerpromoter proximity [START_REF] Lewis | Transcriptional control by enhancers and enhancer RNAs[END_REF] . The selectivity of enhancerspromoters communication are driven by topologically associating domains (TADs), promoterproximal tethering elements (PTEs), and the promoter DNA accessibility. More details can be found in [START_REF] Zabidi | Regulatory Enhancer-Core-Promoter Communication via Transcription Factors and Cofactors[END_REF] .

Procedure of RNA Pol II transcription

Processes of transcription can be divided into three stages, initiation, elongation, and termination (Figure 6). Transcription initiation is the process where RNA polymerase II Termination of transcription is less well-understood compared to initiation and elongation.

Once RNA polymerases pass through Poly A signal (5'-AAUAAA-3'), newly synthesized RNA will be released from RNA Pol II, and RNA Pol II per se is released from the DNA template.

Possible mechanisms are that gradual changes of CTD phosphorylation at distinct sites (e.g., Ser2 increase while Ser5 decrease) displaces RNA Pol II from DNA. Then recruited exonuclease Rat 1 pulls uncapped RNA out from RNA polymerase and degrades it. Transcription initiates with the assembly of Pre-initation complex (PIC) and the synthesis of nascent RNAs (A).

RNA pol II is then phosphorylated and escapes from promoter to start transcriptional elongation (B). Shortly after elongation (30-50nt), RNA pol II pauses, and the processive elongation requires its CTD phosphorylated by P-TEFb (C). Through all these processes, enhancer can access promoters and regulate each step of transcription.

TFs: Transcription factors. COFs: Cofactors. MED: Mediator. From 20 .

Histone post-translational modifications impact chromatin signaling

Chromatin serves as the template for various biological processes such as transcription, DNA repair, replication, and so forth. Modifications of histones, which are the key component of chromatin, are the major regulators in such processes. Histone post-translational modifications (PTMs) refer to the chemical moieties covalently added to amino acids on histone tails or histone folds. There are various types of histone PTMs, including the well-characterized acetylation, methylation, phosphorylation (Figure 7), and the atypical modifications, including short chain acylation (e.g., crotonylation, butyrylation, glutarylation, succinylation, etc.), serotonylation, lipidation, etc. Histone PTMs are reversible, with certain enzymes responsible for adding (termed as "writers") or removing the groups (termed as "erasers"). Histone PTMs can directly alter chromatin structure and organization, or more importantly, recruit effector

A B C
proteins (termed as "readers"). In this section, I'll have a general introduction about different modifications, their corresponding writers, erasers, readers, and the functions they exert.

Methylation

Histone methylation mainly occurs on the side chains of lysines or arginines on all histones (H1, H2A, H2B, H3, and H4). Lysines can be mono-(me1), di-(me2), or tri-methylated (me3) while arginines can be mono-or di-methylated. In the case of arginine di-methylation, two methyl groups can possibly be added symmetrically or asymmetrically to amino groups.

Histone methylation are catalyzed by histone methyltransferases (HMTs) with Sadenosylmethionine (SAM) as a group donor and can be removed by histone demethylases (HDMs) assisted by certain cofactors. Although histone methylation was identified in the 60s of last century, it was not until 40 years later when the first methylation modifier, SUV39H1

(KMT1A) was identified by Jenuwein and colleagues [START_REF] Rea | Regulation of chromatin structure by site-specific histone H3 methyltransferases[END_REF] . Today, two classes of lysine methyltransferases (KMTs) and one class of arginine methyltransferases (protein arginine methyltransferases, PRMTs) have been identified. Depending on the catalytic domains they possess, KMTs can further be divided into 1) SET domain containing methyltransferases, which defines the first and the major class of KMTs, and 2) KMTs which does not have a SET domain and is represented by DOT1L protein in human [START_REF] Black | Histone lysine methylation dynamics: establishment, regulation, and biological impact[END_REF] .

The first member of lysine demethylases (KDMs) LSD1/KDM1A was identified by Yang Shi and colleagues and was characterized as a FAD-dependent amine oxidase that demethylates H3K4me1 and K3K4me2. A few years later the second class of KDMs were identified and designated as jumonji C (JmjC)-domain containing protein, which use α-ketoglutarate (α-KG), oxygen and Fe (II) as cofactors to oxidize methyl groups and release formaldehyde.

Catalytic activity of HMTs and HDMs is dependent on both the site and the degree of methylation. For instance, KMT1A/B (SUV39H1/2) catalyze di-and tri-methylation of histone H3 at K9 from monomethylated state, while G9a and GLP (G9a-GLP) catalyzes mono-and dimethylation of H3K9 [START_REF] Hu | Large chromatin domains in pluripotent and differentiated cells[END_REF] . Moreover, since some of the HMTs and HDMs are formed in protein complexes, the additional components might indeed impact the activity or specificity of the catalytic subunit. For example, SET domain containing core enzymes can only catalyze monoand di-methylation of H3K4, while the catalysis of higher level of methylation, H3K4me3, requires the presence of holo-enzymatic complexes, which are comprised of additional interacting partners (e.g., WRAD) [START_REF] Ernst | WRAD: enabler of the SET1-family of H3K4 methyltransferases[END_REF] . In addition, Polycomb repressive complexes 2 (PRC2) is a well-studied polycomb group (PcG) family protein that serve as writer for mono-, di-, and trimethylation of H3K27. Apart from the catalytic subunit EZH1/2, PRC2 complexes comprise additional components, including SUZ12 and EED are required for the enzymatic activity, as well as accessory subunits which are either important for enhancing activity of PRC2 complexes (e.g., AEBP2 and JARID2) or might play a role in the recruitment of PRC2 to specific genomic loci (e.g., PCL protein) [START_REF] Holoch | Mechanisms Regulating PRC2 Recruitment and Enzymatic Activity[END_REF][START_REF] Healy | PRC2.1 and PRC2.2 Synergize to Coordinate H3K27 Trimethylation[END_REF] . Histone methylation does not alter the charge of histone protein but can impact the size and hydrophobicity of the amino acids, thereby directly influencing nucleosome structure by interfering with histone-histone or histone-DNA interactions. More importantly, histone methylation has been suggested to recruit effector proteins. "Readers" of methylation include PHD, chromo, PWWP, WD40, etc. domains containing proteins that recognize the specific methyl groups and mediate certain outcomes. To speak roughly, in the case of gene transcription, methylation at H3K4 and H3K79 marks active transcription, in contrast, methylation at H3K9, H3K27, and H4K20 is associated with gene silencing.

Different histone methylation marks are distributed in different regions of the genome.

Thanks to the advancement of high-throughput sequencing technology during the last two decades, researchers have mapped the genomic distribution of various site-specific methylations and have summarized their coverage patterns in cis-elements (e.g., promoters, enhancers), gene regions (e.g., exons, introns, TSS, repetitive regions), and their onsite specific roles. For instance, it is well-known that H3K4me3 is enriched in active and poised promoters, whereby it could help to recruit RNA pol II [START_REF] Van Ingen | Structural insight into the recognition of the H3K4me3 mark by the TFIID subunit TAF3[END_REF] . In addition, H3K27me3 and H3K9me3 are hallmarks of facultative and constitutive heterochromatin respectively, and are associated with gene silencing [START_REF] Hu | Large chromatin domains in pluripotent and differentiated cells[END_REF][START_REF] Wiles | H3K27 methylation: a promiscuous repressive chromatin mark[END_REF] .

Furthermore, mechanisms concerning the methylation targeting have begun to emerge.

Specific KDMs and KMTs could directly be recruited to certain loci by transcriptional factors or RNA pol II, DNA elements, noncoding RNA etc. [START_REF] Black | Histone lysine methylation dynamics: establishment, regulation, and biological impact[END_REF] . Functionally, histone methylation can either activate or repress gene transcription depending on the different types and their genomic locations, as well as can play a role in RNA splicing, genomic stability, and DNA damage response etc. [START_REF] Hyun | Writing, erasing and reading histone lysine methylations[END_REF] . The graph summarizes the genomic distribution of different histone methylation, methylation modifiers, and their regulation in transcription. In general, H3K9me3, H3K27me3, H4K20me3 marks inactive genes. In contrast, H3K27me1 enriches in gene bodies of active genes. H3K36me1 is distributed in intergenic regions whereby it might confer to the repression of cryptic transcription. More details are reviewed in [START_REF] Black | Histone lysine methylation dynamics: establishment, regulation, and biological impact[END_REF] . From [START_REF] Black | Histone lysine methylation dynamics: establishment, regulation, and biological impact[END_REF] .

Of note, different histone methylation marks can interfere with the establishment of the other methylation marks, as well as other epigenetic modifications. For example, H3K27 methylation can be disturbed by active histone marks (e.g., H3K27ac, H3K4me3, H3K36me3 etc.). The underlying mechanism is the exclusion of PRC2 from specific chromatin loci [START_REF] Wiles | H3K27 methylation: a promiscuous repressive chromatin mark[END_REF][START_REF] Laugesen | Molecular Mechanisms Directing PRC2 Recruitment and H3K27 Methylation[END_REF] . In addition, DOT1L-dependent H3K79me and SET1-dependent H3K4me can be activated by H2BK120 mono-ubiquitination [START_REF] Worden | Activation and regulation of H2B-Ubiquitin-dependent histone methyltransferases[END_REF] . Such crosstalk adds complexity of interpreting the function of histone code.

Phosphorylation

Histone phosphorylation corresponds to the transfer of phosphate group from ATP to amino acids, in most cases, threonine and serine of histone tails. Not surprisingly, histone phosphorylation is established by kinases and is removed by phosphatases. Histone phosphorylation has been extensively studied and has been implicated in DNA damage repair, cell cycle progression, gene transcription and chromatin architecture.

H2AX phosphorylation at Ser139 (γH2AX) is one of the early events being identified in DNA damage response process [START_REF] Sharma | Histone H2AX phosphorylation: a marker for DNA damage[END_REF] . Upon DNA double strand breaks (DSBs), H2AX is phosphorylated by PI3K family members (e.g., ATR, ATM), serving as platform for the accumulation and retention of MRN, MDC1, and 53BP1 components at DSB sites, which are responsible for DNA damage repair. After proper DNA repairment, surviving cells should exit checkpoint and recover cell cycle progression. This process is possibly regulated by the phosphatase Wip1 which dephosphorylates γH2AX and could be activated by phosphorylation of H4 threonine 80 (H4T80P) in S.cerevisiae [START_REF] Millan-Zambrano | Phosphorylation of Histone H4T80 Triggers DNA Damage Checkpoint Recovery[END_REF] .

Another important function of histone phosphorylation is chromosome architecture, which is exemplified by well-characterized H3S10P as well as H3S28P, which are catalyzed by Aurora B, IKK, Rsk2, AKT and are important for chromatin condensation and segregation during mitosis or meiosis. In addition, phosphorylation at H3T118 has been reported to destabilize nucleosomes [START_REF] North | Histone H3 phosphorylation near the nucleosome dyad alters chromatin structure[END_REF] . In addition to core histones, phosphorylation of H1 was believed to influence nucleosome structure and higher-order chromatin compaction [START_REF] Izzo | The role of linker histone H1 modifications in the regulation of gene expression and chromatin dynamics[END_REF] .

Histone phosphorylation is also involved in gene transcription through an interplay with other histone marks. For example, H3S10P contributes to gene transcription of 14-3-3 family proteins through promoting histone acetylation [START_REF] Li | The histone modifications governing TFF1 transcription mediated by estrogen receptor[END_REF] . Furthermore, recent study revealed that H3.3 phosphorylation can activate p300 in trans, which is responsible for H3K27ac at enhancers and regulates differentiation-specific gene expression [START_REF] Martire | Phosphorylation of histone H3.3 at serine 31 promotes p300 activity and enhancer acetylation[END_REF] . H4Y88P, which is catalyzed by ACK1/TNK2, is crucial to promote androgen receptor -mediated transcription by recruiting WDR5/MLL2, which subsequently catalyzes H3K4me3 [START_REF] Mahajan | ACK1/TNK2 Regulates Histone H4 Tyr88-phosphorylation and AR Gene Expression in Castration-Resistant Prostate Cancer[END_REF] .

Histone phosphorylation is also associated with cell stress and apoptosis. H2BSer36

phosphorylation was suggested to be involved in certain genes transcriptional regulation responsive to cell stress, while H2BSer14 phosphorylation catalyzed by PKA and PKC mediates DNA fragmentation and cell apoptosis [START_REF] Banerjee | A peek into the complex realm of histone phosphorylation[END_REF] .

Acetylation

Acetylation occurs at the ε-N of lysine side chains. While all the histones can be acetylated, acetylation on H3 and H4 is more extensively studied than on H2A or H2B. Traditionally, most of the modifications were considered at the lysine residues of N-terminal tails. However, recent studies have also included the globular domains of histones, where acetylation could probably mediate a different functional output.

Histone acetylation exhibits a high turn-over rate, with half-life (t1/2) ranging from minutes to hours [START_REF] Jackson | Studies on highly metabolically active acetylation and phosphorylation of histones[END_REF][START_REF] Duncan | Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts[END_REF] . The dynamics of histone acetylation is regulated by histone acetyltransferases (HATs), which add acetyl groups onto histones using acetyl-CoA as donors, and by histone deacetylases (HDACs), which remove acetyl group from modified lysine and release acetate, as well as their cofactors.

Histone acetylation is one of the most prominent epigenetic code underpinning active transcription and is involved in various biological processes. In general, acetyl group neutralizes the positive charge of lysine residue and reduces interactions between histones and DNA as well as between adjacent nucleosomes. More importantly, histone acetylation recruit chromatin binding factors that either lead to transcriptional outputs or chromatin remodeling.

Herein, I'll give a brief introduction about the HATs, HDACs, readers, and the functional outputs of histone acetylation.

HATs

Histone acetyltransferases (HATs) are mainly classified into GNAT (GCN5-related Nacetyltransferases), MYST (MOZ, Ybf2/Sas3, Sas2, Tip60), p300/CBP as well as other undefined families according to their sequence homology and structural similarity. In metazoan,

HATs often form complexes with other protein partners, wherein the classical HATs serve as the catalytic center. HATs complexes can have distinct specificity over nucleosomal histones compared to the free histones. It is noteworthy that different HAT complexes often have overlapping substrates in vitro and in vivo. Or JADE1/2/3.

HBO1-BRPF complex

preferentially acetylate H3, especially at K14 and K23.

HBO1-JADE complex have specificity for H4K5, K8, and K12 [START_REF] Doyon | ING tumor suppressor proteins are critical regulators of chromatin acetylation required for genome expression and perpetuation[END_REF][START_REF] Tao | Structural and mechanistic insights into regulation of HBO1 histone acetyltransferase activity by BRPF2[END_REF][START_REF] Lalonde | Exchange of associated factors directs a switch in HBO1 acetyltransferase histone tail specificity[END_REF] .

DNA replication licensing and transcription activation, through promoting pre-replicative complexes loading or by acetylating histones at TSS and intragenic regions respectively [START_REF] Macpherson | HBO1 is required for the maintenance of leukaemia stem cells[END_REF][START_REF] Lan | Deciphering structure, function and mechanism of lysine acetyltransferase HBO1 in protein acetylation, transcription regulation, DNA replication and its oncogenic properties in cancer[END_REF] . KAT3B p300, EP300 H3K18, H3K27, H4 N-terminal tail / All four core histones, among which H3K18ac, H3K27ac are preferred [START_REF] Weinert | Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome[END_REF] .

Transcriptional activation, the presence of p300 is one of the prominent features of active enhancers [START_REF] Wang | Structures of KIX domain of CBP in complex with two FOXO3a transactivation domains reveal promiscuity and plasticity in coactivator recruitment[END_REF][START_REF] Zhang | The ZZ domain of p300 mediates specificity of the adjacent HAT domain for histone H3[END_REF][START_REF] Holmqvist | Genomic occupancy of the transcriptional co-activators p300 and CBP[END_REF] . Acetylate a variety of non-histone substrates. Interact with a broad list of proteins 66 .

KATA3A

CBP, CREBBP 1.3.3.2 HDACs Histone deacetylases (HDACs) remove acetyl group from ε-amino of lysine chain, producing unmodified lysines and acetate. Mammalian HDACs consist of 18 members, which can be classified into four families based on their sequence similarity and their yeast orthologues (Figure 10). Class I HDACs contains HDAC1, 2, 3, 8, which possess homology to yeast RPD3 protein; Class II HDACs are homologous to yeast HDA1, and can be further subclassified into IIa (HDAC 4, 5, 7, 9) HDAC1/2 (Class I) are exclusively located in nucleus due to the presence of nuclear localization signal (NLS) but lack of nuclear export signal (NES). HDAC3 who possesses both NLS and NES and HDAC8, although located predominantly in nucleus, can be found in cytoplasm [START_REF] Chini | HDAC3 is negatively regulated by the nuclear protein DBC1[END_REF][START_REF] Yang | Functional domains of histone deacetylase-3[END_REF] . HDAC1 and 2 share common domains such as IAC (E/D)E motif, which is important for interacting with pocket proteins. HDAC 1 and 2 can dimerize and co-exist in many multiple protein complexes such as SIN3, NuRD, CoREST etc., HDAC3 has been characterized to interact with N-CoR/SMRT complexes through their deacetylase-activating domain (DAD), which is required for activating its inert catalytic activity [START_REF] You | Nuclear receptor co-repressors are required for the histone-deacetylase activity of HDAC3 in vivo[END_REF][START_REF] Brunmeir | Histone deacetylase HDAC1/HDAC2-controlled embryonic development and cell differentiation[END_REF] . HDAC8 is Xlinked in human and can deacetylate both histone and non-histone substrates. Its catalytic activity has been characterized to be retained with many other divalent metal ions in addition to Zn 2+ 71, 72 .

Class IIa family of HDACs possess both NLS and NES and shuttle dynamically between nucleus and cytoplasm. Whereas, class IIb HDACs are predominately located in cytoplasm due to NES. Subcellular localization of class IIa HDACs are regulated by phosphorylation of several conserved sites and the interaction proteins. For instance, phosphorylation of HDAC4 (at Ser246, 467, 632), HDAC5 (at Ser259, 497, 661), HDAC7 (at Ser155, 178, 181, 321, 344, 446,479) and HDAC9 (at Ser220, 451, 611) promotes binding of 14-3-3 family proteins, which in turn either mask NLS from importin α or unmask NES from Exportin 1 receptor (CRM-1), thereby promotes cytoplasmic localization [START_REF] Clocchiatti | Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis[END_REF] .

Class IIa HDACs exhibit very weak deacetylase activity in vitro and their natural substrates are yet to be determined. The reduced activity is due to the amino-acid substitution from conserved histidine to tyrosine within the catalytic domain compared to class I and IIb [START_REF] Asfaha | Recent advances in class IIa histone deacetylases research[END_REF] . Despite their weak deacetylase, class IIa HDACs interact with many transcriptional factors such as MEF2 proteins, Runx, NF-AT3c etc…, thereby act as transcriptional corepressors [START_REF] Yang | Class II Histone Deacetylases: from Sequence to Function, Regulation, and Clinical Implication[END_REF] .

HDAC6 contains tandem catalytic domains, designated as CD1 and CD2, which display distinct catalytic activity. CD2 has broad substrate specificity over acetylated lysines, while CD1 prefers C-terminal acetyl-lysine residues [START_REF] Hai | Histone deacetylase 6 structure and molecular basis of catalysis and inhibition[END_REF] . Other domains possessed by HDAC6 include a ZnF ubiquitin binding domain, which binds ubiquitin and regulates various ubiquitindependent functions, and a SE14 which promotes cytoplasmic retention of HDAC6. HDAC6

can deacetylate important non-histone proteins such as α-tubulin, HSP90 and cortactin [START_REF] Li | Histone deacetylase 6 in cancer[END_REF] .

HDAC10 is located both in nucleus and cytoplasm. It contains a conserved HDAC domain similar to CD1 of HDAC6 and a leucine rich motif with elusive function. HDAC10 is characterized as a weak lysine deacetylase but has a robust polyamine deacetylase activity with preference for N8-acetylspermidine [START_REF] Hai | Histone deacetylase 10 structure and molecular function as a polyamine deacetylase[END_REF] . Among the nuclear members, SIRT1 is the most well-studied member of sirtuin family and was firstly identified to deacetylate H3K9ac, H4K16ac and H1K26ac [START_REF] Vaquero | Human SirT1 interacts with histone H1 and promotes formation of facultative heterochromatin[END_REF] . Later studies characterized SIRT1 as a deacetylase of non-histone substrates, including transcriptional regulators, chromatin modifiers, oncoproteins etc… SIRT1 also plays a role in gene transcription, cell senescence, stress response and energy homeostasis [START_REF] Chen | SIRT1 and aging related signaling pathways[END_REF] . SIRT2 preferentially deacetylates H3K56ac and H4K16ac and other non-histone substrates (e.g., Tubulin, FOXO3A, p53 etc.) [START_REF] North | The Human Sir2 Ortholog, SIRT2, Is an NAD+-Dependent Tubulin Deacetylase[END_REF][START_REF] Vaquero | SirT2 is a histone deacetylase with preference for histone H4 Lys 16 during mitosis[END_REF] . SIRT3-5 are located in mitochondria and are controlling the mitochondrial metabolism by deacetylating many enzymes involved in TCA, OXPHOS etc.

Protein members of

SIRT6 was first identified as a mono-ADP-ribosyl-transferase using NAD+ as a substrate.

Its known substrates are SIRT6 itself, PARP1 and KAP1. Its auto-deacetylation leads to autoregulation of its own activity in DNA damage repair. As a deacetylase, current evidence shows that H3K9ac, H3K18ac and H3K56ac are the targets of SIRT6, the removal of these marks is associated with gene silencing and chromatin compaction [START_REF] Chang | SIRT6, a Mammalian Deacylase with Multitasking Abilities[END_REF][START_REF] Tasselli | SIRT6: Novel Mechanisms and Links to Aging and Disease[END_REF] .

SIRT7 has high specificity for H3K18ac but displays weak deacetylase activity compared to other nuclear members [START_REF] Wu | Advances in Cellular Characterization of the Sirtuin Isoform, SIRT7[END_REF] .

HDAC11 is the most recently discovered histone deacetylase and is designated to a new distinct class of HDACs, class IV. Studies found that location of HDAC11 varies in different cell type and with response to environmental cues. Although identified as robust deacetylase, HDAC11 displayed much more efficient deacylase than deacetylase activity. Recently increasing evidence has highlighted its potential role in physiology and pathophysiology [START_REF] Liu | HDAC11: a rising star in epigenetics[END_REF][START_REF] Gao | Cloning and functional characterization of HDAC11, a novel member of the human histone deacetylase family[END_REF][START_REF] Yanginlar | HDAC11 is a regulator of diverse immune functions[END_REF] .

Readers of histone acetylation

Histone acetylation was traditionally viewed as a robust chromatin regulator altering the electrostatic potential of nucleosomal histones. However, latter studies argued that this mechanism is far from enough to explain the diverse functions of histone acetylation. Indeed, later studies revealed that the major players mediating functional output of histone acetylation are factors recruited by histone marks, which are called "readers". According to the modular domains they have, readers for acetylated histones are classified into several families, including bromodomain (BrD), PHD finger domain, YEATS (Yaf9, ENL, AF9, Taf14, and Sas5) domain, and non-canonical bromodomain-containing proteins [START_REF] Gong | Acetylation Reader Proteins: Linking Acetylation Signaling to Genome Maintenance and Cancer[END_REF] .

Bromodomain is the principal structural module that recognizes acetyl-lysine which can be found in many chromatin and transcription associated proteins. Although present in diverse proteins and have sequence variations, BrD modules share conserved structural fold comprising a four-helical (αZ, αA, αB, αC) bundle linked by highly variable loops (ZA and BC), which form a hydrophobic pocket (Figure 12) [START_REF] Dhalluin | Structure and ligand of a histone acetyltransferase bromodomain[END_REF][START_REF] Filippakopoulos | Histone recognition and large-scale structural analysis of the human bromodomain family[END_REF] . Binding between bromodomain and acetylated lysine occurs in the way that N-acetylated lysine inserts into the pocket formed by ZA and BC loops. This interaction is stabilized by the hydrogen bond between the amide nitrogen of the conserved asparagine (Asn803 in PCAF) and the carbonyl oxygen of acetyl-lysine. Water molecules participate in the formation of hydrogen bond network as well, especially between carbonyl group of acetyl-lysine and conserved tyrosine of BrD [START_REF] Owen | The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase Gcn5p[END_REF] . Some of these BrD containing proteins harbor atypical bromodomains (aBrD), where the conserved Asn is replaced by Tyr, Thr or Asp, and these aBrDs may not be able to bind acetyllysine [START_REF] Zaware | Bromodomain biology and drug discovery[END_REF][START_REF] Mujtaba | Structure and acetyl-lysine recognition of the bromodomain[END_REF] . In human genome, 61 individual BrDs were found in 42 bromodomain containing proteins [START_REF] Filippakopoulos | Histone recognition and large-scale structural analysis of the human bromodomain family[END_REF] .

Based on their major functions, bromodomain-containing proteins can be classified into 9 BrDs in tandem could present an increased affinity for multiple acetylated sites. For example, two bromodomains in TAF1 form a V-shaped structure, with two binding pockets packed close (~25Å) to each other and thereby enhances the affinity to di-acetylated or tetra-acetylated histone (H4K5/8/12/16) than mono-acetylated histone [START_REF] Jacobson | Structure and function of human TAFII250 double bromodomain[END_REF] . BET family proteins, which possess double BrDs preferentially bind hyperacetylated chromatin, with each BrD recognizing distinctive acetylated sites [START_REF] Leroy | The double bromodomain proteins Brd2 and Brd3 couple histone acetylation to transcription[END_REF][START_REF] Schroder | Two-pronged binding with bromodomain-containing protein 4 liberates positive transcription elongation factor b from inactive ribonucleoprotein complexes[END_REF] . Of note, the first bromodomain (BD1) of Brdt can accommodate di-acetylated lysines (H4K5acK8ac) simultaneously, while the second bromodomain (BD2) interacts with another acetylated site within the same or in different proteins 100 .

PHD is a well-known histone methylation reader domain, therefore PHD-BrD tandem containing proteins tend to associate with chromatin presenting specific multivalent patterns.

For example, it is reported that coupling of the second PHD and the BrD of BPTF, enhances its binding to bivalent H4K16ac and H3K4me3 in a trans-histone way within the same nucleosome compared to H3K4me3 alone or to the combination of H3K4me3 and H4K12ac/H4K20ac 101 . In addition, PHD-BrD region of TRIM24 recognizes combined unmodified H3K4 and H3K27ac

in cis respectively, within the same histone tail 102 . Moreover, ZMYND8 has triple PHD-BrD-PWWP reader domains, which form a structural cassette that concomitantly interacts with histone and DNA driven by H3K14ac 103 .

BrD-containing proteins such as p300/CBP, MLL, ASH1L, GCN5L2 possess additional catalytic domains and are designated as "writers that read". Catalytic modules like acetyltransferase (ATs), methyltransferase (e.g., SET) domains facilitate the introduction of histone modifications to specific genomic sites directed by BrD recognition. For instance, bromodomain of p300/CBP binds acetylated histone tails, facilitates their recruitment to chromatin and enhances diverse core histone acetylation and gene expression [START_REF] Zhang | The ZZ domain of p300 mediates specificity of the adjacent HAT domain for histone H3[END_REF][START_REF] Filippakopoulos | Histone recognition and large-scale structural analysis of the human bromodomain family[END_REF]104,105 .

Additionally, GCN5 bromodomain was required to direct its site specific histone acetylation 106 .

As a subunit of MOZ HAT complexe, the bromodomain containing protein BRPF directs the catalytic core of the complex, MOZ, to histone tails through interaction with acetylated histones via its BrDs 107 . BrDs also present in proteins involved in chromatin structural regulation. These involve chromatin remodelers and AAA ATPase proteins (e.g., ATAD2, ATAD2B). For examples, SMARCA2, SMARCA4, BRD7, BRD9 and PBRM1 are components of SWI/SNF complexes while BAZ1A/B, BAZ2A/B, BPTF and CECR2 present in ISWI complexes. BrDs in chromatin remodeling complexes associate with chromatin regions harboring acetylated histone and might contribute to their recruitment and local chromatin remodelling 108 . Bromodomain of ATAD2 was uncovered to bind diacetylated newly synthesized histones (H4K5acK12ac) and might play a role in the replication-coupled chromatin reassembly 109 . Study on Abo1 (yeast homologue of ATAD2) revealed that BrD and AAA+ pore of Abo1 bind with H3 N-terminal tail and load histone H3-H4 onto DNA for nucleosome assembly 110,111 .

Plant homeodomain (PHD) finger domain is a small module comprising of around 50-80 amino acid residues and a zinc binding motif. This domain is known to bind the N terminal tail of histone H3, especially its methylated sites 112 . However, evidence suggests that PHD finger also displays acetyl-lysine recognition capability, as was first exemplified by double PHD finger domain of DPF3b binding to H3K14ac 113,114 . Additionally, PHD domains in MOZ/MORF are able to recognize H3K9ac and H3K14ac, which facilitates their association with chromatin, which is important for gene transcription 115,116 . Lately, PHD domains of MLL4 and MLL3 were identified to specifically recognize H4K16ac. This domain might mediate functional link between these proteins, MOF and H4K16ac 117 . YEATS (Yaf9, ENL, AF9, Taf14, Sas5) domain is an evolutionally conserved module from yeast to human and is found in many chromatin-associated protein complexes. The structure of YEATS domain was characterized by Yaf9, a yeast member of YEATS domain containing protein, where an immunoglobulin, β sandwich fold comprising of 8 antiparallel β-strands capped by 2 short α-helices at one end is adopted 118 . YEATS domain recognizes acetyl-lysine through a serine/threonine-lined aromatic sandwiching cage, whose binding are facilitated by relayed hydrogen bonding and multiple sets of CH-π interactions (Figure 13) 119 . Human genome encodes four YEATS domain-containing proteins, including AF9, ENL, GAS41 (also known as YEATS4) and YEATS2, all of which are implicated in cancer development. Specificity for acetylated histone H3 was observed among these members, with AF9/ENL binding H3K9ac, H3K18ac and H3K27ac, YEATS2 recognizing H3K27ac, and GAS41 preferentially binds acetylated H3K27, H3K14 and H3K18 as well as diacetylated H3, following protein dimerization [119][120][121][122][123] . Of note, recent studies found that YEATS domains act as specific acylation readers (e.g., crotonylation) and this will be discussed later in this essay. 1.3.3.4 Functional output of histone acetylation Since its identification, histone acetylation has been extensively implicated in transcription, replication, and damage repair by directly regulating chromatin structure or by serving as platforms for recruiting chromatin modifying complexes.

Histone acetylation and Chromatin structure

Histone acetylation can alter the biochemical or physical properties of histones, therefore impacting DNA-histone, histone-histone interaction, and nucleosome dynamics as well as higher-order chromatin compaction. For instance, H3K56ac, H3K64ac, H3K122ac were suggested to disfavor the histone-DNA contacts within nucleosomes and enhance chromatin dynamics 124 . In addition, histone tails play a crucial role in inter-nucleosome interaction and chromatin fiber compaction. Bascom and colleagues revealed that acetylation at tails of all four core histones further abolishes tail-tail interactions and decreases long-range contacts 125 . Indeed, the most prominent role of H4 tail in nucleosome structure is that it interacts with acid patch of adjacent nucleosomes, acetylation at H4 tail in turn blocks such interaction and inhibits the compaction of 30-nm fiber 126,127 .

Histone acetylation further recruits and cooperates with chromatin interacting complexes to modify chromatin structure. H4K16ac inhibits nucleosome mobilization by ATP-utilizing chromatin assembly and remodeling enzyme ACF 127 . H3K56ac attracts histone chaperones such as CAF1 and Nap1, facilitating nucleosome assembly or enhancing nucleosome opening respectively 128 . H3K115 and H3K122 acetylation cooperate with SWI/SNF and RSC complexes to enhance nucleosome disassembly 129 . These are some non-exhaustive examples of the impact of histone acetylation on nucleosome/chromatin structure/organization, but there are other mechanisms that would be too long to discuss here.

Transcription activation

In general, most TFs binding free DNA need to overcome the chromatin barrier to gain access to DNA template. Chromatin structure modifications that modulate DNA accessibility can largely influence transcription initiation. Not surprisingly, less compacted chromatin at promoter and enhancer regions, which for example, are covered by specific histone modifications (e.g., H3K27ac, H4K16ac, etc.) or constituted of certain histone variants (e.g., H3.3, etc.), promotes TF binding and transcription initiation. Indeed, histone acetylation is viewed as general mark of active transcription. Consistently, nucleosomes flanking TSSs are in hyperacetylated states in actively transcribed genes 130 .

In addition, histone acetylation can recruit transcriptional factors and cofactors, thereby contributing to transcriptional activation. For example, BRD4, an important histone acetylation reader, can bind promoters and recruit P-TEFb, thereby promoting CDK9-dependent, and, transcription elongation 131,132 . Additionally, H3K9ac was described as recruiting super elongation complex and enhancing Pol II pause release 133 . Histone variants and their modifications also play a role since for instance, H2A.Zac was found essential for enhancer RNA transcription, enhancer promoter interaction and RNA Pol II recruitment 134 .

Histone acetylation is a highly dynamic mark Histone acetylation is a highly dynamic modification. Early in the 1980s, researchers found that the half-life (t1/2) of histone acetylation ranged from minutes to hours [START_REF] Duncan | Kinetics of histone hyperacetylation and deacetylation in human diploid fibroblasts[END_REF] . Lately, using timeresolved approach, Weinert and colleagues identified that a subset of p300/CBP regulated sites of histone acetylation has very fast turnover rate, with half-lives of less than 1 hour for acetylation on 12 histone sites (including H2B N-terminal acetylation, H3K18/36ac and H4K8ac) [START_REF] Weinert | Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome[END_REF] . Although theoretically this dynamics could result from histone turnover or continuous action of HATs and HDACs, the latter was acknowledged as the predominant cause 135 . Indeed, histone modifications and histone turnover seem to be relatively independent events 136 .

Acetylation dynamics seems to be important for active transcription. Through genome-wide mapping, Wang and colleagues identified that both HATs and HDACs are enriched on highly active genes in human cells. They demonstrated that, at active genes, HDACs could indeed, remove acetylation marks in order to keep the acetylation at levels supporting transcription, while preventing promiscuous intiation 137 . Gryder and colleagues further illustrated that H3K27ac distribution at enhancer regions is balanced by an interplay between p300 and HDAC, and this balance is required for core regulatory transcription factors (CR TFs)-mediated active transcription in rhabdomyosarcoma. Hyperacetylated state might disrupt core gene regulatory network by removing RNA Pol II from core regulatory genetic elements, thereby decreasing CR TFs-mediated transcription 138 .

Acylation

Apart from the aforementioned classical well-studied modifications, a growing body of studies have uncovered a repertoire of short chain non-acetyl acylations during last two decades, including propionylation (pr), crotonylation (cr), butyrylation (bu), succinylation (succ), βhydroxylbutyrylation (bhb), 2-hydroxyisobytyrylation (hib), malonylation (mal), glutarylation (glu), formylation (fo), lactylation (lac) etc…, which will be designated as acylation hereafter (Figure 14) [139][140][141][142] . Histone acylations are dynamic and evolutionally conserved modifications, which can be detected in various species ranging from yeasts to human. Compared to acetylation, these acylations are much less abundant, with most of them covering robustly 1-5% of histone H3 and H4, in striking contrast to 15-30% by acetylation 143 .

Similar to acetylation, these novel acylations appear to be driven by the corresponding CoA thioesters (acyl-CoA). Not surprisingly, acyl-CoAs level is only 1/10-1/100 of acetyl-CoA in vivo, in agreement with the low abundance of histone acylations. Acyl groups can be added onto histones through both enzymatic and non-enzymatic chemical reactions in vitro. Of those types of acylations, acidic (malonyl, succinyl and glutaryl) and β-hydroxybutyryl moieties were reported to favor non-enzymatic reaction to a greater extent than the others.

Common HATs and HDACs can add or remove acylations as well, but display different (lower, in general) efficiency compared to acetylation. An additional similarity between these acylations and acetylation is that, these acylations are distributed at TSS and enhancer regions and are associated with active gene transcription 139,144,145 . Currently, the function of acylations are, to a great extent, far from being understood. Apart from acetylation, lysines can be modified by other groups including formyl, propionyl, butyryl, crotonyl, βhydroxybutyryl (bhb), 2-hydroxyisobutyryl, benzoyl, malonyl, sccuinyl, glutaryl, etc. Among them bhb can be R or S forms of enantiomers. From 146

HATs for acylations

Simithy and colleagues assessed the capability of seven common HATs (i.e., p300/CBP, GCN5, PCAF, Tip60, MOF, NatA) catalyzing diverse acylations in vitro using mass spectrometry and found that HATs have lower affinity for longer chain acylations compared to the two-carbon acetylation. Among the acylations they tested, most HATs preserve catalytic ability for butyrylation and propionylation, but are less efficient in producing acidic acylations (e.g., malonyl, succinyl and glutaryl), in branched chain acylations (e.g., β-hydroxybutyryl) and in crotonylation 143 propionylation) can stimulate transcription to a comparable extent as acetylation 144,145,169 . Such correlation with active transcription is also observed in vivo, where studies documented that acylations (i.e., butyrylation, crotonylation, β-hydroxybutyrylation, 2-hydroxyisobutyrylation, lactylation) are mainly enriched at active TSS regions and enhancer regions 140-142, 144, 145 .

Accordingly, to increase the level of acylations by ectopic expression of HATs, or by exposure to higher concentration of acyl-CoAs, or to decrease them by HDACs or by decreased acyl-CoA concentration resulted in enhanced transcriptional activation, or gene silencing respectively 152,154 .

Although acylations were firstly depicted to co-occur with acetylation and have similar implications in active transcription, these marks were suggested to play active roles in gene transcription, and are not merely by-products of active transcription. In spermatid cells, 29% of H4K8hib marks testis specific genes and better defines highly-expressed genes than H4K8ac 142 .

In spermatogenic cells, Kac and Kbu are found to co-occur at TSS regions of highly expressed genes, however Kbu can compete with Kac and prevent Brdt from binding and might fine-tune both specific gene expression program as well as histone eviction mode during late spermatogenesis 145 . In a study of histone crotonylation by p300/CBP, introduction of p300/CBP mutants, which retain HCT but not HAT can enhance transcriptional activation under the physiological condition of low concentration of crotonyl-CoA 152 . However, another study of histone crotonylation by p300/CBP indicates that HCT and HAT activities of p300 could not be easily separable 148 .

The non-redundant role of acylations is further supported by the identification of preferential histone acyl readers and writers. For instance, YEATS domain of AF9 preferentially recognizes crotonylation that links Kcr with transcriptional activation 163 . Furthermore, Taf14 YEATS domain is essential for transcriptional repression of growth-related genes under nutrient limitation in parallel with increased H3K9cr 170 . GCN5 (also known as KAT2A) preferentially binds succinyl-CoA over acetyl-CoA. When in complex with α-KGDH (α-ketoglutarate dehydrogenase), it can act as succinyltransferase and catalyze H3K79succ, which is enriched on TSS regions and is required for gene expression and cancer proliferation 151 .

Other types of modifications

In addition to the above-mentioned modifications, histones can be subjected to other modifications such as ubiquitination (ub), sumoylation, serotonylation (ser), glycation, ADPribosylation, citrullination, long chain fatty acylations (lipidation) that play a role in diverse biological processes (Figure 15).

Ubiquitination

Histone ubiquitination is to add a ubiquitin peptide to histone lysines mainly on H2A and H2B

histones. Ubiquitination is catalyzed by E3 ligases including RNF168, RING1B (RNF2, member of PCR1 complexes), BRCA1/BARD1 and RNF20/40, which are responsible for ubiquitinating H2A at K13/K15, K119, K127/129, and H2BK120 respectively. Ubiquitin can be removed by deubiquitinating enzymes (DUBs), such as ubiquitin-specific protease (USP)3/11/44/51 etc. Histone ubiquitination is predominantly involved in DNA damage repair and transcriptional regulation through recruiting and organizing DNA repair factors (e.g.,

53BP1

) and through cooperating with other chromatin interaction proteins 171,172 . Of interest, there is an interplay between histone ubiquitination and other histone marks. This is exemplified by H2BK120, where mono-ubiquitination at this site activates DOT1L-dependent H3K79me and SET1-dependent H3K4me respectively [START_REF] Worden | Activation and regulation of H2B-Ubiquitin-dependent histone methyltransferases[END_REF] .

Sumoylation

Histones are also substrates for small ubiquitin-related modifier (SUMO) proteins, generating sumoylation on all core histones. SUMO is added to substrates through E1-E2-E3 enzymatic cascade similar to ubiquitylation and can be cleaved by SUMO proteases. Histone sumoylation is generally associated with transcriptional repression. Shiio Y. and Eisenman R.N. reported that H4 sumoylation could recruit HP1 and HDACs, therefore mediates gene scilencing 173 .

Investigators also found an interplay between histone sumoylation and other histone marks, such as histone acetylation and ubiquitination. For instance, histone sumolyation was found to occur at or adjacent to acetylation sites in yeast, suggesting a role for this modification in counteracting acetylation-mediated gene transcription 174 . Histone sumoylation can be stimulated by H2B ubiquitylation and in turn inhibits Ctk1-mediated RNA Pol II phosphorylation and transcriptional elongation 175 . Of note, functions of histone sumoylation should be interpreted cautiously since SUMO occurs on a number of nuclear proteins other than histones, including chomatin remodelers and transcriptional coregulators 176,177 .

Serotonylation

Recently Farrelly and colleagues identified that histone tails can be serotonylated (5hydroxytryptamine). They uncovered that transglutaminase 2 can serotonylate histone H3 that carries H3K4me3 mark, which results in the combination of H3K4me3Q5ser marks on histone tails. H3K4me3Q5ser is associated with active transcription, which could be explained by the potentiation of H3K4me3 activity in recruiting transcriptional machineries, and the fact that these dual marks enhance interactions of TFIID complexes with chromatin. H3K4me3Q5ser mark is closely linked with neuronal cell differentiation 178 . The discovery of histone serotonylation has linked the chromatin regulatory networks into neurotransmitter-dependent cellular signaling. This discovery might pave path for understanding many pathophysiological conditions concerning serotonin 179, 180 .

ADP-ribosylation

Histone ADP-ribosylation refers to the addition of ADP-ribose onto a repertoire of target residues including aspartate, glutamate, lysine, arginine, serine and tyrosine and has been found

to occur on all types of histones 181,182 . Histones can be modified by single or several ADP-ribose molecules, termed as mono-ADP ribosylation (MARylation) or poly-ADP ribosylation (PARylation) respectively. Several ADP-ribose can form either linear or branched chain in the case of MARylation. Histone ADP-ribosylation is a reversible modification, which can be added by Diphteria toxin-like ADP ribosyltransferases (ARTDs; also called PARPs), and removed by ADP-ribosyl hydrolases (ARHs) and poly-ADP-ribose glycohydrolase (PARG).

Domains that read ADP-ribosylation include PAR-binding motif, PAR-binding zinc finger, Macrodomains, WWE domain etc. There is also an interplay between histone ADP-ribosylation and other PTMs including acetylation, methylation, phosphorylation etc… which have been implicated in chromatin dynamics and cell fate determination 181,183 .

Citrullination

Protein citrullination is the conversion of arginine side chain to citrulline by protein (peptidyl) arginine deiminases (PADs). In human, there are 5 PAD homologues designated as PAD1-4

and PAD6 whose activity is regulated by calcium ions. Among them, PAD4 is located in nucleus and catalyzes citrullination on H2A, H3, and H4. Besides, PAD4 was also reported to catalyze demethylimination, where Arg monomethylation is hydrolyzed into citrulline and methylamide. Histone citrullination might be involved in chromatin structure regulation and is associated with transcriptional regulation in combination with other histone PTMs such as methylation and acetylation 184,185 . 1.3.5.6 Glycation Histone tails are susceptible to non-enzymatic covalent modifications (MECMs) on their nucleophilic side chains. One of the most prevalently occurred NECMs is glycation, where sugar (e.g., glucose, fructose) and their derivatives (e.g., ribose, methylglyoxal, MGO) are attached to histones 186,187 . Latest studies indicate that histone glycation is reversible, with the identification of DJ-1 to be a potential histone deglycase. Histone glycation has been suggested

to regulate nucleosome stability and assembly, and alter the biophysical properties of chromatin [188][189][190] .

Lipidation

Protein lipidation (also known as fatty acylation) is the attachment of fatty acids (e.g., myristoyl (14:0), palmitoyl (16:0), stearate (18:0), oleate (18:1), arachidonate (20:4)

Combinatorial histone modifications

Apart from the increasing number of the types of histone modifications, an additional complexity of signaling by histone PTMs is the combination of different histone marks. A special example is the occurrence of bivalent histone marks at primed promoters, which was believed to prime the status of the subsequent active or repressed transcription. With the usage of tandem mass spectrometry, sequential MNase based ChIP, the combinations of different histone modifications have been characterized in a genome-wide scale. One study comes from

Wang and colleagues, who performed a comprehensive study about the distribution of 39 histone PTMs on human genome using CD4+ T lymphocytes. They identified a common modification 'backbone' comprised of 17 modifications that coexist in 3286 active promoters and pointed out that these marks tend to colocalize and correlate with each other 193 .

The functional interpretation of combinatorial histone marks can be tackled by reader proteins who recognize multiple modifications. The combinatorial readout of histone marks by readers comprises: 1) some reading modules recognizing several modification types within the same histone protein; 2) reader proteins possessing tandem reading modules, which facilitate the recognition of different marks on the same histones (cis-histone), distinct intranucleosomal (cis-nucleosome) or internucleosomal histones (trans-nucleosome). 3) reader proteins that might co-exist with additional readers or other proteins within a complex (e.g., chromatin modifier complexes), which facilitate a series of functional outputs, including the establishment of combinatorial pattern of histone marks. More information is provided in 194,195 and summarized as Figure 16. Indeed, it has been proposed to cause tumorigenesis especially in those cancer contexts where the driver genetic aberrations are lacking 198 . Since epigenetics is relatively dynamic because of the functions of writers, erasers, readers and remodelers and its link to cell metabolism, investigators have envisioned cancer therapeutics by targeting epigenetic enzymes and interacting proteins to restore the disrupted epigenetic profiles, and to break the resultant oncogenic transcriptional circuitry.

Herein, I'll summarize the cancer-related altered epigenetic profiles. I will also give a brief overview of the currently-identified aberrations of epigenetic players and their possible roles in cancer. Finally, I will summarize the current therapeutic approached to impact the cancer epigenome and their clinical efficacy. Since noncoding RNA, DNA methylation and chromatin remodelers are not the main topics in this thesis, I will leave out most of the details relating to them and will focus on histone modifications.

Disrupted epigenetic mechanisms contribute to oncogenesis

Altered epigenetic signatures in cancer

Cancer cell epigenome displays distinct patterns compared to their normal counterparts.

Alterations of cancer epigenomic signatures are typically exemplified by the well-characterized DNA methylation, which shows global hypomethylation (including intergenic regions and repetitive elements) and regional hypermethylation (e.g., CpG islands) in cancer. These alterations contribute to cancer establishment and development. For instance, the hypermethylation at CpG islands, which are present in most gene promoters, can repress gene transcription including that of tumor suppressors. In special cases such as colorectal cancer, CpG islands methylator phenotype (CIMP) can be observed, where a set of tumor suppressor genes could be repressed simultaneously. Additionally, hypomethylation at repetitive elements might lead to genomic instability, which might disrupt normal self-constraint program and envision genetic aberrations leading to oncogenesis 199 . Finally, spontaneous deamination of 5methylcytosine (5mC) to thymine is likely to be responsible for generating mutation hotspots in somatic cells, which might increase the risk of tumorigenesis.

Histone modifications alterations are extensively implicated in cancer, both at a global level of the whole genome and at specific loci. For instance, an overall loss of acetylated and trimethylated forms of H4 (predominantly H4K16ac and H4K20me3) was detected in several cancers. Loss of these histone marks co-occurs with DNA hypomethylation at repetitive regions 200 . Furthermore, changes in histone modification patterns have been reported in a variety of tumors and are associated with prognosis [201][202][203] . Similar to DNA methylation, histone modifications may repress or activate oncogenic transcriptional circuits and might act as epigenetic drivers of cancer 204 . Certain histone modifications are associated with chromatin alterations and genetic mutations, suggesting the existence of a crosstalk between chromatin organization and genetic abnormalities 205,206 .

Epigenetic players in cancer

In addition to the general alterations of the epigenetic patterns, genetic studies and exosome sequencing projects have led to the discoveries of writers, erasers, and readers of histone PTMs among the frequently altered genes in cancers. Alterations of epigenetic modifiers in different cancer contexts can result from gene mutation (gain-of-function mutation or loss-of-function mutation), translocation, gene amplification, overexpression and silencing from non-genetic causes, etc. [207][208][209] . In many cases, epigenetic modifiers can be either oncogenic or tumorsuppressive depending on the cell contexts. An additional layer of histone modification signaling circuits is through reader proteins. Aberrations of readers proteins have been reported in cancer, exemplified by BRD4-NUT fusion protein that acts as the oncogenic driver in midline carcinoma 210 . It is worth noting that, although numerous aberrations concerning epigenetic players have been identified in cancer, many of their roles are not fully understood. Moreover, in many cases, the understanding of the precise role of epigenetic players in cancer can be complicated due to their direct involvement in many intracellular signaling networks (e.g., act on non-histone substrates) other than via histone modulation and gene transcription. Table 3. Common KDMs and their implications in cancer.

Name Histone targets Implications in cancer LSD1

H3K4me1/2, H3K9me1/2

Overexpression of LSD is associated with cancer aggressiveness in neuroblastoma, leukemias [285][286][287] . LSD1 is required to maintain oncogenic program controlled by driver oncogenes in acute lymphoblastic leukemias [288][289][290] . LSD1 dysregulation is directly involved in activating oncogenes such as p21, CCNA2, E2F1, E-cadherin etc. in solid tumors 286,291 .

LSD1 was also characterized as a tumor suppressor in breast cancer [292][293][294] .

UTX/KDM6A

H3K27me2/3 UTX/KDM6A mutations (e.g., missense, truncating and indels mutations), gene amplifications, and gene fusions, dysregulation are found in various solid tumors and hematological malignancies 295 . Tumor suppressive or oncogenic in different types of cancers 296- 300 .

JMJD3/KDM6B

Loss of JMJD3 is associated with poor prognosis in colorectal cancer and is associated with aggressive pancreatic ductal adenocarcinoma 301,302 .

JMJD3/KDM6B is oncogenic in leukemias 303 . Overexpression of JMJD3 is associated with poor prognosis in esophageal squamous cell carcinoma 304 , clear renal cell carcinoma, glioma, non-small cell lung cancer [305][306][307] , multiple myeloma 308 .

KDM5A

H3K4me2/3 NUP98-KDM5A was detected in acute megakaryoblastic leukemia and is associated with worse outcome 309,310 . Expression of KDM5A promotes cell proliferation in lung, ovarian cancer 311 312 .

KDM5A is downregulated in metastatic glioma 313 .

KDM5B

Oncogenic in various solid cancers whose expression is associated with poor prognosis [314][315][316][317][318][319] KDM5B promotes the drug tolerance of melanoma propagating cells 320 .

KDM5C

KDM5C mutation has been discovered in clear cell renal carcinoma cells 245,321 . KDM5C overexpression was identified in breast, gastric, and prostate cancer and is associated with metastasis and poor prognosis [322][323][324] . KDM5C point mutation was identified in acute myeloid leukemia and is associated with chemo-resistance 325

KDM5D

KDM5D is frequently deleted in metastatic types and its low level is associated with drug resistance and poor prognosis in prostate tumors [326][327][328] . KDM5D can also play a role in metastasis of gastric cancer 329,330 .

Table 4. Common HATs and their implications in cancer.

Name Targets

Implications in cancer p300/CBP Histones and non-histone substrates Loss of heterozygosity (LOH) mutations concerning p300/CBP have been identified in gastric, colon, cervical cancers, leukemia and lymphomas and are markers for poor prognosis [331][332][333][334] .

MLL-CBP, MLL-p300, MOZ-p300, MOZ-CBP translocations can be found in acute myeloid leukemia, chronic myelomonocytic leukemia and myelodysplastic syndrome [335][336][337][338][339][340][341] .

Generally p300/CBP displays tumor-suppressive functions [342][343][344][345] .

Mutation or translocation mediated oncogenic activities can be attributed by their regulation of non-histone proteins (e.g., BCL6, p53, AML1, NF-κB) [345][346][347][348][349] .

GCN5/PCAF

Histones and non-histone substrates Somatic mutations and deregulation were identified in many tumors 204,350,351 .

The major oncogenic role of GCN5/PCAF is to cooperate with bona fide oncoproteins (e.g., Myc, E2F1 etc.) and promotes their activities 352- 356 .

PCAF/GCN5 are also suggested to exert tumor-suppressive functions 357,358 .

MOZ

Histones and non-histone substrates

The oncogenic roles of MYST family are mainly through transcriptional activation, DNA damage repair, DNA replication etc. dependent or independent of their acetyltransferase activity towards histones [START_REF] Lan | Deciphering structure, function and mechanism of lysine acetyltransferase HBO1 in protein acetylation, transcription regulation, DNA replication and its oncogenic properties in cancer[END_REF]359 .

MOZ-CBP, MOZ-p300, MOZ-TIF2, MORF-CBP can be found in acute leukemias 360 .

HBO1

Histones and non-histone substrates HBO1 can be oncogenic in cancer cells and its overexpression has been identified in many cancers due to gene amplification [361][362][363][364] .

Can be tumor-suppressing through upregulation of tumor suppressor 365 .

NUP98-HBO1 chimera could induce chronic myeloid monocytic leukemia 366 .

TIP60

Histones and non-histone substrates Has bivalent roles in human cancer [START_REF] Judes | A bivalent role of TIP60 histone acetyl transferase in human cancer[END_REF] . TIP60 acetylates p53 and regulates cell cycle progression and apoptosis 367,368 . TIP60 was also found to promote androgen receptor, c-Myc and NF-κB mediated gene transcription and cell transformation 369,370 .

HDACs

HDACs are involved in a variety of cancers including hematological and solid tumors 204 . A high level of HDACs is generally associated with aggressive disease and inferior outcomes of patients [371][372][373] . In fewer cases, HDACs have putative tumor-suppressive effects, as exemplified by the identification of HDAC2 loss of function mutation in epithelial cancers, where its reexpression causes a reduced tumor growth 374,375 . Deregulated HDAC activity in cancer results mostly from overexpression, which has been detected in cohorts of primary samples and diverse cancer cell lines, presenting a specific application domain for HDAC inhibitors in cancer treatment 376,377 . In many cases, aberrant enzymatic activity has also been identified and accounts for altered epigenomic profiles and the oncogenic roles of HDACs 378,379 . Additionally, oncogenic effects of HDACs can be mediated by abnormal recruitment by key regulators. For example, in acute leukemia, HDAC-containing repressor complexes can be recruited by PML-RARα, AML1-ETO, which in turn contribute to the oncogenic chimera proteins-induced leukemogenesis 380,381 .

Mechanisms of HDAC contribution to tumorigenesis are diverse and to a large extent, context-dependent. This is partially due to the fact that HDACs can impact various key SIRT3-5 SIRT3 could deacetylate p53 and is antiproliferative. In contrast, it could also regulates energy metabolism and promotes proliferation 395,396 .

SIRT6

Activation of SIRT6 inhibits cell proliferation and induces cell cycle arrest in hepatocellular carcinoma 397 . HDAC3 deacetylase activity and NCoR/HDAC3 complex were shown critical in regulating differentiation in rhabdomyosarcoma via blocking myoblast determination protein 1 (MYOD1)-mediated myogenic differentiation 408 .

Apoptosis

HDAC6

HDAC6 depletion was shown to decrease cancer stem cells pluripotency and promote differentiation in tetratocarcinoma 409 .

DNA-Damage

Response

HDAC1, 2 HDAC1 and HDAC2 promote nonhomologous end-joining (NHEJ) 410 . HDACs were also reported to regulate proteins in DNA damage response, such as ATM, ATR, FUS, KU70, Rad52 etc. 411,412 .

Class IIb HDACs (HDAC6, 10)

HDAC6 and HDAC10 have been shown to deacetylate DNA mismatch repair protein 2 (MSH2), leading to MSH2 ubiquitination and degradation and therefore impair mismatch repair 413,414 .

SIRT1

SIRT1 was reported to interact with several DDR proteins, including KU70, APE1, XPA, PARP-1 etc. 371 .

SIRT6

SIRT6 was found to regulate base excision DNA repair 415 and contribute to the activation of PARP1 and therefore promotes both HR and NHEJ 416,417 .

Metastasis

HDACs

HDACs were found to silence epithelial markers and promotes epithelial-mesenchymal transition (EMT) [418][419][420] .

SIRT1

SIRT1 could be recruited to CDH1 promoter and regulate cell migration in prostate cancer cells 421,422 . SIRT1 was also reported to inhibit metastasis via deacetylating Smad4 in breast epithelial cells and in oral squamous cell carcinoma 423,424 .

SIRT2

SIRT2 is required for gastric cancer metastasis. SIRT2 inhibitor, SirReal2 could inhibit SIRT2 activity and decrease cell migration, which is associated with impaired mitochondrial metabolism and RAS/ERK/JNK/MMP-9 425 . SIRT6 SIRT6 could promote EMT via interacting with Snail and through suppressing TET1 transcription by removing H3K9ac at its promoter regions 426 .

Angiogenesis

Class I HDACs upregulated in hypoxia and could interact with HIF-1α 427,428 , promote HIF-1α protein stability [429][430][431] .

HDAC2 and HDAC3 act as co-repressor of VEGF 432 .

Class IIa HDACs

Increase HIF-1α transcription and repress pro-angiogenic genes 433,434 . HDAC7 could repress matrix metalloproteinase10 (MMP10), therefore promote blood vessel development and vascular integrity maintenance 435,436 .

Class IIb HDACs HDAC6 regulates HIF-1α and VEGF and positively 437,438 or negatively regulate angiogenesis 439 .

SIRT1

Deacetylates HIF-1α and inhibits its activity 440 . SIRT1 activation could downregulate VEGF expression and suppress angiogenesis in human osteosarcoma 441 . SIRT1 was also reported to decrease VEGF biological activity through promoting PAR modification of VEGF 442 .

SIRT6

SIRT6 was reported to exert anti-angiogenic effect by repressing the expression of VEGF and FGF-2 in endothelia cells 443 .

Autophagy

HDAC1

Induce autophagic cell death by promoting accumulation of LC3-II in liver cancer cells 444 .

HDAC3 could promote the expression and the activity of proteasome and induce autophagy 445 .

HDAC6

An important regulator of basal autophagic flux as well as a central component for ubiquitin proteasome system 409,446 , mitophagy 447 etc. HDAC10 HDAC10 could ensure efficient autophagosome-lysosome fusion and protect cells against cytotoxic drug in neuroblastomas 448 .

SIRT1

Directly deacetylates autophagy-associated proteins, including Atg5, Atg7, Atg8, and LC3 and contributes to the induction of autophagy [449][450][451] .

Directly upregulate transcriptional level of autophagy associated proteins (e.g., LC3-II, beclin-1) 452, 453 .

SIRT3, 5

Trigger autophagy in osteosarcoma, mesothelioma, breast cancer, colorectal cancer, and diffuse large B cell lymphomas [454][455][456][457] .

SIRT6

Regulate autophagy in melanoma, esophageal cancer, hepatocellular carcinoma [458][459][460][461] .

Required for the activation of the AMPK-ULK1-mTOR signaling in ROS triggered autophagy.

Pharmacological activation of SRIT6 could confer to autophagy-related cell death in cancer cell lines 462 .

READERS

As discussed above, histone modifications, especially acetylation and acylation signaling largely relys on readers and their impact on chromatin structure and gene transcription. Indeed, numerous studies depicted histone PTM readers as key players in oncogenic transcription program, chromatin structure regulations, DNA damage response, genome integrity. For example, BRD4 is well-known for its oncogenic role in regulating Myc transcription in diverse cancers 463 . In addition, YEATS domain-containing protein ENL, links histone acetylation to cancer-driving genes' expression (e.g., MYC, HOX) in acute leukemia as well as in Wilms tumor The functional specificity of BRD4 in mediating oncogene expression in cancer has been partly explained by its specific action on the super-enhancers (SEs). SEs are large clusters of enhancers featured by enriched transcription machineries and chromatin remodelers, possibly compartmentalized by liquid-liquid phase separation 481,482 , often marked by H3K27ac and H3K4me1, and lineage-specific transcription factors and oncogenic factors. The involvement of BRD4 in SEs has been characterized in multiple myeloma, where BRD4, Mediator, and P-TEFb were found to be enriched on SEs. Inhibition of BRD4 using BETi leads to loss of BRD4 preferentially at SEs, thereby disrupting SE -mediated oncogene expression including c-MYC in multiple myeloma 483 .

Apart from acting as master regulator of transcription, BRD4 has also been implicated in RNA splicing, DNA damage response and telomere regulation 463 . BRD4 was found to interact with splicing machinery and to regulate alternative splicing 484,485 . Several studies reported that BRD4 is required for DNA damage repair (e.g., NHEJ, HR) in cancer cells [486][487][488] .

Mechanistically, BRD4 can be recruited by increased H4 acetylation that occurs at DNA double strand breaks and serves as platform for DNA repair machineries. BRD4 was found to interact with 53BP1 and might stabilize its binding with DNA repair complex at break sites 487 . In addition, BRD4 as discussed above, might have intrinsic acetyltransferase and kinase activities, which might acetylate or phosphorylate DNA repair proteins and regulate their functions 467,481 .

BRD4 could also positively regulate DNA damage checkpoint activation by interacting with components including DNA pre-replication factor CDC6, chromatin condensing II complex (SMC2) or by transcriptional regulation of damage response protein TopBP1 [489][490][491][492] .

Recently, BRD4 has been also implicated in telomere maintenance in cancer cells 493 . BRD4 was found to be involved in regulation of telomerase complex component TERT in TERT promoter mutated cancers 494,495 . Inhibition of BRD4 with BET-inhibitors or genetic depletion of BRD4 reduces TERT expression in TERT promoter mutated cancers 496 .

Other readers

In addition to BRD4, other readers have also been implicated in cancers. For example, BRD3

was also identified as fusion partner of NUT, with the fusion protein functioning similar to BRD4-NUT. BRD7 has been characterized as a tumor-suppressive factor in nasopharyngeal carcinoma, breast cancer, prostate cancer, etc 497 . Mechanistically, BRD7 can act as a coactivator of p53 and its target genes, thereby promoting p53-dependent senescence and suppresses tumorigenicity 498 . In breast cancer, BRD7 interacts with BRCA1 and regulates estrogen receptor gene expression 499 . Besides, it could decrease the expression of transcriptional activator YB1, therefore blocks epithelial-mesenchymal transition and tumor metastasis 500 . BRD9 is the subunit of SWI-SNF chromatin-remodeling complex. BRD9 plays an oncogenic role in acute myeloid leukemia (AML) and squamous cell lung cancer by regulating MYC transcription 501,502 . YEATS domain-containing protein ENL has been implicated in leukemia and Wilms tumor, where it regulates oncogene expression (e.g., MYC, HOX) and the mediated oncogenic program 120,[464][465][466] . Another member of YEATS protein, GAS41 was found to bind H3K27ac, H314ac and to promote H2A.Z deposition. GAS41 is necessary for cell growth and survival in non-small cell lung cancer 122 . YEATS4 was reported to promote cell proliferation by activating Wnt/β-catenin in gastric cancer cells 503 . p300/CBP possesses both bromodomains, transcription binding domains as well as a acetyltransferase module. p300/CBP has been implicated as a critical mediator of histone marks in actively transcribed regions. p300/CBP bromodomain is required for sustaining cell typespecific gene expression, inhibition of bromodomain of p300/CBP decreases H3K27ac and chromatin accessibility at target genes, thereby accelerating induced pluripotent stem cells (iPSCs) cellular reprogramming 105 .

Histone PTMs' therapeutic implications in cancer

Epigenetic drugs have been developed to target DNA/histone-modifying enzymes, histone readers, or other chromatin-associated proteins. Some of these drugs have displayed promising efficacy in treating cancers in research and in clinical usage. For example, DNMTi (azacytidine and decitabine) has been approved by US Food and Drug Administration (FDA) for treatment of myelodysplastic syndrome (MDS) or AML, and has achieved 15% response rate in these patients 504,505 . Following DNMTi, a variety of epigenic drugs were developed, some had been approved by US FDA and entered clinical trials or are still under research (Table 6). In general, there are three major classes of epigenetic drugs according to the enzymes they target and the potential mechanisms they exploit: 1) those who impact deregulated epigenetic profiles, including HDACi, DNMTi; 2) those who disrupt specific key oncogenes and oncogenic programs, represented by BETi, HDACi, DNMTi; 3) those that are exploited in specific cancer types, including DOT1Li, LSD1i. Epigenetic drugs have been tested either as monotherapy or in combination with additional epigenetic drugs or traditional chemotherapies to achieve therapeutic efficacy 204 .

Here, I describe the therapeutic strategies by targeting epigenetic players and their working mechanisms in cancers.

Impact of deregulated epigenetic profiles in cancer establishment and develoment

As introduced above, cancer cells tend to have altered epigenetic profiles including DNA methylation and histone modifications. These changes, could on one hand directly deregulate the expressions of key oncogenes or tumor suppressors, and on the other hand allow for oncogenic genome reprogramming. A permissive chromatin state is required for oncogenic transcription factor activity and for the occurrences of mutations 506,507 . Therapeutic strategies targeting the cancer-specific altered epigenetic profiles are well documented by the use of DNMTi (e.g., azacytidine, decitabine) 507 . In the case of HDAC, an increasing body of studies has focussed on the development and characterization of various generalist or class-specific HDAC inhibitors.

Over last decades, dozens of HDAC inhibitors have been evaluated on tumors, among which pan-HDACi Vorinostat (SAHA), belinostat and romidepsin have been approved for treating T cell lymphomas, and another pan-HDACi panobinostat was approved for treating refractory/relapsed multiple myeloma. Lately, more selective HDACi, for example, class I HDACs inhibitor entinostate (MS-275), HDAC1, 2, 3, 10 inhibitor chidamide have been developed.

HDACi have been tested as monotherapy or in combination with hypomethylating agents azacytidine, histone demethylating agents (named as KMTi hereafter) as well as chemotherapeutic regimens in cancer treatment 508 . In general, HDACi displayed higher efficacy in hematological malignancies than in solid tumors, possibly in part due to the short half-life of the molecules that prevent these agents to reach therapeutic concentrations in solid tumors 509 .

HDACi could act at multiple levels given that they increase global histone acetylation and enhance chromatin decompaction. Additionally, they also act on numerous non-histone proteins 510 . It is clear that several studies have indicated that HDACi treatment efficacy could at least in part be attributed to its impact on chromatin landscape. For example, using ATAC-seq technology, Qu and colleagues reported that clinical response to HDACi in cutaneous T cell lymphoma is associated with a gain in DNA accessibility 511 . In addition, using high-grade gliomas, which possess a reshaped chromatin landscape (i.e., loss of H3K27me3 and gain of H3K27ac) as models, Krug and colleagues demonstrated that DNA methyltransferases and histone deacetylase inhibitors could exaggerate the increased H3K27ac at and repeat elements, leading to their induced activation and thereby conferring to therapeutic vulnerability 512 .

Epigenetic drugs regulate crucial oncogenic or tumor-suppressive signaling

In addition to their impact on global epigenetic pattern, HDACi or DNMTi could also exert in 204,523 .

The most prominent epigenetic drugs that control oncogene expression is exemplified by BET factors bromodomain inhibitors (BETi). I have already mentioned that BETi inhibiting BRD4 disrupts BRD4-mediated SEs. BETis (e.g., birabresib, venetoclax, molibresib) have been tested in several small cohorts in solid and hematological tumors, where they achieved some responses in clinical studies. Preclinical studies also indicated that a combined treatment of BETi with additional epidrugs/other therapies including HDACi, CDKi, bcl2 inhibitors, hormone therapy, standard chemotherapy etc,.. could be advatageous. [524][525][526] . Additionally, BETi resistance have been reported in several researches, which had prompted investigators searching for specific combinatorial strategies 527, 528 .

Epigenetic drugs targeting specific types of cancers

Some types of cancers are known to be driven by specific aberrations of epigenetic modifying enzymes (e.g., MLL rearranged leukemia), making them the most promising targets for treating the corresponding cancers. Small molecules inhibitors targeting interaction between MLL and Menin has been developed and has displayed effective anti-tumor effects in MLL-rearranged leukemia cells and in mouse models 529 . DOT1L is aberrantly regulated in MLL-rearranged leukemia and is required for leukemogenesis of this cancer subtype, small molecules inhibitors targeting DOT1L were shown to be effective in MLL-rearranged leukemia [530][531][532] .

In addition, inhibitors of LSD1 are active in a subset of small cell lung carcinoma with specific DNA methylation patterns 533 . In AML, LSD1i displayed antileukemic effect through enhancing PU.1-and C/EBPα -dependent differentiation program in MLL-AF9 leukemia cells 534 . IDH mutations have been identified and characterized as key oncogenic events in glioma, leukemia etc…. In these cases, IDH inhibitors could be used as a targeted therapy 535,536 . EZH2 gain of function mutations have been identified in lymphoma, and the inhibition of EZH2 exerted cytotoxic effects in cancer cells carrying these mutations 208,537,538 .

Of note, the situation could be very complex and it is not always easy to define a precise therapeutic strategy based on the use of epidrugs. For example, DOT1L inhibition could also lead to the deregulation of oncoproteins including Myc, which can be detrimental in a variety of cancers [539][540][541][542] . Note: This table is adapted from 507, 543 .

Cell metabolism is a major driver of histone modifications

As noted above, histone PTMs are dynamic and are constantly regulated by counteracting enzymes designated as writers and erasers. Most of these enzymes utilize metabolic cofactors to exert their catalytic reactions. This situation hence creates a tight interplay between cell metabolism and histone modifications. For instance, KMTs, PRMTs and DNMTs utilize SAM, KATs utilize acetyl-CoA or acyl-CoA, and kinases utilize ATP respectively as co-substrates to transfer the chemical moieties to DNA or to the amino acid residues of histones. In contrast, JmjC family demethylases (JMJD or JHJM for short) require α-ketoglutarate, SIRT family deacetylases require NAD as cofactors to remove the modifications.

In this chapter, I will introduce the compartmentalization and maintenance of several key epimetabolites pools, as well as the biochemical pathways that generate or consume these molecules. I will also give examples of how these metabolites change in response to nutritional perturbations and how they impact histone modifications. Finally, I will briefly discuss how the resultant epigenetic alterations regulate nuclear gene transcription programmes, which in turn impact other biological processeses such as cell fate determination.

SAM

One-carbon metabolism

S-adenosylmethionine (SAM) is involved in the methionine cycle (Figure 17). SAM is synthesized from methionine and ATP, a reaction which is catalyzed by methionine adenosyltransferase (MAT). After the methyl group is transferred for methylation usage, SAM is converted to S-adenosyl homocysteine (SAH), which then is hydrolyzed to homocysteine

SAM in histone methylation

SAM is the methyl donor for histone and DNA methylation processes. As co-product of methylation reaction, SAH is a potential inhibitor of methyltransferases. Thus, the level of SAM and the ratio of SAM/SAH tunes the methylation state of chromatin. Indeed, the modulation of SAM levels and SAM/SAH ratio through changes of methionine supplementation affects global DNA/histone methylation status, with H3K4me3 displaying the most prominent change. ChIPseq analysis of H3K4me3 revealed that only the level of this mark but not the genomic distribution altered upon methionine restriction. This correlation between H3K4me3 level and methionine availability was observed both in cell line and in the mice liver 545 . SAM generation and SAM/SAH ratio maintained by glucose and serine metabolism were also suggested to support H3K36me3, which mediates IL-1β production for inflammation response in macrophages 546 .

Methionine supplement

As an essential amino acid, methionine can be absorbed from the environment and not surprisingly, its intracellular level is affected by amino acid transporter (e.g., LAT1).

Knockdown of LAT1 results in methionine as well as SAM reduction, which is concomitant with decreased H3K27me3, H3K4me and H4K20me marks 547 . In addition to the uptake of methionine, dietary intake of other nutrients that participate in SAM production and maintenance (e.g., SAM, folic acid, vitamin B12, Choline and its metabolite, betaine) might modulate histone methylation 548, 549 .

Serine metabolism

Serine synthesis pathway (SSP) starts with the glycolytic intermediate 3-phosphoglycerate (3-PG) being converted to 3-hydroxypyruvate by phosphogycerate dehydrogenase (PHGDH). 3hydroxypyruvate is then used to produce phosphoserine through a transamination reaction catalyzed by phosphoserine aminotransferase (PSAT1), phosphoserine is dephosphorylated by phosphoserine phosphatase (PSPH) and produces serine 550 .

Since serine is a one-carbon source that contributes to folate cycle, serine metabolism can impact SAM production and might regulate histone methylation. In addition, serine can support de novo ATP synthesis, which is required for SAM production from methionine 551 . Indeed, elevated serine synthesis pathway mediated by loss of LKB1 leads to high SAM level through upregulation of PSAT1 and PSPH, which results in DNA hypermethylation 552,553 .

SAM compartmentalization

In addition to the global intracellular level, the subcellular compartmentalization of SAM also impacts histone methylation because of different accessibility to the chromatin-modifying enzymes. For example, SAM was found in the nuclear compartment in yeast and was suggested to control specific gene expression. Li and colleagues identified that, in yeast, SESAME (Serine-responsive SAM-containing Metabolic Enzyme complex) interacts with Set1 and is recruited to target genes 554 .

Acetyl-CoA

Production and utilization of acetyl-CoA

Acetyl-CoA can be produced in mitochondrion, cytoplasm and nucleus and are generated from diverse nutrient sources, including pyruvate, fatty acid, amino acids, acetate, and citrate (Figure 18).

In mitochondrial compartment, pyruvate generated from glycolysis enters into mitochondrion and is converted to acetyl-CoA by pyruvate dehydrogenase complex (PDC).

Acetate is ligated to CoA by acyl-CoA synthetase short-chain family members 1 and 3 (ACSS1/3). Fatty acids undergo β-oxidation through rounds of dehydrogenation, hydration, dehydrogenation and thiolase, producing one acetyl-CoA and one fatty acyl-CoA (with a reduction of 2C) during each round. Amino acids (e.g., tryptophan, lysine, phenylalanine, tyrosine etc.) can be converted to acetoacetyl-CoA and acetyl-CoA through multiple steps.

Acetyl-CoA generated in mitochondrion enters into tricarboxylic acid (TCA) cycle and is completely oxidized, releasing ATP through coupling with oxidative phosphorylation.

For the moment there is no known acetyl-CoA carrier in the mitochondrial membrane, therefore acetyl-CoA must be converted to citrate before it shuttles to cytoplasm. Acetyl-CoA and oxaloacetate are then regenerated in cytosol by ATP citrate lyase (ACLY). Another source of acetyl-CoA outside of mitochondrion is through acetyl-CoA synthase (AceCS1/ACSS2), which is located in cytosol/nucleus and uses acetate as substrate. In addition, PDC can be translocated to nucleus, where it can produce acetyl-CoA for nuclear usage. Acetyl-CoA in cytoplasm and nucleus is used for histone and non-histone protein acetylation as well as de novo fatty acid and cholesterol biosynthesis. Acetyl-CoA pool for histone acetylation is replenished by various nutritional sources.

Glucose is one of the major sources that fuels cell growth of tumor cells. A large body of evidence suggests that glucose derived acetyl-CoA is the major driver of histone acetylation during cell proliferation. Consistently, limitation of glucose or inhibition of glycolysis leads to decreased acetyl-CoA production and hence histone acetylation [559][560][561] . However, when supplied with medium-chain fatty acid, McDonnell and colleagues reported that lipid-derived acetyl-CoA contributes to 90% of certain sites of histone acetylation, even supplied with abundant glucose. Considering that fatty acid oxidation occurs in mitochondria, theoretically, the generated acetyl-CoA needs to be exported from mitochondria via citrate shuttle in order to be used for histone modification in the nucleus. However, in the above-mentioned experimental setting, the knock down of ACLY and ACSS2, did not affect the increased histone acetylation by octanoate supplement, suggesting that the fatty acids derived acetyl-CoA might be exported through a yet unknown major pathway different from the citrate cycle 562 . In addition, in the case of ACLY deficiency, acetate was found to compensate for acetyl-CoA production through upregulation of ACSS2, which supported histone acetylation and lipid synthesis 563 . These studies, in addition to documenting metabolic adaptation, also implicate a delicate choice of priority for metabolites contributing to histone acetylation, and have raised the question of whether there are other undetermined possibilities for transporting acetyl-CoA across subcellular compartments (e.g., acetylcarnitine-carnitine system) 564,565 .

Acetyl-CoA pool homeostasis for histone acetylation is sustained by catabolic and anabolic

Spatiotemporal control of acetyl-CoA

The cytoplasm and nucleus are likely to constitute a single compartment with respect to acetyl-CoA, because of the existence of nuclear pores that are large enough for small molecules to pass through. This supposition implies the occurrence of an interplay between nuclear and cytoplasmic acetyl-CoA pool. Recent evidences, however, suggest that acetyl-CoA may not exchange freely between nuclear and cytoplasmic compartments. Mammalian cells are filled with macromolecules (e.g., protein concentration range from 50 to 250g/L, nucleic acid 20-50g/L), and these molecules are not homogenously distributed and could cause macromolecular crowding, therefore the diffusion of molecules might be largely perturbed especially between sites that are spatially far away 574 . This principle points to the possibility of a local compartmentalization in nucleus where small metabolites are generated close enough to where they are consumed (onsite generation) 575 . To this end, PDC, ACLY and ACSS2 have been found

to be present in nucleus. For example, under oxygen and serum limitation, nuclear ACSS2 can capture acetate released from histone deacetylation to maintain histone acetylation 576 . In neuronal cells model, Mews and colleagues have identified that ACSS2 increases in the nucleus of differentiating neurons and is recruited to chromatin, where it directly regulates histone acetylation and controls memory-related neuronal genes expression 577 . ACLY was found to be phosphorylated in response to DNA damage, whereby it orchestrates acetyl-CoA production and enables H4 acetylation near DNA double-strand breaks, therefore enhances BRCA1 mediated homologous recombination of DSB repair 578 . PDC was found to be translocated from mitochondrion to nucleus in response to serum, growth factor, or mitochondrial stress and in a cell-cycle-dependent manner, where it catalyzes synthesis of acetyl-CoA for acetylation of core histones 579 . In mammals, nuclear PDC accumulation was found to be crucial and indispensable for zygotic gene expression by maintaining histone acetylation 580 . The spatiotemporal control of epimetabolites has yielded an exciting perspective for metabolic signaling to epigenome and gene regulation specificity. More comprehensive studies are needed to address the current questions, such as the existence of microcompartments for metabolites, the molecular mechanism of nuclear translocation of acetyl-CoA producing enzymes. It is also important to identify proteins that interact with metabolite-producing enzymes and understand how cells sense environmental cues and initiate the process of enzyme translocation etc. 555 . Thirdly, acetyl-CoA can regulate specific sets of genes through transcriptional coregulators.

Transcription

For instance, BET family proteins which read acetylated lysine can recruit Mediator complex and regulate a set of genes which are dependent on locus control regions 463 . In leukemia cells, acetyl-CoA reduction mediated by AMPK depletion can reduce BET-dependent gene expression program by decreasing histone acetylation and therefore displacing BET protein from chromatin 583 .

Acyl-CoAs

Similar to acetyl-CoA, acyl-CoAs are key factors linking cellular metabolism to chromatin regulations. Recent studies documented that many HATs (e.g., p300/CBP, GCN5) can also utilize acyl-CoAs to generate respective histone modifications, and that the concentrations of acyl-CoAs are correlated with the level of respective histone acylations. However, while acetyl-CoA metabolism and compartmentalization are extensively studied, the process of acyl-CoA metabolism and their signaling to chromatin modifications remain poorly understood. From a biochemical point of view, non-acetyl acyl-CoAs are generated during lipid metabolism, amino acid catabolism, ketogenesis and can be directly converted from short chain fatty acids provided by intestinal microbiota fermentation. It is postulated that, since many of the biochemical processes generating acyl-CoAs occur in mitochondrion, the export of these metabolites is needed for nuclear usage, via yet unknown mechanisms. Similar to the compartmentalization of acetyl-CoA, the production and maintenance of acyl-CoAs in cytoplasmic and nuclear compartments could also exist, but this is in need of further studies.

Here I will introduce the metabolic pathways involving acyl-CoA generation. I will emphasize on the current evidences about their role in histone acylations. I will discuss the possible mechanisms contributing to compartmentalization and enabling their nuclear availability. Finally, I will also give examples of their functional output considering chromatinbased biological events.

Lipid metabolism

Many acyl-CoAs are present as intermediates in lipid metabolism, including β-oxidation, fatty acid and cholesterol synthesis. keep growing by rounds of condensation, reduction, dehydration, and reduction until it reaches 16 or 18. The three differences between FAS and FAO are, 1) ACP is used for stabilizing acyl esters; 2) NADPH instead of NAD system is used; 2) D (R)-form rather than L (S) enantiomers are generated during the spiral. In mammals, cytoplasmic FAS is catalyzed by a single complex called fatty acid synthase (FASN), comprising of 6 catalytic subunits and an ACP. mtFAS was firstly characterized in yeasts where it mainly produces octanoate and then converted to lipoic acid, which is an important cofactor for a number of mitochondrial enzymes. In mammalian cells however, mtFAS is less characterized 586 .

Evidences suggests that lipid metabolism might be an important source of acyl-CoA for histone acylations. For instance, crotonate supplementation can stimulate histone crotonylation, which is due to the increased conversion to acyl-CoA by ACSS2 144,587 . Depletion of PCC, which consumes propionyl-CoA, causes an increase in propionyl-CoA and histone propionylation (H3K14pr) in mouse livers. However, SCAD knockout mice did not show increased H3K14bu, although an increase of butyryl-CoA and non-histone protein butyrylation were observed in liver cells 169 . Using highly synchronized yeast metabolic cycle, Gowans and colleagues reported that histone crotonylation coincides with expression of genes in β-oxidation in the metabolic cycle 170 . In mitochondrion, short chain acyl-CoA can be converted to acyl-carnitine by carnitine acetyltransferase (CrAT), indicating the possibility of acyl-CoA being exported out of mitochondrion and this could support its export and the subsequent histone propionylation 588 .

Lately, Choi and colleagues identified that many FAO enzymes including ACSL1, HADHA and ACAA2 are located in the nucleus, implicating the generation of acyl-CoA in nuclear compartment 589 . Besides, the cytosolic FAS process and peroxisomal FAO are two other possibilities to fuel cytocylic/nuclear compartment of acyl-CoA pools. For example, succinyl-CoA, succinate and glutarate might be produced through dicarboxylic metabolism in peroxisome 590 . Fang and colleagues observed that the expression of peroxisomal acyl-CoA oxidase ACOX3 is increased during endoderm differentiation of embryonic stem cells, which along with other crotonyl-CoA producing enzymes (e.g., ACSS2) enhances histone crotonylation and control the gene expression program of endodermal differentiation 587 . The contribution of ketogenesis to histone acylations is documented in several studies. For example, mice liver cells exhibited higher intracellular acyl-CoA levels and increased histone butyrylation, β-hydroxybutryrylation after fasting 141,591 . It is worth noting that ketone bodies might contribute to histone modifications through increased acyl-CoA generation or through directly inhibiting HDAC activity. β-hydroxybutyrate, as well as other short chain fatty acids (e.g., butyrate) which is predominantly produced by intestinal microbiota, are known as HDAC inhibitors 592,593 .

Ketogenesis, TCA cycle and amino acid catabolism

TCA cycle links acetyl-CoA oxidation with energy production, which is coordinated by several metabolic intermediates including oxaloacetate, citrate, isocitrate, α-KG, succinyl-CoA, succinate, fumarate, malate. Succinyl-CoA is generated from α-KG, which is catalyzed by αketoglutarate dehydrogenase (α-KGDH) and is hydrolyzed to succinate by succinyl-CoA synthetase. The resultant succinate can subsequently be converted to fumarate by succinate dehydrogenase (SDH).

Although succinylation occurs prominently on mitochondrial proteins through nonenzymatic reaction, recent studies revealed that histones and extramitochondrial non-histone proteins could also be succinylated. Indeed, TCA cycle enzymes have been suggested to impact histone succinylation. For example, it is documented that SDH depleted MEF cells, which have TCA cycle defect, displayed higher intracellular succinyl-CoA concentration and increased histone succinylation 594 . In addition, α-KGDH complex was found to be present in nucleus where it produces succinyl-CoA for chromatin succinylation 151,589 . Succinyl-CoA is an abundant metabolite with its concentration ranging 0.1 -0.6 mM inside mitochondria 595 . Succinyl-CoA might be exported through carnitine / succinyl-carnitine system and produce a cytosol/nuclear succinyl-CoA pool, as suggested by the evidence that succinate can be exported from mitochondrion through dicarboxylate carrier SLC25A10 596 . Amino acid catabolism might be another source for acyl-CoAs. Ketogenic amino acids such as lysine, leucine and tryptophan can be converted to acetoacetyl-CoA and participate in ketogenesis. Methionine and branched chain amino acids (BCAA, e.g., valine, isoleucine) can be converted to propionyl-CoA through multiple steps. The most abundant amino acid, glutamine is subjected to glutamate production catalyzed by glutaminase. Glutamate can then be converted to α-ketoglutarate (α-KG) through a reversible reaction catalyzed by glutamate dehydrogenase and therefore participates TCA cycle, which as described above, might impact acyl-CoAs pool.

Glycolysis

Lactate is produced from pyruvate in the last step of glycolysis catalyzed by lactate dehydrogenase (LDH). In response to hypoxia or mitochondrial complex I inhibition, which induces glycolysis and lactate production, macrophages displayed increased level of histone lactylation. Consistently, inhibition of glycolysis using 2-DG or inhibiting PDH (pyruvate dehydrogenase) and LDH by sodium dichloroacetate (DCA) and Oxamate respectively, reduce lysine lactylation 140 .

FAD

FAD is synthesized from riboflavin (vitamin B2). The reaction starts from generation of riboflavin mononucleotide from riboflavin catalyzed by riboflavin kinase, resultant riboflavin mononucleotide can then be converted to FAD by FAD synthase (FADS). FAD is a cofactor for numerous biochemical processes, including histone demethylation. Lysine demethylases LSD1/KDM1A and LSD2/KDM1B are FAD-dependent oxidase that remove methyl group with the conversion of FAD to FADH2 602 . FADS was found to be located in nucleus, although less abundant than its mitochondrial counterpart, which might contribute to nuclear pool of FAD 603 .

α-KG

α-KG pool is maintained through several nutritional pathways. It is generated through the action of glutamate dehydrogenase (GDH, GLUD) from glutamate or from isocitrate by isocitrate dehydrogenase (IDH), and can be converted to succinyl-CoA by α-KGDH during TCA cycling.

Therefore, metabolites such as fumarate and succinate are TCA products downstream of α-KG can competitively inhibit the generation of α-KG. As a cofactor for DNA demethylase TET and JmjC family of histone demethylases, α-KG has been widely studied for its role in histone methylation and gene expression regulation. For example, intracellular α-KG/succinate ratio was shown to regulate H3K27me3 and DNA methylation, which in turn impact pluripotencyassociated gene expression 604 . Fumarate and succinate accumulation by dysfunctional fumarate hydratase (also known as fumarase) and succinate dehydrogenase leads to genome-wide increase of histone and DNA methylation by inhibiting α-KG dependent dioxygenases 605 .

PKM2

Pyruvate kinase M2 (PKM2) is a tumor specific kinase and is well-established for aerobic glycolysis. PKM2 was found to bind to and phosphorylate histone H3, which promotes HDAC3 dissociation and CCND1, MYC gene expression 606 . PKM2 was also suggested to directly phosphorylate H2AX and promotes genomic instability in tumor cells 607 .

IDH

Isocitrate dehydrogenase (IDH) catalyzes reversible conversion of isocitrate to α-KG in TCA cycle. IDH1 and IDH2 were found frequently mutated in glioblastoma and in myeloid leukemia patients. Mutated IDH1/2 could produce 2-hydroxyglutarate (2-HG) from α-KG, which competes with α-KG for the active site of demethylases and subsequently inhibits α-KG-dependent dioxygenases, including histone demethylases and TET family of DNA methylcytosine hydroxylases 608 . Consistently, IDH1/2 mutated cancer cells have altered genome-wide histone and DNA methylation that contributes to oncogenic programs (e.g., suppression of differentiation associated genes) 609,610 .

In conclusion, metabolic state impacts the nuclear genome in many ways and hence tunes all chromatin-based biological activities in response to nutritional cues, adding a new layer of complexity to intracellular signaling. The metabolism-nucleus crosstalk is, to a large extent, mediated by metabolites, which act as cofactors for epigenomic modifications. Unraveling the regulation of nuclear accessibility to epigenomic metabolites is an important challenge in modern biology. For instance, it would be critical to know what are the nuclear compartments or sub-compartments of metabolites and how they are impacted by fluctuations of other nonnuclear pools and by nutrient signaling.

Additionally, the question of how metabolites specifically impact specific genomic outputs, is a very important one. Answer to these questions would undoubtedly help us to solve many issues in modern biology and to provide the community with a deep understanding of what we call epigenetics. Given the fact that alterations of the cellular metabolism have been observed in many diseases including cancer, understanding of the metabolism-nucleus signaling and the key players would also shed a new light on new therapeutical approaches.

Metabolism-driven histone modifications contribute to cancer biology 4.1 Cancer cells have reprogrammed metabolism

Cancer cells depend on their reprogrammed metabolism to maintain unrestricted proliferation and to survive environmental stress (e.g., hypoxia, nutrients restriction). This metabolic rewiring also allows to meet the demand for energy supply, macromolecules biosynthesis and redox homeostasis. Pavlova and Thompson summarized six metabolic hallmarks harbored by cancer cells, including deregulated glucose and amino acid uptake, increased demand for nitrogen, plastic modes for nutrient acquisition, increased biosynthesis using metabolites, metabolism driven gene regulation and cell signaling and metabolic interactions with microenvironment 611 . As described above, since chromatin signaling is impacted by cellular metabolism, the specific crosstalk between metabolism and chromatin should be commonly adopted in all cancers and hence this crosstalk should be considered as a pillar of cancer biology. Herein I will firstly introduce metabolic characteristics of cancer cells, including the altered metabolism of glucose, amino acid and then the enhanced biosynthesis of intracellular biomass.

In the second part of this chapter, I'll briefly introduce metabolic signaling to cell nucleus, give examples of how metabolic alterations impact or even dominate cell behavior through epigenetic modulation.

Enhanced glucose uptake and glycolysis

Cancer cells require continuous supply of nutrients to build cytoskeleton and macromolecules for assembling new cells. In mammalian cells, glucose and glutamine are two important nutrients that are used for macromolecules biosynthesis. Indeed, increased glucose uptake was observed in cancer cells by Warburg more than a century ago. Compared with nonproliferating adult cells, cancer cells prefer to use glycolysis even when supplied with abundant oxygen (termed as aerobic glycolysis) 612 . Although carbon sources produce much less ATP (reducing equivalents) through glycolysis than coupled with the TCA cycle. Based on this, positron emission tomography (PET) imaging that determines glucose uptake of tissues using radioactive glucose has been used in the clinics for tumor staging and diagnosis.

Increased glucose uptake and utilization in glycolysis had been proposed to result from dysfunctional mitochondria following early studies. However, later researches identified that many cancer cells did have functional mitochondria and intact oxidative phosphorylation machineries. One hypothesis of such metabolic switch is to enhance glycolytic intermediates production which fuel various biosynthetic pathways, including nucleosides and amino acids. Indeed, such a metabolism is also adopted by rapidly dividing embryonic cells, demonstrating its role in support of cell proliferation. Moreover, elevated pentose phosphate pathway (PPP), which deviates from lactate production after the first rate-limiting step of glycolysis, has been observed in cancer cells. PPP supplies ribose for nucleotides synthesis as well as generates NADPH for fatty acid synthesis 613 . An additional glycolytic intermediate which is required for biomass production is dihydroxyacetone phosphate (DHAP). DHAP is produced from glyceraldehyde-3-phosphate, and is utilized in the biosynthesis of phospholipids when converted into glycerol-3-phosphate.

Besides biosynthesis, enhanced glucose uptake and glycolysis protects cancer cells from excessive oxidative damage. On one hand, reduced use of glucose to fuel the TCA cycle and hence the reduced coupled oxidative phosphorylation, restrict the production of reactive oxygen species (ROS) from mitochondrial respiration 614,615 . On the other hand, increased production of NADPH from alternative pathway (e.g., PPP) helps to maintain the redox homeostasis in response to oxidative stress. Consistently, inhibition of glucose uptake and glycolytic enzymes have been considered as serious anti-cancer strategies 616,617 .

Enhanced glucose uptake and glycolysis are mediated by altered cell signaling. PI3K-Akt signaling pathway is a master regulator of glucose uptake. PI3K-Akt signaling promotes expression of the major glucose transporter GLUT-1 and its translocation to the cell surface 618,619 . In addition, Akt can activate hexokinase (HK) and phosphofructokinase (PFK), which catalyze rate-limiting steps in glycolysis 620,621 . Other oncogenic pathways (e.g., RAS, MYC) and mutations of tumor suppressors (e.g., TP53) have also been reported to be associated with glycolysis 197 . Enhanced glycolysis is also coupled with changes in expression/activity of key metabolic enzymes. Cancer cells predominantly express M2 form of pyruvate kinase (PK), which depending on its homo-multimerizations PKM2 could possess limited catalytic activity compared to M1, expressed in most normal cells. PKM2 in its less active form induces the accumulation of glycolytic intermediates which could be used in synthetic processes, such as nucleic acid-, phospholipid-, and amino acid synthesis. Besides, the expression of pyruvate dehydrogenase kinase 1 (PDK1), which phosphorylates and inhibits pyruvate dehydrogenase, is also increased in cancer cells and hence further restricts the use of pyruvate into TCA cycle 615,622 .

The Warburg effect is likely to be triggered by limited supply of oxygen and nutrients in tumor microenvironment. Under hypoxic condition, hypoxia-inducible factor 1α (HIF-1α) is stabilized, which in turn induces the expression of metabolic enzymes involved in glycolysis, including GLUT, PKM2, PDK1 and LDHA, and leads to glycolytic reprogramming [623][624][625] . Furthermore, acidic environment resulted from glycolysis could stabilize HIF-1α and couid reinforce its function 626 . In addition, PKM2 is activated by metabolites (e.g., fructose bisphosphate, serine), therefore the nutrient-depleted tumor microenvironment disfavors glucose entry into TCA cycle 627,628 .

Interestingly, solid tumors have been described to contain metabolically distinct subpopulations, those who adopt glycolysis and produce lactate, and those who utilize lactate as major carbon source 629,630 . The coexistence of both lactate producing and lactate consuming cells might somehow reflect the cooperation of tumor cells and the adaptation to intratumoral environment 197 . During last few decades, a subpopulation of slow cycling cells has been identified in many types of solid tumors as well as in hematological malignancies. Those slowgrowing, stem-like cells might constitute a minor fraction of the cells. In contrast to bulk tumor cells, these cells could be more resilient to environmental stress (e.g., chemotherapies, nutrients limitation, etc.), therefore they could be responsible for replenishing bulk tumors after treatment and disease relapse 631,632 . Interestingly, in contrast to rapidly dividing bulk tumor cells, quiescent cells display higher dependence on oxidative phosphorylation 633-635 .

Increased demand for glutamine and elevated glutaminolysis (Glutamine addiction)

Increased demand for glutamine by proliferating cells was firstly described by Eagle in the 1950s, when he demonstrated that cultured HeLa cells require 10-to 100-fold excessive of glutamine in culture medium relative to other amino acids 636 . Later studies found that glutamine is depleted from the environment in tumor samples compared to normal tissues 637,638 . Indeed, deprivation of glutamine could lead to cell cycle arrest and cell death in certain cellular contexts 639,640 . Such dependence of glutamine is known as glutamine addiction.

Glutamine addiction ensures glutamate-derived macromolecules biosynthesis. Glutamine is transported across cell membrane via amino acid transporters, ASCT2 (also known as SLC1A5) and SN2 (also known as SLC38A5). Once imported into cytoplasm, glutamine can be converted into substrates through multiple steps that fuel the TCA cycle. Glutamine is firstly converted to glutamate by glutaminase (GLS). Glutamate is then converted into α-KG either by glutamate dehydrogenase (GLUD), which generates ammonium and NADH/NADPH, or by transaminases (oxaloacetate transaminase, GOT; glutamate-pyruvate transaminase, GPT; phosphoserine transaminase, PSAT). In addition, glutamine is the important nitrogen donor for the biosynthesis of nitrogen-containing molecules (e.g., bases of nucleotides, nonessential amino acids, polyamines etc.). Transaminases in glutaminolysis promote the generation of nonessential amino acids (NEAAs) from glutamine, including aspartate, alanine, and phosphoserine. The deamination of glutamine into glutamate donates an amide group to enable nucleotides synthesis de novo and the synthesis of amino sugars and NAD+ cofactors 641 .

Glutamine is also involved in oxidative stress by serving as substrate for glutathione (GSH) synthesis. L-glutamate and cysteine form γ-glutamylcysteine (GCL) catalyzed by glutamatecysteine ligase (GCLC), which together with glycine, generates GSH by glutathione synthase (GSS). Glutathione is an important antioxidant used to deplete peroxide and is vital to multiple cellular processes.

Similar to glucose, positron emission tomography (PET) imaging has also exploited radio labeled glutamine ( 18 F or 11 C) to assess glutamine uptake and utilization by cancer cells, which has shown great potential in cancer diagnosis and therapeutic monitoring in some tumor contexts [642][643][644] . This is especially interesting for tumors that cannot be effectively distinguished by glucose uptake, for example, tumors in brain tissues where physiological heavy glucose utilization is found.

Glutamine metabolism is strictly controlled in cancer. C-Myc is the major driver of glutamine utilization by various cancer cells 645,646 . It increases the expression of glutamine transporters ASCT2 and SN2, therefore enhances glutamine uptake. Furthermore, it can upregulate GLS1, phosphoribosyl pyrophosphate synthetase (PRPS2), carbamoyl-phosphate synthetase 2 (CAD) and promote the utilization of glutamine to glutamate [647][648][649] . Other factors that regulate glutamine metabolism include oncogenes KRAS, PI3K/Akt/mTOR, and tumor suppressors such p53, Rb, LKB1etc. LKB1 and Rb have been reported to inhibit ASCT2 expression and reduce glutamine uptake 650,651 . KRAS decreases the expression of GLUD but enhances the expression of GOT and therefore increases NADPH production 652 . mTOR can repress SIRT4 expression, which is responsible for inhibition of GDH 653 . PI3K/Akt and p53 were reported to promote the generation of GSH and therefore protect cells from ROS damage 654, 655 .

Altered metabolism of other amino acids

Increased biosynthesis of serine

Mammalian cells can acquire serine from the environment via alanine/serine/cysteine/ threonine (ASCT) transporters (e.g., ASCT1, ASCT2), system A transporters (e.g., SAT1, SAT2) and alanine/serine/cysteine (ASC) transporter system 656 . Serine can be biosynthesized from glycolytic intermediate 3-phosphoglycerate (3-PG) via the phosphorylated pathway, which requires the action of several enzymes including phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1), and phosphoserine phosphatase (PSPH).

Additionally, serine can be generated from glutamate transaminase reaction by the catalysis of PSAT1 and PSPH.

Serine is consumed in folate cycle. The generation of 5,10-methylenetetrahydrofolate (CH2-THF) from tetrahydrofolate (THF) requires the donation of one carbon unit by serine accompanied by the production of glycine with the action of serine hydroxymethyltransferase (SHMT). The produced CH2-THF is needed for thymidine synthesis, and when converted to 10-formyl-tetrahydrofolate (CHO-THF) through folate cycle, is needed for purine synthesis. In addition, folate cycle provides carbon source for SAM pool through methionine cycle.

Collectively, serine contributes to nucleoside biosynthesis as well as supports SAM production and protein methylation. As noted above, serine can be used for GSH production and thus plays an important role in redox homeostasis.

Increased serine metabolism has been observed in tumor cells 657 . Some tumors depend on the availability of serine supply, depletion of serine suppresses cell proliferation in those cancer contexts 658 . Elevated expression of enzymes involved in the serine metabolism, including PHGDH amplification and increased activity of SHMT1 (cytosolic SHMT) have been identified in a variety of cancers [659][660][661] . Furthermore, increased serine synthesis pathway (SSP) such as PHGDH overexpression is associated with higher tumor grade in gliomas 662 . The contributions of serine metabolism to tumor cells proliferation and survival could, at least partially, provide carbon for nucleotide synthesis and might involve other biological processes utilizing one carbon substrates including protein methylation and might also involve redox homeostasis 551 .

Serine metabolism is regulated by various factors (e.g., p53, c-Myc, TAp73, ATF4, G9a, NRF2, PKCζ) in tumors 613 . Asparagine serves as amino acid exchange factor for serine and threonine transport. In human cervical cancer cell lines, inhibition of asparagine synthase or deprivation of asparagine supplement impair the uptake of serine, leading to an increased expression of SSP enzymes to compensate for decreased serine uptake 663 . p53 is a critical regulator for cells to deal with extracellular depletion of serine. Upon serine starvation, p53 activates p21 to promote GSH production to limit ROS stress 664,665 . In addition, the oncogene c-Myc has been found to regulate serine and glycine metabolism (e.g., increases PSPH expression and enhances SSP) under normal as well as starvation conditions 666 .

Addiction to branched-chain amino acids

Branched-chain amino acids (BCAAs) including leucine, isoleucine and valine are essential amino acids that must be acquired from diet or from protein degradation. BCAAs are metabolized in cancer cells by branched-chain aminotransferase (BCAT1 in cytosol, BCAT2 in mitochondrion) into branched-chain α-keto acids (BCKAs). In this process α-KG is converted to glutamate by receiving the amide group. The produced glutamate could be used in nucleotide and nonessential amino acid synthesis as aforementioned. BCKAs can be further catabolized to produce acetyl-CoA or succinyl-CoA 613, 667 . To this end, BCAAs metabolism are enhanced in certain types of cancer and are required for cancer growth 668 . Furthermore, BCAT1 was found to be associated with wild-type IDH expression and balance α-KG with 2-HG oncometabolites in glioblastoma and ovarian cancer 669 . In addition, accumulated intracellular BCAAs levels, especially leucine, can activate mammalian target of rapamycin (mTOR) via cytosolic leucine sensor proteins and promote cancer progression 670 .

Increased catabolism of tryptophan has also been observed in some types of cancer.

Tryptophan-2,3-dioxygenase (TDO), which is responsible for the conversion of tryptophan to kynurenine was found to be overexpressed in various tumors 671,672 . Tryptophan catabolism can modulate immune cell function including promoting Treg and inhibit effector T cell function, therefore contributes to tumor invasion 673,674 .

Asparagine metabolism plays a crucial role in tumor survival, although the mechanism has not been clearly elucidated. Asparagine is produced from aspartate by asparagine synthetase (ASNS) using glutamine as amide donor. Acute lymphoblastic leukemia cells lack ASNS and rely on exogenous asparagine. Based on this fact, L-Asparaginase that can deplete extracellular asparagine, has been used as an anticancer treatment in ALL 675, 676 .

De novo lipid synthesis

While the de novo biosynthesis of fatty acids is low in normal adult tissues, tumor cells possess increased lipid synthesis 677,678 . Lipid synthesis enzymes are upregulated in tumor samples and their inhibition reduces tumor growth [679][680][681] . The increased lipid synthesis might not only facilitate the formation of lipid bilayers but also enables the alteration of cell membrane composition with increased oxidative damage-resistant saturated fatty acids 682 .

Utilization of acetate, ammonia, ketone bodies, lactate

Cancer cells face deficient nutrients supply. In addition to glucose and glutamine, tumor cells are able to take advantage of nonconventional nutritional sources including acetate, ammonia, ketone bodies and lactate.

Lactate:

As noted, cancer cells have increased aerobic glycolysis as well as lactate production. Lactate has been found to be absorbed by cancer cells and be oxidized in mitochondrial respiration for ATP production in several cancer cells 683 . Lactate might also be the carbon source for lipid synthesis in lung cancer cell lines (i.e., HeLa and H460) 684 .

Acetate:

ACSS2 enzyme, which utilizes acetate for the production of acetyl-CoA, was found to be upregulated in breast, ovarian and lung tumor samples 613 . Acetate-deriven acetyl-CoA contributes to cancer growth through modulating histone acetylation as well as providing carbon for fatty acid and phospholipid.

Keton bodies:

Ketone bodies are mainly synthesized by liver cells (ketogenesis) and are transported and utilized by extrahepatic tissues such as brain and skeletal muscle (ketolysis). The normal hepatocytes per se are not able to perform ketolysis due to the lack of the key ketolytic enzyme OXCT1. However, the reactivation of ketolysis resulting from increased OXCT1 expression has been discovered in liver cancer cells in response to starvation 685 .

Ammonium ions:

Free ammonium ions often accumulate in tumor environment. It has been found that ammonia can be used for glutamine production by cancer-associated fibroblasts and then transferred to and be used by cancer cells 686 . Ammonia might also be a source for proline, aspartate, glutamate and BCAA generation in breast cancer cells 687 . In addition, cancer cells have been reported to use exogenous proteins, including living cells, apoptotic bodies under nutritional stress 611 .

Cell metabolism signals to nucleus via epigenetic modifications

Emerging evidences suggest that cellular metabolism is not merely the adapted outcome of extracellular nutrition, but can in turn transmit the information of nutritional availability to nucleus via metabolites and metabolic enzymes. Accordingly, they coordinate chromatin behavior and gene expression and the metabolic state. This coordination is highlighted in cancer, as cancer cells have metabolic reprogramming and display metabolic plasticity to survive nutritional perturbations. The crosstalk between cell metabolism and chromatin, and their contribution to cell biology have received much attention.

As described above, acetyl-CoA is the central metabolite bridging multiple biosynthetic and catabolic pathways, and is the substrate for protein acetylation. Various studies have documented that, the abundance of acetyl-CoA could link cellular metabolism to the cancer cell behavior via epigenetic modifications of histone and non-histone proteins.

Akt phosphorylates ACLY and enhances its activity 688 . Increased glucose availability as well as introduction of Akt increase acetyl-CoA abundance, thereby leading to increased global histone acetylation, which in turn promotes gene expression and tumor development 556 . AMPK was shown to be required for acetyl-CoA homeostasis and histone acetylation. Depletion of AMPK reduces histone acetylation and displaces BET protein, the combination of BET inhibitor and AMPK inhibitors suppresses the development of MLL-rearranged AML 583 . ACC1 catalyzes carboxylation of acetyl-CoA to malonyl-CoA for de novo fatty acid synthesis. TGFβactivated kinase mediated phosphorylation of ACC1 inhibits its activity, leading to increased acetyl-CoA levels and the acetylation of transcription factor SMAD2, thereby conferring to increased metastasis in breast and lung cancer 567 . Similarly, metformin treatment results in AMPK-dependent ACC phosphorylation, leading to increased acetylation of histone and nonhistone proteins (e.g., NF-kB p65 subunit) and enhanced expression of NF-kB target genes 689 .

In addition to acetyl-CoA, a number of studies reported that DNA and histone methylation is sensitive to intracellular SAM levels [690][691][692] .

Metabolic enzymes have been implicated in tumorigenesis as well. IDH1/2 mutations have been identified in glioblastoma and in leukemia cells. IDH1/2 mutants can produce oncometabolite 2-HG from α-KG, which competitively inhibit α-KG dependent demethylases and result in DNA and histone hypermethylation that contribute to tumorigenesis 693 . In addition, α-KGDH binds GCN5/KAT2A and produces succinyl-CoA at local sites, which promotes GCN5 dependent H3K79 succinylation and facilitates gene expression and tumor growth 151 .

Collectively, these findings suggest that metabolic reprogramming might play an important role in cancer biology through epigenetic modulations.

Identification of a gene associated with poor prognosis in ALL

Overview of ALL

Acute lymphoblastic (lymphocytic) leukemia (ALL) is a hematological malignant disorder which derives from clonal lymphoid precursors. More than 75% of ALL patients are B cell type (B-ALL). ALL affects mostly children, with about 60% of the patients are below 20 years 694 .

Typically, the clinical manifestations include hematopoietic failure, e.g., anemia, thrombopenia and leukopenia, and the resultant signs and symptoms, e.g., fatigue, dyspnea etc. Patients can also display splenomegaly, hepatomegaly, or lymphadenopathy when leukemic blasts infiltrate the corresponding organs. Diagnosis is defined by cell smear showing that leukemic blasts comprise > 25% of nucleated cells in bone marrow. The more precise diagnosis of ALL relies on the definition of characteristic molecular and cytogenetic alterations as well as on immunophenotyping, which not only provides information that is useful for diagnosis and risk stratification, but also contributes to treatments guidance.

Traditional treatment strategy corresponds to multiagents-based chemotherapy, with the conventional backbone consisting of cyclophosphamide, anthracyclines, and corticoids.

Currently, the treatment strategy is strictly dependent on patients' risk stratification in order to orient intensive chemotherapy towards the high-risk patients and to save the low-risk patients from excessive toxicity of chemotherapy. Thanks to the adoption and development of riskadapted treatment strategies, the last 5 decades have seen the improvement of patient managment so as ALL is now one of the most curable cancer, with long term survival of pediatric patients approaching 90%. However, around 10-15% of pediatric patients still undergo relapse. Traditionally, the salvage therapies are Allogenous hematopoietic stem cell transplantation (Allo-HCT) following second complete remission (CR). However, with the adoption of multiple agents-based chemotherapy during the initial treatment, the relapsed patients are often resistant to the chemotherapy, therefore it could be difficult to reach second CR. Besides, a small percentage of patients develop resistance at the early onset of treatment (designated as refractory cases). Indeed, relapse and refractory (r/r) are still the main cause for treatment failure in childhood patients.

Additionally, the treatment for adult patients is much more challenging, with nearly half of adult patients relapse and 5-year overall survival (OS) falls to less than 50% 695 . The main reasons of dismal outcome of adult patients are higher frequency of cytogenetic abnormalities (such as Ph-like, hypodiploidy, near-haploidy, complex karyotype etc.), less tolerance over childhood regimens (such as the usage of asparaginase), less treatment adhesion and obedience, higher relapse and resistance rate, etc. Hematologists and researchers are trying to improve the adult patients' outcome. Inspired by the success of childhood ALL treatment, adolescents and young adults (AYA, 15-39 years) are also prescribed with pediatric ALL regimens now. However, older patients cannot tolerate childhood regimen especially the frequent applications of L-asparaginase. Another promising approach is the use of targeted therapy. Indeed, targeted therapy increases the response rate of older adults who cannot tolerate multi-agents based chemotherapy. Lately, new therapies, such as monoclonal antibodies (e.g., CD19, CD20, CD22 antibodies, etc.), bispecific anti-T receptor/CD19 antibody blinatumomab, and chimeric antigen receptor (CAR) T cell therapy (e.g., CD19 CAR) have brought additional choices for treating r/r patients [696][697][698] . However, there are limitations associated with these approaches such as immune escape, less persistence, the high expenses of antibodies, and the less experienced techniques etc. 699 Prognostic factors are the main bases for risk stratification and treatment choices.

Traditionally, prognostic factors include age, immunophenotype, WBC count at disease onset, the infiltration of central nervous system (CNS), morphological CR status etc. Adults (>35 years), T cell subtype, high WBC (>30×10 9 in B-ALL, >100×10 [START_REF] Jerkovic | Higher-Order Chromosomal Structures Mediate Genome Function[END_REF] in T-ALL), delayed remission (>4 weeks) or not remission are poor prognostic factors. More recently, minimal residue diseases (MRD), which refers to the residual blasts after initial therapy has been incorporated to the evaluation for prognosis and used to monitor the relapse.

Cytogenetic and molecular abnormalities are important prognostic factors. In B-ALL, the Ph chromosome, MLL rearrangement, hypodiploidy, etc. are associated with poor outcome. In contrast, ETV6-RUNX1 fusion, hyperdiploidy, E2A-PBX1 translocation, triosmy (+4, +10, +17) etc., are associated with good prognosis. Notably, the novel Ph-like subtype whose transcriptional profile is much alike those seen in the Ph positive blasts, but lack of BCR-ABL1 oncoprotein, has been identified. Ph-like cases are featured by frequent mutations of B cell development and differentiation genes, activation of cytokine receptor genes and kinase pathways, and is associated with persistent MRD and higher relapse rate compare to non Phlike counterparts 700 . Besides, MEF2D rearrangement, DUX4 rearrangement, ZNF384 rearrangement, PAX5 mutation, IKZF1deletion etc., have been reported to predict prognosis.

Prognostic factors involving genetic abnormalities might provide evidence for the development of targeted therapies. The most successful example is the exploitation of tyrosine kinase inhibitors (TKIs). Nowadays, TKIs combined with chemotherapy has been the standardized treatment strategy for BCR-ABL1 positive patients. In addition, the inhibitors targeting JAK-STAT have entered clinical trials.

In conclusion, the treatment of high-risk and r/r ALL cases remains challenging. Currently, risk-adapted chemotherapy remains the first-line treatment strategy. Identifying prognostic factors and understanding the corresponding pathophysiology could not only minimize relapse rate by early application of intensified chemotherapy but also could help to decide Allo-HCT at CR1 for high-risk patients. In addition, this would guide the clinicians to prescribe the optimal treatment and to avoid the unnecessary toxicity of the intensified chemotherapy for standard risk patients. This would also point to new targeted therapies.

Ectopic activation of testis-specific genes is associated with cancer aggressiveness

Tissue development and differentiation is a programed process. Mature tissues composed of differentiated cells are generated by successive expression of specific genes. Under these conditions many tissue-specific genes remain silent. These genes' expression activation and silencing are precisely controlled by the cell-type specific epigenetic profile. Malignant transformed cells have a life-sustaining capability, which is facilitated by the activation of genes underlying the so-called cancer hallmarks.

The abnormal signaling in cancer cells is to some extent established and stabilized by an altered epigenetic landscape. One common feature displayed in multiple cancers is a genomewide DNA hypomethylation and locus-specific hypermethylation (e.g., CpG islands).

Methylation-mediated repression of critical tumor suppressor genes has been well documented to be oncogenic events 701 . Additionally, global DNA hypomethylation contributes to a higher genomic instability (e.g., chromosome rearrangements) which is a frequent cancer-associated event. However, an unexplored feature of cancer is the oncogenic activity of the many genes that are aberrantly activated due to DNA hypomethylation.

Most of the cancer-specific hypomethylated genes are testis-specific genes (termed as cancer-testis, C/T, also termed as cancer-germline, C/G), whose expression is restricted in normal adult tissues. Generally, testis-specific factors play a role in spermatogenesis. It is postulated that the ectopic activation of C/T genes might contribute to the signaling network required for cell transformation and/or malignancy maintenance 702,703 . This theory has been documented by several studies. For instance, Janic and colleagues have identified a soma-togermline transformation with an ectopic expression of germline genes during malignant brain tumorigenesis in Drosophila, while inhibition of these germline genes suppress tumor growth 704 .

There are many examples of the functional studies of the C/T genes in cancer. One interesting example is the aberrantly activated PRAME gene, which was found to be an antagonist for retinoic acid receptor (RAR), inhibiting the RAR-dependent cell differentiation program 705 .

The ectopic activation of C/T genes can be associated with cancer behavior and patients' prognosis. In most cases, the frequent activation of C/T genes indicate higher tumor grade and poor outcome 706 . For instance, in a cohort of 293 lung cancer cases, Rousseaux and colleagues have identified an ectopic gene expression signature associated with a subset of highly aggressive tumors, predictor of patient's outcome 707 . In addition, Emadali and colleagues discovered that an ectopically-expressed gene CYCLON, is associated with cancer aggressiveness and resistance to anti-CD20 antibody in high-risk lymphoma. CYCLON gene expression is driven by MYC and could be sensitive to the BET inhibitor JQ1. The depletion of CYCLON could reverse drug resistance 708 .

Higher expression of C/T genes could occur in cancer stem cells (CSCs) compared to the bulk cells. For example, MAGEA9 is highly expressed in CSCs in hepatocellular carcinoma.

In addition, compared to the bulk of leukemic blasts, CD34+CD38-cells (known as leukemic stem cells) have higher expression of MAGEA1.

C/T genes are plausible candidates for immunotherapy since they could be immunogens. For instance, CD4+ T cell immunotherapy targeting MAGEA3 has achieved partial response in advanced esophageal cancer, urothelial carcinoma and osteoblastoma 709 . Anti-C/T tumor vaccines have been tested in multiple types of cancers, some of the corresponding clinical trials show the low-toxicity and promising efficacy of this approach 710 . Indeed, increasing evidence indicate that C/T genes can be important in cancer development, with demonstrated oncogenic roles in establishing/promoting multiple cancer hallmarks.

Examples are C/T genes role in growth signaling, metabolism reprogramming, resisting apoptosis etc… Some of these mechanisms have been reviewed by Tongelen and colleaguess and are summarized in (Figure 21) 703 . What is fascinating about C/T genes is that, because of their limited expression in normal tissues, they could be promising therapeutic targets in cancer with minimized side effects 710 .

Figure 21. C/T genes contribute to various pro-tumoral processes.

Examples of C/T genes sustaining cancer hallmarks, including cell proliferation, angiogenesis, metastasis, apoptosis, senescence etc... For more details, please refer to 703 . From 703 .

Identification of six genes' signature associated with patients' outcome in ALL

Previously, a work conducted in collaboration between the Grenoble team and the Shanghai Institute of Hematology identified six genes whose combined expression is associated with prognosis in ALL 711 . In this work, they firstly screened genes that are exclusively expressed in testis/placenta/embryonic stem (ES) cells but not in normal adult tissues. In the next step, genes that are sporadically expressed in normal bone marrow were filtered out. The rest of the remaining genes were subjected to screening for ectopically activated genes in ALL. Among the imposed criteria, was an expression above mean values + 3 standard deviations of 112 normal adult tissues and occurring in more than 10% of the ALL blasts. Finally, genes whose expression was associated with prognosis in ALL were screened out and were ranked by the pvalue of their association with prognosis. This leads to the identification of 6 genes (AK022211, FASTKD1, STARD4, CAMSAP1, PCGF6 and SH3RF) (Figure 22). The figure shows the workflow to screen genes ectopically expressed in ALL that are associated with prognosis in two ALL cohorts, GSE11877 and GSE7440. After ranking the p-value of the gene's association with prognosis, the top six genes were screened out and used for further study. ES: Embryonic stem cells; BM: Bone marrow. From 711 Among these 6 genes, expression of AK022211, FASTKD1 and STARD4 is associated with poor prognosis, termed as "negative genes", while the expression of CAMSAP1, PCGF6 and SH3RF is associated with good prognosis, termed as "positive genes". Further, they designed an algorithm combining these 6 genes, which can stratify childhood ALL. Patients with blasts expressing one or more of the "negative genes" plus none or only one of the "positive genes" are assigned as group P3, while the others are assigned as P1 and P2 based on the expression state of "negative genes" plus at least two "positive genes". These assignments successfully stratified patients into relatively good (P1&P2) and poor (P3) outcomes. Furthermore, the six gene's algorithm was applied to another two adult cohorts and successfully predicted patients' outcome as well. Notably, compared with childhood patients, adult cases identified higher frequency of patients that have been assigned to P3 group, which is in accord with the overall worse prognosis of the adult patients observed in the clinics.

Following a supervised analysis of the global transcriptomic data, the gene expression profile of the aggressive P3 group was identified. The authors of this work found that in childhood and in the adult cohorts, P3 group exhibited similar expression pattern. One interesting feature of this gene expression profile is that a group of hematopoietic stem cell genes was activated, while genes involved in proliferation and cell cycle were inhibited. These data suggest that the aggressive leukemia might acquire non-dividing and stem-like properties. Indeed, standard treatments of ALL are based on cytotoxic agents, many of which target important steps in cell division (such as DOX inhibits DNA topoisomerase II) and metabolism (MTX inhibit dihydrofolate reductase and thus purine and pyrimidine synthesis), thereby cause severe effects in rapidly proliferated cells, low-cycling cells are documented to be less sensitive to chemotherapies and are viewed as the leading causes for refraction/relapse 632 . These authors further checked ectopically activated genes individually with respect to their association with prognosis. Among which FASTKD1 alone was significantly associated with shorter survival in two ALL cohorts (GSE11855 and GSE7440) (Figure 24) 711 .

FASTKD1 is a potential regulator of mitochondrial activity

Mitochondrial genome

Mitochondrial genome (mtDNA) is alike bacterial DNA in that it is compacted and organized into nucleoid structures. Mitochondrial nucleoids are genetic units of mtDNA segregation and allocation to the separate mitochondria during mitochondrial fission 712 . Each mitochondrial nucleoid contains 1-2 copies of mtDNA (1.4 on average in human cells and 1.1-1.5 on average in mouse cells) 713 . mtDNA is a double-strand circular DNA comprising of approximately 16.5 kb. mtDNA genes are encoded by the two strands named here chains: the heavy chain (H) encodes 2 rRNAs, 14 tRNAs and 12 mRNAs, and the light chain (L) that encodes ND6 mRNA and 8 tRNAs (Figure 25). All of the 13 polypeptides are enter the respiratory complexes, also known as oxidative phosphorylation (OXPHOS) complexes, including complex I, III, IV and V.

mtDNA transcripts processing and maturation

mtDNA is transcribed from both strands, producing polycistronic transcripts corresponding to almost the entire length of mtDNA. mtDNA transcripts (mtRNAs hereafter) are subsequently processed and matured giving rise to functional mtRNAs. Primary mtRNAs are processed in dynamic protein structures called RNA granules (MRG), which are composed of mtRNAs and the associated RNA-binding proteins. Most of the mtRNAs are processed via tRNA punctuation, where tRNAs flanking most of the mt-mRNAs and mt-rRNAs coding regions are cleaved by RNase P and RNase Z at the 5' ends and 3'ends of ORFs, respectively. The resultant mRNAs will be polyadenylated with shorter poly-A tails than nuclear mRNAs, and are capped with NAD+. This cap influences mRNA stability and protein translation. The mtRNAs processing, maturation, degradation and translation are tightly regulated by a repertoire of factors associated with MRGs 712 .

OXPHOS complexes

In mammalian cells, OXPHOS complexes are consisted of multiple subunits encoded by In eukaryotes, OXPHOS is the major source of energy. OXPHOS complexes consume NADH, FADH2, which are mainly produced by the TCA cycle and β-oxidation of fatty acids and produce ATP. Besides ATP production, OXPHOS contributes to the generation of harmful superoxide ions prominently by complex I and III and therefore contributes to the cell oxidative stress. OXPHOS-mediated oxidative stress was traditionally proposed to be harmful to cancer cells survival. However, increasing evidence shows that some types of cancers rely on OXPHOS, leading to the OXPHOS as therapeutic targets in these cancer contexts 717,718 .

FASTKD1 regulates mtRNAs biology

FASTKD1 is one member of the FAS-induced serine/threonine kinase domain containing protein family (FASTK) which consists of five other members, FASTKD2-5 and FASTK.

FASTK family protein were reported to localize in mitochondria 719 . These proteins share FAST_1, FAST_2 domains, which are of unknown function as well as RAP domain which is predicted to be a RNA binding module (Figure 27) 720,721 . While FASTK has been documented to be a kinase, the other members do not appear to ensure any catalytic activity because of the lack of a conserved active site. Studies revealed that these proteins interact with the newly synthesized mitochondrial RNAs (mtRNAs) in mitochondrial RNA granules (MRGs), which are sites for posttranscriptional mtRNAs processing, maturation and mitochondrial ribosome biogenesis. Therefore they could regulate mitochondrial gene expression and the activity of respiratory complexes 719,722,723 . Indeed, FASTKD1 is documented as a negative regulator of ND3 gene 721 . A few studies uncovered the role of FASTK family proteins in cell biology and diseases.

FASTK was found to be a survival protein, which is displaced from mitochondrion under stress and protect cells from UV-induced apoptosis. FASTKD2 is the only member that has been linked with mitochondrial encephalomyopathy 724 . In addition, FASTKD1 was reported to decrease complex I-dependent respiration and protect mouse embryonic fibroblasts (MEFs) from oxidative stress induced death 725 .

THESIS OBJECTIVES

FASTKD1 is a potential regulator of mitochondrial activity and according to the team's previous study, is associated with poor prognosis in acute lymphoblastic leukemia. Based on this, my thesis project aims to characterize its oncogenic role in ALL. Since it is established that cell metabolism could regulate histone PTMs, this project was built on the link between mitochondrial metabolism and histone PTMs.

The preferential impact of FASTKD1 on mitochondrial activity and hence on histone nonacetyl acylations prompted us to investigate their roles in transcriptional regulations. A previous study from our laboratory suggested that the balance of histone acyl/acetyl marks impacts BET -chromatin interaction dynamics. Therefore, taking FASTKD1 as a tool, we also considered this hypothesis as well. Following this hypothesis, a role for acyl/acetyl-driven alteration of BRD4 dynamics and its impact on gene expression was considered.

Specifically, the main body of thesis project aims to:

1) characterize the function of FASTKD1 in regulating mitochondrial activity 2) document the correlation between mitochondrial activity and histone PTMs 3) substantiate the hypothesis that the relative levels of acyl/acetyl influence BETchromatin interaction dynamics 4) address the role of mitochondrial metabolism-driven alteration of histone acyl/acetyl in transcriptional regulation

RESULTS

FASTKD1 represses mitochondrial activity in ALL 1.1 Exogenous expression and depletion of FASTKD1 in B-ALL cell lines

To study the function of FASTKD1 in ALL, we carried out the 'loss of function' and 'gain of function' experiments. I first tested several commercial antibodies from Abcam, Abclonal and Origine company, none of them displayed specific or stable signal in various ALL cell lines.

We also generated our own antibody in house but still failed to get valid signal. Therefore, during all the following project, I either used flag-tag for exogenously expressed protein, or use qPCR to determine transcriptional level of this gene.

After introducing LeGO-FASTKD1-flag recombinant DNA into REH and NALM6, expression of the protein was checked with anti-flag antibody as well as with qPCR, the latter of which displayed 8 (NALM6) or 12 folds (REH) of mRNA upregulated than in empty vector (EV) transfected cells (Figure 28A). In the case of knockdown effect, 5 out of 10 shRNAs successfully knocked down the mRNA level to less than 1/2 in REH, as well as 3shRNAs in

FASTKD1 overexpression or depletion do not cause significant phenotypic alterations in ALL cell lines

Since FASTKD1 is associated with inferior outcome in ALL, and was reported to confer a protective role in MEF cells under oxidative stress conditions 725 , we also tested if overexpression or depletion of FASTKD1 cause any changes in ALL cell lines. For this purpose, we counted the cells for cell proliferation, performed CCK-8 assay to measure cell viability after exposure to chemotherapy (DEX, Ara-C), PI staining to detect cell cycle. Unfortunately, none of these experiments gave stable or significant differences between control and genedepleting or gene-overexpressing cells (Figure 29).

FASTKD1 represses mitochondrial activity

Following the previous studies uncovering that FASTKD1 plays a role in mitochondrial biology 721 , its role in mitochondrion was also checked in our ALL cell lines. Firstly, with FASTKD1 overexpressing cell line, we confirmed that this protein is located in mitochondrion in ALL cell lines (Figure 30A). RNA-seq analysis on FASTKD1 knockdown and knockout cells revealed that depletion of FASTKD1 led to the upregulation of all mtRNAs encoded by mtDNA (Figure 30B). Repressive function of FASTKD1 in mitochondrial transcription (with ND2 and ND3 as the most affected genes) was also validated with qPCR (Figure 30C). Protein level of respiratory complexes were checked with cocktail OXPHOS antibody, demonstrating that FASTKD1 represses the expression of OXPHOS complexes, especially complex I in protein level (Figure 30D). Finally, we measured the oxygen consumption rate (OCR) using seahorse XFe96 analyzer and uncovered that both the basal level and maximal level of OCR increased in KO cells (Figure 30E).

To confirm that increased mitochondrial activity is mediated by depletion of FASTKD1, we re-expressed FASTKD1 in ko-1 cells, which in turn repressed the mitochondrial gene expression (Figure 30F). In addition, mtDNA copy number showed no significant difference between control and knockout cells, which is consistent with previous study uncovering that FASTKD1 regulates post-transcriptional processes of mitochondrial genes (Figure 30H).

Mitochondrial activity and β-oxidation drive histone PTMs

Mitochondrial metabolism drives histone modifications

It is well established that cell metabolism could influence nuclear gene expression by regulating DNA methylation or histones acetylation. Indeed, as previously discussed, metabolites can act as substrates (SAM, acetyl-CoAs etc) or cofactors (FAD, α-KG etc) for enzymes capable of adding or removing histone post-translational modifications. More emerging evidences suggest that in cancer cells, metabolic alterations and subsequent changes in chromatin modifications could potentially modulate cellular characteristics and impact tumor establishment and growth.

Since our previous data demonstrated that FASTKD1 could regulate the mitochondrial activity, we postulated that depletion of FASTKD1 might also affect mitochondrial metabolic activities and hence affect histone PTMs, specially histone acylation. To this end, we performed MS analysis in an unbiased manner the change of histone acylation with a focus on propionylation and butyrylation. As shown in Figure 31A, there is an increased level of H4K5 and H4K8/12bu in two ko cells compared to WT cells. We performed western blot analysis to validate these findings by an independent approach. After the knockout of FASTKD1, we observed an overall change in various site-specific of histone modifications, especially affecting non-acetyl acylations: butyrylation, beta-hydroxybutyrylation, crotonylation, lactylation and succinylation (Figure 31B). Since many of the novel acylations are of unknown function, we focused our latter research on histone crotonylation and butyrylation, which have been reported to mark active transcription. Interestingly, our data showed that there was an increase in H4 lysine 5/8 butyrylation/crotonylation in FASTKD1 KO cells, while no significant change was observed in acetylation of the same sites (Figure 31B). We further treated the cells with OXPHOS inhibitors, rotenone, which is known to inhibit complex I, and discovered a decrease of histone acylation (Figure 31C). They are also involved in ketogenesis, ketolysis and amino acid catabolism. Since in KO cells, several acylations marks (bu, cr, bhb) changed, from a biochemical point of view, we hypothesized that fatty acid metabolism could be involved. To study the role of fatty acid metabolism in histone acylation, we take advantage of some compounds that either fuels or inhibit fatty acid metabolism (Figure 32A).

Octanoate is an 8-carbon fatty acid which is known to fuel beta-oxidation. Addition of octanoate to the cell culture medium drastically enhanced the overall level of histone modifications, including acetylation, butyrylation, and crotonylation (Figure 32B). Additionally, treatment of cells with crotonate (4 carbon non-saturated fatty acid) not only increased histone crotonylation but also acetylation (Figure 32 Figure 32C). Repressing acyl-CoA thiolase (KAT) involved in β-oxidation spiral with ranolazine attenuates the basal, as well as octanoate induced acetylation and acylation (Figure 32D). Furthermore, inhibition of long chain fatty acid-CoA synthetase (ACSL) enzymes with Triacsin C, or of acetyl-CoA carboxylase 1 (ACACA, producing malonyl-CoA from acety-CoA in lipid de novo synthesis) with ND-630 reduced histone acylation (Figure 32E&F). Finally, we also knocked down key enzymes in β-oxidation. These data suggest that β-oxidation indeed might be the important pathway in driving histone acylation and acetylation. Western blot analysis of H4K5ac and H4K5bu were performed in urea cell extracts from cells pre-treated with the indicated chemical compounds. REH (upper panel) and SEM (lower panel) cells were treated with octanoate for 24 hours with the indicated concentrations (B). REH was treated with crotonate for 6 hours with the indicated concentrations before western blot analysis (C). REH WT and two ko cell lines were exposed to 0.3 mM ranolazine with and without 2 mM octanoate for 24 hours (D), or with100 nM ND-630 for 6 hours (E) , or with 3 μM Triacsin C for 16h (F).

A C B

Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients' samples

Inspired by our data one cell lines, we come back to ALL patients' samples. Bone marrow samples from a cohort of 31 B-ALL patients were acquired and protein and RNA were extracted in parallel and used to monitor specific gene expression by RT-qPCR and the occurrence of histone PTMs by ELISA. First, we found that CPT1A mRNA level is highly correlated with ND2 (rs = 0.531) and ND3 (rs = 0.508) (Figure 33A,B

Acyl/acetyl determines the dynamic interaction of BET with chromatin

3.1 BRD4 binds acetylated but not K5 acylated histones BET family proteins are important readers that link acetylation with transcriptional regulation.

Previous study indicated that first bromodomain (BD1) of BRDT accommodates diacetylated lysine residue (H4K5acK8ac) but not when the K5 residue is butyrylated (i.e., H4K5buK8ac).

Therefore the ratio of acyl/acetyl might impact BET-chromatin interaction dynamics: higher ratio disfavors BRDT-chromatin interaction, resulting in a loose and dynamic bound state, while a decreased ratio favors a tight binding and leads to a stable interaction 145 . We noticed that FASTKD1-mediated mitochondrial activity prominently impacts non-acetyl acylations but not acetylations, therefore we decided to take this cell model as a tool to test such hypothesis.

In this setting, we considered BRD4, the ubiquitously expressed BET protein. We tested BRD4's ability to bind H4K5 and K8 ac/bu peptides following a pull down experiment. As expected, BRD4's binding to an acetylated H4 tail peptide can be inhibited by butyrylation, especially H4K5bu (Figure 34). Cell extracts were prepared from REH WT cells and was incubated with beads conjugated with the indicated modified peptides. BRD4 was visualized after the pull-down assays.

Acyl/acetyl ratio determines BRD4 binding affinity to chromatin in REH cells

As described in the introduction part, we proposed the hypothesis that BRD4 binds to chromatin in a dynamic manner, furthermore, such binding dynamics is determined by acyl/acetyl ratio.

Since our previous data suggested that the depletion of FASTKD1 increases dramatically histone butyrylation and crotonylation but not significantly acetylation. This situation should in turn result in an increased acyl/acetyl ratio. We decided to take use of these cell line models to verify our hypothesis. Firstly, we confirmed that the altered acyl/acetyl did not change the amount of total BRD4, nor the ability of BRD4 binding with chromatin in control and KO cell lines (Figure 35A). To visualize the bound state of BRD4 to chromatin, we took advantage of salt elution assay and JQ1 dissociation assay. Indeed, we obtained more soluble BRD4 in KO cells using a lysis buffer containing 200 mM NaCl compared to WT cells, which suggest less tightly bound state of BRD4 to chromatin in KO cells (Figure 35B). Similarly, when treating cells with low dose of JQ1 to competitively dissociate BRD4 with chromatin, we obtained more soluble BRD4 in KO cells, which is in accord with a more dynamic binding in vivo (Figure 35C). Furthermore, we treated REH cell nuclei with butyryl-CoA to increase butyryl/acetyl ratio and performed salt elution assay to obtain BRD4. As shown in Figure 35D, we eluted more BRD4 in butyryl-CoA treated compared to control cell nuclei. Collectively, these data suggest that increased acyl/acetyl ratio contributes to loosely bound state between BRD4 and chromatin in REH cells. A. REH WT and two KO cell lines were extracted with lysis buffer containing 500 mM salt, the supernatants containing total BRD4 were then collected and diluted to reach a concentration of 250 mM, which used in a peptide pull-down assay with the indicated peptides. BRD4 was then eluted from beads and probed following a western blot analysis.

B. REH WT and two KO cells were used to extract proteins with lysis buffers containing increased concentrations of NaCl. After centrifugation to pellet nuclei, supernatants were used to detect soluble BRD4. In this figure BRD4 appeared to be less tightly bound to chromatin in KO cell groups when salt concentrations reached 200mM (red text). (concentrations as indicated) before cell lysis in a buffer containing 100 mM NaCl. Less tightly bound BRD4 was dissociated from chromatin in KO cells when treated with 0.5 μM JQ1.

D.

Nuclei from REH cells were used for acylation reaction. Left panel shows the H4K5bu and H4K5ac level after acylation reaction. Acylated nuclei were used in a salt elution assay similar to B (right panel).

Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells

Previously, Pr. Heinz Neumann and Dr. Martin Spinck constructed several mutants of CobB which displayed distinctive deacetylase, decrotonylase or debutyrylase activity 730 . We therefore transfected this protein and its mutants in order to change the balance of acylation/acetylation status. Unfortunately, we failed to establish stable changes of de-ac/bu/cr marks after transient transfection of either wild type or mutated CobB following different series of experiments (data not shown). We also established stable cell lines expressing wild type CobB and managed to obtain a stable increased acyl/acetyl ratio in CobB-expressing cells. The increased acyl/acetyl ratio was observed both with western blot analysis and ELISA (Figure 36A&B). Next, we performed salt elution assay in control and CobB-expressing cells. Similarly, we obtained more soluble BRD4 in CobB expressing cells, which is in accord with a more dynamic binding of BRD4 in this group of cells. To visualize the dynamics of BRD4 in live cells, we decided to perform FRAP experiment on BRD4. According to our previous experience, BRD4 was found to be very dynamic in COS cells. Therefore we reasoned that it might be difficult to record an even more dynamic movement by FARP on BRD4 in COS-CobB cells. To solve this problem, we performed FRAP experiment on BRD4-NUT, given that it forms stable foci on chromatin dependent on histone acetylation and BRD4 bromodomains 471 . Using this system, we observed faster recovery after photobleaching, with shorter t1/2 of recovery time in CobB transfected cells (6.26 ± 0.33s VS 8.94 ± 0.73s, and a higher mobile fraction in CobB expressing cells (0.84 ± 0.05 VS 0.76 ± 0.02), indicative of a more dynamic binding of BRD4 in CobB expressing group (Figure 36C&D). These data suggest that acyl/acetyl ratio determines binding dynamics between BRD4 and chromatin. The t1/2 values were calculated from the FARP curve.

Acyl/acetyl drives BET redistribution across genome 4.1 H4K5cr is enriched on highly acetylated chromatin and is associated with active transcription

We performed ChIP-seq on H4K5cr, H4K5ac to visualize the distribution of these marks across the genome. RNA-seq analysis was also performed to determine the transcriptional output of these alterations. First, we confirmed the correlation between histone acylation (H4K5cr in our case) with transcriptional activation, as it has been characterized and reported in previous publications 142,144,167 . Furthermore, we identified that higher H4K5cr/ac ratio marks the most active genes (Figure 37A). Ever since the discovery of short chain histone acylations, these marks have been suggested to be redundant with acetylation, given that they overlap with acetylation and are relatively of low abundance. However, in our work, we uncovered that the correlation between histone acetylation and crotonylation does not always follow a linear correlation. Indeed, by comparing all the genes TSSs' coverage of these two marks, we identified that at low level of histone acetylation, there is a linear correlation between H4K5ac and H4K5cr, whereas, at high levels of acetylation, a 3 rd degree polynomial correlation better fits their correlation (Figure 37B, right panel). Besides, we found that in FASTKD1 KO cells which harbor increased acyl/acetyl, such deviation between the fitted linear correlation and 3 rd degree polynomial correlation is even more obvious (Figure 37B, left panel). Collectively, these data suggest that, H4K5cr preferentially occurs on highly acetylated area and covers the highly active genes.

Altered H4K5cr/ac drives a redistribution of BRD4 genomic localization

As described above, our previous results suggested that a higher acyl/acetyl ratio favors the dynamic movement of BRD4. Since the total amount of BRD4 does not increase in KO cells,

A B

we hypothesized that the increasing acyl/acetyl ratio might result in a re-distribution of BRD4 on genomic regions. To characterize the genomic distribution of BRD4, we performed ChIPseq analysis. We could identify that BRD4 is enriched over the nucleosome free region (NFR)

surrounded by the +1 and -1 nucleosomes (Figure 38A, left panel). When grouping gene TSS regions (± 2000bp) according to their transcriptional activity, we better visualized that H4K5cr, H4K5ac and BRD4 preferentially cover active genes (Figure 38A). In the case of BRD4, the highly active genes are covered by high levels of BRD4 on the NFR-associated regions (Figure 38B). In addition, cells with an increased acyl/acetyl ratio (FASTKD1 KO), we observed an increased enrichment of BRD4 at the NFR regions. These data suggest that, upon increased acyl/acetyl marks, BRD4 can be released from various genomic regions towards the NFRs of active genes. Anti-BRD4, H4K5ac and H4K5cr ChIP were performed in WT, ko-1 and ko-2 cells.

A B

A. (Left panel) Distribution of BRD4 on all TSS regions (TSS ± 2000). A peak on nucleosome free regions (NFRs) other than -1/+1 nucleosome was identified at TSS (shown as blue). TSS regions were then divided into four groups according to their expression state (right panels). H4K5ac, H4K5cr and BRD4 distributions were indicated in each group of genes. As shown in the figure, the most highly expressed genes are associated with high H4K5cr

and H4K5ac, and are mostly enriched with BRD4 at NFRs.

B.

ChIP signals of BRD4 were mapped on TSS regions in different groups described in A. As shown in the figure, the most highly expressed genes are mostly enriched with BRD4 at NFRs, in ko-1 and ko-2, such enrichment is more obvious.

Genomic redistribution of BRD4 regulates the expression of sets of genes in REH cells and ALL patients

BRD4 is a master regulator of gene transcription, therefore the redistribution of BRD4 should result in an alteration of specific gene transcription. To uncover the transcriptional changes mediated by BRD4, we treated the WT cells with BRD4 bromodomain inhibitor, JQ1 and performed RNA-seq analysis. Using GSEA, we identified that a subset of genes upregulated in FASTKD1 KO cells are enriched in genes downregulated in JQ1 treated cells. Of note, we also identified a subset of genes upregulated in FASTKD1 KO cells enriched in downregulated genes in JQ1 treated cells (Figure 39A,B). This data demonstrate that these two sets of genes in FASTKD1 KO groups are JQ1 sensitive, and the differential expression of these genes in KO compared to WT group is likely attributed to the redistribution of BRD4 in KO cells.

Furthermore, we also identified that genes downregulated and upregulated in FASTKD1 KO groups are also enriched in different groups of B-ALL patients samples with high or low acylation level respectively (Figure 39C,D). We further performed Gene Ontology analysis and identified that genes that encode mitochondrial membrane protein, cell cycle are upregulated, while genes that encode stemness are downregulated in FASKTD1 KO cells or in JQ1 treated cells (Figure 39E). B. The two gene groups defined in A were respectively used as genesets for GSEA plots to test for their enrichment/depletion in the JQ1-treated REH cells. These plots show a significant depletion of genes up or down regulated in FASTKD1 KO cells. These genes are therefore JQ1 sensitive.

D.

The two gene groups defined in C were respectively used as genesets for GSEA plots to test for their enrichment/depletion in the FASTKD1 KO cells. These plots show significant depletion or enrichment of genes respectively down or up regulated in FASTKD1 KO cells suggesting that the transcriptional effect of high acylation in B-ALL blasts is similar to the effect of FASTKD1 ko-induced high acylation in REH cells. FASTKD1 belongs to the FASTK family, which are known to be involved in mitochondrial mtRNA biology, from RNA processing to translation. Among the six proteins of this family, FASTK, FASTKD2-5 have all been reported to be required for the proper expression of various mitochondrial mRNAs and are required for ETC stoichiometry and function 712 . In contrast, FASTKD1 appears to be the only member that negatively regulates mitochondrial mRNAs 721 .

E. GeneSet Enrichment

FASTK family proteins possess distinctive specificities over mitochondrial mRNAs. For instance, FASTK was reported to protect ND6 from mitochondrial degradosome-mediated degradation, while FASTKD1 specifically represses ND3 expression 719,723,738,739 . FASTKD2 and FASTKD5 were characterized to profoundly regulate the overall biogenesis of mitochondrial proteins 722,740 .

In our previous experiment, we confirmed that FASTKD1 is a negative regulator in mtRNA biology. However, in our study, compared to Boehm and colleagues' study 721 , depletion of FASTKD1 leads to an overall upregulation of mitochondrial mRNAs in addition of ND3. This discrepancy might be attributed to the different cell models used in our work. Besides, we uncovered that FASTKD1 does not influence mtDNA copy number of cells, implicating that it is the regulation of mitochondrial mRNAs. Indeed, our data indicate that FASTKD1 should act at transcriptional or post-transcriptional levels, such as RNA processing and degradation.

Previous studies indicate that OXPHOS activity is associated with cancer cell survival or tumor progression, although it remains enigmatic whether it is oncogenic or tumorsuppressive [741][742][743] . Traditionally, high OXPHOS activity generates high reactive oxygen species (ROS), which in turn damage the tumor cells. Indeed, this has been viewed as one of the major mechanisms underpinning dysfunctional mitochondrial activity in solid tumors. We also measured the ROS in FASTKD1-depleted cells but found no significant changes (data not shown), indicating that the redox balance could be well-maintained in our model system. As FASTKD1 represses mitochondrial respiration, we assumed that it might protect cells from stress. However, although we monitored various phenotypic changes after depletion or overexpression of FASTKD1, no impact on the cell proliferation, chemotherapeutic responses and the cell cycle were observed. We also cultured FASTKD1-deleted and overexpressing cells to FBS starvation but still no significant difference was observed compare to control cells (data not shown). Such observations implicate that the oncogenic role of FASTKD1 in ALL is either modest or more delicate to detect.

Cancer aggressiveness relays on a series of pathological events that favor resilient cellular characteristics in cancer as well as the manifestation of refractory or relapsed diseases in the clinics. In general, aggressive cancers can be classified as: 1) cancer cells that are primarily resistant to therapies, i. e., the percentage of blasts barely reduces after the initial therapy; 2)

the bulk of blasts is eliminated after initial therapy, but a small fraction of cells survive and induces the development of a tumor in a short term; 3) patients achieve morphological complete remission but develop relapse in the long run.

The primary resistance or short-term relapse might result from the enhanced ability of the cells to export chemotherapeutic drugs, or to acquired anti-apoptotic capabilities, etc. In contrast, long-term relapse is mediated by persistent cancer re-initiating cells. Such cancer reinitiating cells possess some common characteristics, such as dormancy and stemness 632 . Indeed, in the previous work leading to the identification of the six marker genes, thanks to their cell stratifying ability, we could uncovered that tumoral cells from patients who have poor outcome have acquired "stem-like" characteristics 711 .

However, it is noteworthy that relapsed or refractory diseases are complex traits and could depend on may linked or independent oncogenic events. Indeed, we know that 1) the in vivo environment (e.g., bone marrow microenvironment) of tumoral cells is much more complicated than that of cell lines; 2) the relapse-inducing cells are minor clones; 3) the acquisition of cancer aggressiveness could be a multiple-step process. These considerations might partially explain why in our in vitro studies we did not detect any frank oncogenic activity associated with FASTKD1 expression.

In conclusion, this part of work addressed the repressive role of FASTKD1 on mitochondrial activity, and uncovered that FASTKD1 does not influence the proliferation nor the viability of REH cells in vitro. We also identified a correlation between the mitochondrial gene expression and prognosis by reviewing clinical data of B-ALL and T-ALL cohorts (not shown). Supporting the conclusion of our molecular studies. Collectively, this part of my work uncovered the correlation between mitochondrial activity and cancer prognosis in ALL.

Mitochondrial activity affects histone PTMs

It has long been established that cell metabolism affects histone modifications. Since more than two decades ago, the perturbation of acetyl-CoAs has been documented to impact the protein acetylation, including histone acetylation 744,745 . With the identification of novel types of modifications, including crotonylation, butyrylation, succinylation etc., more evidences have emerged concerning the process of cellular metabolism-driven histone modifications in physiology and diseases, including cancer.

For the last few decades, studies have revealed the spatiotemporal control of metabolite production. Indeed, metabolites, including acetyl-CoA, non-acetyl acyl-CoA have different subcellular compartments. Normally, cytosolic and nuclear compartments are believed to share the same metabolite pool because of the existence of permissive nuclear pores. Mitochondrion, however is a distinctive compartment because of its non-permissive membranes. Although transport systems have been characterized for the export of acetyl-CoA and import of fatty acids, there are still many obscure aspects of CoA derivatives' export that need to be understood.

Lately, more studies have described metabolite compartmentalization and the underlying mechanisms. Two new concepts have been introduced regarding the metabolism-driven histone modifications. Firstly, various metabolic enzymes, including the TCA enzymes have been to

found to be located in the nucleus, whereby they produces the corresponding metabolites for histone modifcations 594 . Secondly, several studies indicate that other than the citrate shuttle, there might be unknown mechanisms regarding the metabolites cross-talk between mitochondrial and nuclear compartment. For instance, McDonnell and colleagues have reported that fatty acid is the major fuel of histone aceylation and the produced acetyl-CoA could impact histone acetylation in a manner that is independent of the citrate shuttle 562 . In addition, Murphy and colleagues have pointed out that sccinyl-CoA might be shuttled through carnitinesuccinylcarnitine system, while succinate can be exported from mitochondrion through dicarboxylate carrier SLC25A10 and supplies the cytosol/nuclear succinyl-CoA pool 596 . With our cell model and in patients tumoral cells, we established the link between mitochondrial activity and histone PTMs, prominently non-acetyl acylations, where an increased mitochondrial activity drives a higher level of histone H4 acylations (e.g., crotonylation, butyrylation, β-hydroxybutyrylation, etc.).

As cancer cells have almost ubiquitously reprogrammed metabolism, metabolism-driven histone modifications could be a common mechanism underpinning tumor biology. Indeed, although only tested in a small patient samples cohort, our studies uncovered that mitochondrial metabolism-driven histone modifications (H4K5cr and H4K5bu) also occurs in tumoral samples. However, more precise models need to be established to address the functional roles of the interplay between cell metabolism and its epigenome and to precisely characterize the resultant changes (e.g., cell fate), as well as to demonstrate the specific epigenetic patterns underpinning the corresponding functions in cancer biology.

Collectively, metabolism-driven histone modifications have begun to emerge as one of the crucial oncogenic mechanisms. Our work on mitochondrial activityfatty acid metabolism -histone PMTs added one piece of evidence to these mechanisms. For the moment, the precise epimetabolic pathways active in cancer cells remain enigmatic. More specially there are many questions regarding metabolic enzymes translocation, metabolites shuttling etc. In the case of functional output of metabolism-driven epigenome programming, it would be important to define the types of epigenetic patterns that are required for specific aspects of cancer biology.

Furthermore, the mechanisms underpinning specific epigenetic alterations and gene expression programs are to be understood. All these fields await further investigations and might shed light on novel mechanisms underlying cancer establishment and development.

The ratio of acyl/acetyl tunes BET-chromatin interaction dynamics

Previous studies showed that the first bromodomain (BD1) of BET family protein has very weak affinity over monoacetylated lysine. However, this binding is greatly enhanced by multiple acetylated sites, e.g., tetra-acetyllysine (H4K5acK8acK12acK16ac). In contrast, the second bromodomain (BD2) of BET can bind mono and multiple modified histones, including histone H3. Interestingly, the binding of BD2 to tetra-acetylated H4 has only 1/10 the affinity of the binding affinity of BD1 to tetra-acetylated H4 [START_REF] Filippakopoulos | Histone recognition and large-scale structural analysis of the human bromodomain family[END_REF] . Moreover, Morinière and colleagues have identified that BD1 binds simultaneously H4K5ac and H4K8ac and that the affinity of BD1 binding to diacetylated histone H4 tail is almost equal of its binding to tetra-acetylated histone H4 tail 100 . These data suggest that the interaction between BD1 and H4K5acK8ac is the major interaction underlaying the binding of BET to acetylated H4.

Furthermore, it has been suggested that the butyrylation at H4K5 can preclude the binding of acetylated H4 peptides by BD1. A structural model considering the interaction between BRDT BD1-H4K5buK8ac in comparison with BRD4 -H3K14bu revealed that the butyrylated lysine at H4K5 position results in a steric clash between the histone peptide and the ZA loop of BD1. Furthermore, the conformational adaptation of H4K5buK8ac into BD1-binding pocket might indeed disrupt many of the interactions between acetylated H4 peptides and BD1 100 . Our data was in accord with these findings, where the binding between BRD4 and acetylated H4 is abolished by the butyrylation at H4K5 site (Figure 34).

In the literature there are evidences indicating that H4 hyperacetylation is strictly ordered where histone H4 acetylation occurs from K16 and spreads over N terminal lysine residues ("zipper acetylation"). This theory is supported by the observations that only H4K16 acetylation, H4K16K12 di-acetylaion, K4K16K12K8 tri-acetylation and the tetra-acetylation of all four lysines (H4K5K8K12K16) were identified using mass spectrometry analysis of histone modifications. This situation was observed in a variety of organisms ranging from lower eukaryotes to human cells [746][747][748] . In this context, the presence of K5acK8ac is considered as a mark for H4 hyperacetylation and often indicates active transcription 749 . As demonstrated before, in spermatocytes and round spermatids, there is a co-occurrence of H4 acetylation and H4 butyrylation marks (H4K5K8 ac/bu) around the TSS regions (TSS±5000), of the most active genes. Furthermore, the BRDT bound regions are enriched with a higher degree of the cocoverage of these four marks, even though H4K5bu perturbs BRDT -H4 interaction.

Considering that histone acetylation and butyrylation are dynamic marks which undergo rapid turnover, the authors from my host laboratory proposed the hypothesis that explains such a paradox: BET (BRDT) binds with acetylated chromatin in a dynamic manner. Based on this hypothesis, the dynamics of BET-chromatin is tuned by the relative level of histone butyrylation over acetylation : increased butyrylation disfavors BRDT -H4 interaction and thus contributes to a more dynamic interaction. In contrast, increased acetylation favors a tight binding 145 .

In our work, we brought additional arguments in favor of the above theory which seems to be also valid in a completely different system. Firstly, the above theory applies not only to butyrylation, but also to all other histone acylations (more than 3 carbons, >3C abbreviated).

This is based on the evidence that: 1) short chain acylations, for example crotonylation, βhydroxybutyrylation etc. have similar to butyrylation and longer chain acyl groups and the corresponding histone acylations should be less likely to properly interact with BD1. 2) Similar to acetylation, all these newly identified histone acylations have a rapid turnover rate. 3) These acylations systematically co-occur with histone acetylation (this will be further discussed in the next section). Secondly, the relative level of histone acetylation over acylation is tuned by acetyl/acyl-CoA level, which are themselves controlled by cell metabolism. The point has been covered in the introduction part.

Based on our data, we proposed that the interaction between BET proteins (BRD4 in our case) and chromatin are determined by acyl/acetyl ratio. A higher acyl/acetyl ratio leads to a more dynamic BRD4-chromatin interaction, whereas a lower ratio contributes to a tighter chromatin binding by BRD4. Among different experiments we performed, BRD4 elution assay and FRAP experiments directly support this hypothesis (Figure 35, Figure 36).

In conclusion, we discovered that acyl/acetyl ratio controls BET -chromatin interaction dynamics. As acyl/acetyl marks can be regulated by metabolic state, factors such as FASTKD1 that control the mitochondrial activity, could in turn control the metabolismchromatin signaling. Since cancer cells present a systematic altered metabolism and given that BRD4 is broadly involved in various cancer types, the mechanism we uncovered here might largely contribute to oncogenesis.

In the next section, I will discuss how this dynamic pattern impact the BRD4 mediated biological processes including gene transcription.

Acyl/acetyl ratio controls BRD4 genomic redistribution and gene transcription

Since their identification, functional studies on histone acylations revealed redundant activities.

Although much less abundant than acetylation, histone acetylation co-occurs with histone acetylation on active genes. However, there are some evidences supporting a distinct role for non-acetyl histone acylation in gene transcription. Among these evidences we can enumerate the identification of specific readers and writers for these acylations. However, here also we are missing convincing arguments in favor of the distinctive roles of these factors compared to those known to control histone acetylation.

Herein, we found that histone acylation, H4K5cr for example, is associated with active transcription, as reported in previous studies. Furthermore, we uncovered that, high ratio of cr/ac marks the most actively transcribed genes. Indeed, the ChIP-seq analysis revealed that metabolism-driven H4K5cr preferentially occurs on highly acetylated chromatin. As described in chapter III, longer chain acylations (>3C) preclude BET-chromatin interaction. We therefore reasoned that chromatin loci presenting high acyl/acetyl ratio should favor the release of BET proteins from the corresponding regions. This observation allowed us to propose the reservoir theory for BET factors. This hypothesis was previously proposed for a transcription factor MIFF which is released from low-affinity regions and as a result of its acetylation. Acetylated MIFF becomes then available to bind its high affinity regulatory elements 750 . In our case, BRD4 is displaced from regions gaining a higher acyl/acetyl ratio and becomes available for binding to other genomic regions, i. e., the TSS of highly active genes.

Gene transcription initiates with PICs assembly at the promoter regions upstream of TSS.

Nucleosome free regions (NFRs) were firstly identified on Pol II promoters in yeast upstream of TSS 751,752 and were then found on active gene.TSSs in human cells [START_REF] Baldi | Beads on a string-nucleosome array arrangements and folding of the chromatin fiber[END_REF]753 . Since most of the transcription factors preferentially access free DNA, NFRs could theoretically allow transcription factors to assemble in order to initiate transcription. NFRs were also suggested to modulate enhancer-promoter communication 754 . In our work, using MNase ChIP-seq, we observed that the NFRs could interact with BRD4. Furthermore, the redistribution of BRD4 driven by acyl/acetyl ratio impacts the presence of BRD4 at NFRs, which in turn might control gene transcriptional alterations. Our results identified subsets of both upregulated and downregulated genes in high acyl/acetyl cell group (FASTKD1 KO) that are also among the JQ1 sensitive genes. This observation also comes in support of our model on the regulation of BRD4 activity by the changing acyl/acetyl ratio.

It is noteworthy that we also monitored the acylation-tuned transcriptional output in patients'

samples. Indeed, the gene set enrichment analysis showed that tumors with higher histone acylation groups have enhanced expression of genes involved in mitochondrial component, cell cycle and ribosome synthesis. They also present decreased expression of stemness associated genes in cell lines and in primary blasts.

Introduction

Since the discovery of histone lysine propionylation and butyrylation in 2007 (Chen et al., 2007), an increasing number of different histone acylations has been reported. The vast majority of these histone post-translational modifications (PTMs) occur at sites already known to be acetylated and hence overall these new histone PTMs could collectively be considered as acetylation competing marks. Functional studies carried out on these histone marks in different biological systems have shown that they are all associated with active genes and directly stimulate transcription, similar to histone acetylation (Kebede et al., 2017; Goudarzi et al., 2016; Dai et al., 2014; Sabari et al., 2015; Smestad et al., 2018; Xie et al., 2016; Bao et al., 2019; Huang et al., 2018; Zhang et al., 2019).

Comparative high-resolution genome mapping also revealed that, in the majority of active chromatin loci, these histone acylations co-exist with acetylation (Tan et al., 2011; Dai et al., 2014; Goudarzi et al., 2016; Sabari et al., 2015; Kebede et al., 2017; Crespo et al., 2020). Additionally, although these acylations are mostly mapped at gene transcriptional start site (TSS)-associated regions, as would be expected for active histone marks, their relative abundance corresponds to only a small fraction in comparison with acetylated histones (Simithy et al., 2017). Finally, although different acyl-donor groups may result from different metabolic pathways, their redundant functions do not leave much room for any metabolic-specific action. These observations therefore raise an important unsolved issue in modern biology, which is the specific functional significance of these histone acylations compared to acetylation. An answer to this issue came from the discovery of specific domains in various proteins that show a better affinity for binding to longer chain acyl groups on histones compared to acetylation. For instance, YEATS and DPF domains present a better binding activity for crotonylated histones than for acetylated histones (Andrews et al., 2016a; Li et al., 2016; Xiong et al., 2016).

An unexplored possibility to solve this issue is coming from in vitro data showing that most bromodomains lose their affinity for chromatin regions bearing histones with acyl groups longer than three carbons (Flynn et al., 2015; Goudarzi et al., 2016; Olp et al., 2017). The functional consequence of the differential effects of histone acylation on bromodomainchromatin interaction was first considered in the context of the late stages of differentiation of male germinal cells, during which a large-scale histone hyperacetylation is coupled to a genome-wide histone removal involving the testis-specific BET factor, Brdt (Shiota et al., 2018). Using this specific system, we discovered that H4 bearing butyrylation at K5 and K8 escapes this wave of replacement and survives longer in late spermatogenic cells than histone H4 bearing the corresponding acetylation. This observation supports the hypothesis that, since Brdt's first bromodomain is unable to bind H4 when it is modified by butyrylation specifically at K5 (Goudarzi et al., 2016), the corresponding histones "escape" acetylation and the consequent Brdt-dependent removal.

However, these observations remained correlative and the hypothesis of the modulation of the action of BET factors by an interplay between histone acetylation and acylation, awaited confirmation.

Here, by taking advantage of ALL cell biology, we directly demonstrate that a modified histone acetyl/acyl ratio, specifically at H4K5, controls the dynamics of interaction between chromatin and the ubiquitously expressed member of BET double bromodomain factor, BRD4, whose expression and activity are frequently dysregulated in many unrelated cancers (Fujisawa and Filippakopoulos, 2017). The emerging general concept developed here is that a mixture of finely tuned competing histone acetylation and longer chain acylations defines bromodomain factors functional availability.

Results

Aberrant activation of FASTKD1 is associated with a general shut-down of mitochondrial activity

We previously identified FASTKD1 as an ectopically expressed gene in childhood and adult B-ALL (Wang et al., 2015). FASTKD1 presents a mitochondrial targeting signal and hence is expected to exert its function in mitochondria (Jourdain et al., 2017). To investigate its function, we first identified the established B-ALL cell line REH, expressing FASTKD1 and then generated REH cell lines stably expressing two different anti-FASTKD1 shRNAs (Fig. S1A) or used the CRISPR/Cas9 system to knock-out the gene (Fig. S1B). RNA-seq were generated from the two FASTKD1 knock-down as well as two independent FASTKD1 knock-out clones (Fig. S1A,S1B). Focusing on mitochondrial gene expression, we observed that the inactivation of FASTKD1 leads to a significant enhancement of mitochondrial encoded gene expression (Fig. 1A). This activation of mitochondrial gene expression was also independently confirmed by RT-qPCR, by evaluating the expression of ND2 and ND3 genes after treating cells with a series of five different anti-FASTKD1 shRNAs as well as in the two FASTKD1 ko cell lines (Fig. 1B, upper panels, Fig. S1C). Finally, FASTKD1-Flag re-expressed in the FASTKD1 ko cells resulted in the mitochondrial localization of the ectopically expressed FASTKD1-Flag and the downregulation of mitochondrial gene expression (Fig. S2, Fig. 1B, lower panels).

All these data demonstrate that FASTKD1 should logically down-regulate mitochondrial activity.

To test this hypothesis, mitochondrial respiration was measured from control REH cells or from the two FASTKD1 ko clones. The results show an enhancement of respiration in both FASTKD1 ko clones compared to the control REH cells, in perfect agreement with a role for FASTKD1 in the decrease of mitochondrial activity, more specifically the respiration (Fig. 1C).

Mitochondrial activity is a driver of histone acylation

FASTKD1 appeared to us as an excellent factor to investigate the relationship between mitochondrial activity and histone acetylation/acylation (Matilainen et al., 2017; Lozoya et al., 2019; Haws et al., 2020; Trefely et al., 2020) in the specific context of ALL. We focussed on histone H3 and H4 propionylation and butyrylation, since, while all bromodomains bind acetyllysines and propionyllysines, many of them are unable to bind butyryllysine (Flynn et al., 2015).

Histone extracts from wild-type REH cells and the two derived FASTKD1 ko cell lines were used in an unbiased approach to measure changes in histone butyrylation and propionylation.

After trypsin-digestion of histones, the resulting peptides were directly quantified using a label free mass spectrometry method.

These analyses were performed without prior enrichment of modified histone peptides (with anti-PTM antibodies) to detect and visualize the most abundant modified peptides. This analysis demonstrated that histone H4 K5, K8 and K12 butyrylation is the most responsive to FASTKD1 depletion (Fig. 2A, Table S1, S2 and S3). A more detailed analysis of the mass spectrometry data also highlighted the occurrence of unique peptides with a combination of acetylation and butyrylation-propionylation (data file S1). This observation is indicative of continuing exchange of acylations at a particular lysine site.

We then decided to focus on H4 K5 and K8 to confirm these findings. Immunodetection of histone PTMs in protein extracts showed a clear increase in both butyrylation and crotonylation of H4K5 and H4K8 in the two FASTKD1 ko cell lines. The corresponding histone acetylation level did not show any noticeable change in FASTKD1 ko cell lines (Fig. 2B).

These results suggest the existence of a direct relationship between the studied histone acylations and the extent of mitochondrial activity. To confirm this conclusion, we treated control and FASTKD1 ko cell lines with the electron transport chain poison Rotenone and demonstrated that the impairment of mitochondrial activity severely affects the maintenance of histone H4K5 butyrylation and H4K5 crotonylation (Fig. 2C). This treatment with Rotenone had relatively little effects on acetylation, probably due to the considerably higher levels and more stable pools of acetyl-CoA and acetylated histones.

Overall, these data also suggest that histone butyrylation and crotonylation could be more sensitive to a change in mitochondrial activity than histone acetylation.

Fatty acid synthesis  -oxidation is a major driver of histone butyrylation and crotonylation

Fatty acid synthesis and -oxidation particularly involve the generation of acyl-CoA derivatives that could potentially be used to add the corresponding acyl groups on histones (Pougovkina et al., 2014 ; McDonnell et al., 2016; Gowans, 2019; Tarazona et al., 2020). We therefore first used a competitive inhibitor of acyl-CoA synthetase, Triascin C, to prevent the synthesis of fatty acids and monitored the effect on H4K5K8 aceylation and butyrylation (Fig. 2H, scheme).

The treatment of cells with Triascin C abolished the increase of H4 butyrylation at H4K5 observed in FASTKD1 ko lines, with no remarkable effect on the acetylation of this residue (Fig. 2D). Additionally, we decided to target the enzyme that catalyses the first step of fatty acid synthesis, acetyl-CoA carboxylase (ACC1, Fig. 2H, scheme), by using the ACC1 inhibitor ND-630. The inhibition of acetyl-CoA carboxylase also abolished the increased level of H4K5bu compared to the parental cells, with no remarkable effect on H4K5ac (Fig. 2E).

In order to also test the role of fatty acid oxidation (FAO) in histone acylations, we sought an approach based on treating the cells with octanoate, a molecule directly usable in FAO (McDonnell et al., 2016). The octanoate treatment increases the levels of H4K5 acetylation, butyrylation and crotonylation in REH cells (Fig. 2F andS3). However, compared to acetylation, the increase in H4K5bu-cr is more remarkable (Fig. S3).

In order to further test the role of FAO in histone acylation, we also used Ranolazine, an inhibitor of FAO, and showed that the treatment of cells with this inhibitor abolishes the increase in H4K5bu observed in the FASTKD1 ko clones (Fig. 2G).

-oxidation and mitochondrial activity are major sources of histone acylation in ALL malignancies

To generalize our conclusions on the relationship between mitochondrial activity and histone acylations, we quantitatively measured H4K5cr and H4K5bu by ELISA in 31 B-ALL patient malignant cells samples (Table S4). Using RT-qPCR, we quantified in the same samples, Carnitine Palmitoyltransferase 1A (CPT1A) mRNA, encoding a protein involved in the transport of long chain acyl groups into mitochondria controlling the fatty acids -oxidation potential of the cells, and the mitochondrial gene ND2 mRNA, as an indicator of mitochondrial activity.

Figure 3A shows that there is a tendency for coregulation between the expression of CPT1A mRNA, as a measure of -oxidation potential, and the expression level of ND2, as a measure of mitochondrial activity (rs = 0.531).

From our functional data obtained in REH cells (Fig. 1 and 2), we expected to find a relationship between the level of mitochondrial transcription (ND2 mRNA) and the intensity of H4K5 crotonylation and butyrylation. To test this hypothesis, ALL samples were divided into two groups as a function of the expression of ND2. A ROC curve of the RT-qPCR output values was used to define a cut-off value at -dCt=1.2782, which we used to stratify the ALL into two groups of 13 (42%) low ND2 expressing and 18 (58%) high ND2 expressing samples. The results showed that, similarly to REH cells, patients' ALL cells with higher mitochondrial activity are associated with higher levels of H4K5 crotonylation and butyrylation (Fig. 3B and3C).

All these data support and extend our conclusions on the major role of mitochondrial activity and -oxidation in driving histone acylation.

The H4K5 acyl/acetyl ratio is a major determinant of BRD4 -chromatin interactions

In a previous work, by analysing the function of histone H4K5K8 butyrylation in spermatogenic cells and its impact on the function of the testis-specific BET factor Brdt, we observed that, during late spermatogenesis, H4K5bu-containing nucleosomes escape the acetylation-and Brdt-dependent histone removal (Goudarzi et al., 2016).

Our present data suggest that similarly, in the case of ALL cells, a change in the ratio of H4K5 acetyl/butyryl-crotonyl could also affect the interaction between BRD4 and chromatin.

Using a peptide pull-down assay with a H4 N-terminal tail peptide bearing all combinations of acetylation and butyrylation at K5 and K8, we demonstrate the inability of BRD4 to bind H4, when the peptide is modified by a butyryl group at K5 (Fig. 4A). A depletion of FASTKD1 and a change of mitochondrial activity did not affect the total level of BRD4 in a high salt nuclear extract (Fig. 4B, input, also see Fig. 4C) nor its ability to bind to an acetylated H4 tail peptide (Fig. 4B, pull down). These data suggest that H4 butyrylation at H4K5 should affect the ability of BRD4 to interact with chromatin. Accordingly, an increase in H4K5bu-cr, as observed in our FASTKD1 ko cells, should to some extent loosen the interaction of BRD4 with chromatin compared to the wildtype cells.

In order to test this hypothesis, we used wild-type and FASTKD1 ko REH cells to compare the strength of BRD4-chromatin binding, with the prediction that, in FASTKD1 ko cells, a loose BRD4chromatin interaction should be observed due to the increase in the H4K5 acyl/acetyl ratio. Accordingly, wild-type and FASTKD1 ko cells were lysed in a buffer containing increasing salt concentrations and, after centrifugation, the respective amounts of BRD4 released in the supernatant or remaining bound to chromatin were visualized by immunoblotting. Figure 4C (upper panels) shows that, in the presence of 200 mM NaCl, both FASTKD1 ko clones released higher amounts of BRD4 compared to wild-type cells, suggesting a weaker binding of BRD4 to chromatin.

In order to further support our observation, we also treated our cells with increasing concentrations of the BRD4 small molecule inhibitor, JQ1. We reasoned that a more dynamic BRD4-chromatin interaction should make BRD4 bromodomains more sensitive to an inhibition by JQ1. After treating cells with the solvent, or 0.1, 0.5 or 5 M of JQ1, the respective amounts of BRD4 in soluble protein extracts and in the chromatin-bound fractions were analysed. Figure 4C (lower panels) shows that, at 0.5 M concentration, the JQ1 treatment leads to an increase in the soluble pool of BRD4 in FASTKD1 ko cell, compared to wild-type cells.

Finally, in order to directly test the effect of a change in the histone acetylation/butyrylationcrotonylation ratio on the efficiency of BRD4 binding, we sought another approach to demonstrate a change in the dynamics of BRD4 in live cells. For this purpose, we used a previously characterized COS-7 cell-based system and induced a change in the ratio of histone acetylation/butyrylation-crotonylation in these cells. We had previously shown that the expression of the oncogenic fusion protein, BRD4-NUT, in COS-7 cells creates well-defined nuclear foci, which depend on chromatin acetylation and BRD4 bromodomains (Reynoird et al., 2010). In addition, these BRD4-NUT induced foci are large and stable enough to allow in vivo approaches such as Fluorescent Recovery After Photobleaching (FRAP) assays. First, we stably expressed CobB, a bacterial NAD + deacetylase appropriately modified for expression in mammalian cells (Spinck et al, 2020), in COS-7 cells and visualized the effect of this deacetylase on the acetyl/butyryl-crotonyl ratio. Figures 4D andS4A show that the ectopic expression of CobB, while preferentially decreasing the level of H4K5ac, has no noticeable effect on H4K5bu and H4K5cr in vivo in these cells. Therefore, our prediction was that, in the CobB-expressing cells, this increase in the H4K5acyl/acetyl ratio should be associated with an increased dynamic of the BRD4chromatin interaction. We also performed the salt extraction assay described above, in the control and in CobB-expressing COS-7 cells and observed that, similar to REH cells, an increase in the H4K5acyl/acetyl ratio enhances the salt elution of BRD4 from chromatin (Fig. S4B).

Finally, FRAP was used to precisely measure the dynamics of GFP-BRD4-NUT chromatin interactions in these cells. Figure 4E shows that the ectopic expression of CobB and an increased H4K5acyl/acetyl ratio clearly increases the dynamics of BRD4 -chromatin interaction.

A metabolically driven increase in H4K5 acylations preferentially occurs on highly acetylated chromatin regions

The distribution of H4K5cr-bu/H4K5ac ratio was the investigated at high resolution in a genome-wide manner. The H4K5cr, H4K5bu and H4K5ac ChIP-mapping allowed us to visualize the relationship between H4K5cr and H4K5bu with H4K5ac in each of the cell lines considered. To this end, we plotted the ChIP-seq read counts corresponding to H4K5cr and H4K5bu as a function of the read counts corresponding to H4K5ac on gene TSSs. This representation shows a linear correlation between H4K5cr-bu and H4K5ac on TSSs associated with relatively low levels of H4K5ac. However, at higher levels of H4K5ac, this linear relationship between the considered histone modifications is distorted with an increasing H4K5cr-bu/ H4K5ac ratio, and this part of the correlation plot best fits a non-linear (exponential regression) model (Fig. 5A).

The H4K5cr-bu/H4K5ac ratio controls BRD4 genomic distribution

Two independent anti-BRD4 ChIP-seq were performed to evaluate the impact of the change in H4K5cr-bu/ac ratio on the binding of BRD4 to chromatin at high resolution. Focussing on gene TSS, we found that BRD4 accumulates on the TSS of highly active genes. By comparing the levels of these TSS region-bound BRD4 between wild-type and FASTKD1 ko cells, in both anti-BRD4 ChIP-seq experiments we found significantly higher levels of BRD4 present on the highly active TSSs in the two ko cell lines (see Fig. 5B andC, showing heatmap and metagene profiles for all genes in the first experiment, also see Fig S5A showing heatmaps and profiles for a selection of genes with the highest BRD4 peaks in both anti-BRD4

ChIP-seq experiments). Since there was no change either in the total level of BRD4 (Fig. 4B, input and Fig. 4C) nor in its ability to bind acetylated H4 tail (Fig. 4B, pull down) in our FASTKD1 ko cells compared to wild-type cells, we concluded that the observed increase in BRD4 on active gene TSSs in FASTKD1 ko cells, should be due to a redistribution of BRD4.

Therefore, the increased BRD4 binding to the TSS of highly active genes is very likely a consequence of the genome-wide increase in BRD4 mobility making BRD4 more available for binding active gene TSS regions.

Functional significance of the genomic redistribution of BRD4 in REH cells and in ALL malignancies

We reasoned that the genes that show a change in expression in FASTKD1 ko cells compared to wild-type cells, should to some extent, also be sensitive to the BET bromodomain inhibitor JQ1. To this end, we generated RNA-seq from wild-type REH cells treated with different doses of JQ1 and compared the differentially regulated genes (JQ1-responsive genes) with the genes that are differentially expressed between wild-type and FASTKD1 ko REH cells. Interestingly, a significant number of genes differentially expressed in FASTKD1 ko cells compared to wildtype cells are found among the JQ1-responsive genes (Fig. 6A and6B). Indeed, a subset of genes that are down-regulated in FASTKD1 ko REH cells are also down-regulated in wild-type REH cells treated with JQ1 (Fig. 6B, upper panel). Additionally, a significant subset of genes among those that are upregulated in FASTKD1 ko cells are also down regulated in wild-type JQ1 treated cells (Fig. 6B, lower panel).

The observation that both FASTKD1 ko down-and up-regulated genes are significantly enriched among JQ1-sensitive genes suggests that both up-and down-regulated genes have a BRD4 dependent expression. This is also illustrated by Figure S5 which shows that genes associated with BRD4 peaks are JQ1 sensitive. Indeed, to demonstrate the relationship between the redistribution of BRD4 between wild-type and FASTKD1 ko cells, genes whose TSS regions were associated with high BRD4 peaks and increasing BRD4 binding in ko cells were selected (Fig. S5A). This group of genes was used as a geneset for a GSEA analysis to test for its enrichment/depletion in the transcriptomes of JQ1 treated REH cells. Figure S5B shows a significant depletion of this group of genes, visualizing their down regulation in JQ1 treated cells, which demonstrates that their expression is BRD4 dependent.

In order to show that this H4K5 acylation-and BRD4-dependent regulatory circuit could also be involved in B-ALL tumour cells, we analysed the transcriptomes of a subset of B-ALL tumours (Table S4) for which we had measurements of H4K5bu and H4K5cr levels (Fig. 3). We generated RNA-seq from 25 B-ALL samples and identified the genes differentially expressed between the two groups of H4K5cr-bu high (n=15) and H4K5cr-bu low (n=10) B-ALL (Fig. S6). We then compared the list of differentially expressed genes between these two categories of ALL cells (Fig. 6C), with the list of differentially expressed genes between REH wild-type and FASTKD1 ko REH cells (Fig. 6A), which also showed higher levels of H4K5crbu compared to wild-type cells (Fig. 2). Interestingly, the two transcriptional signatures share similarities since a significant number of genes are regulated in the same manner in FASTKD1

ko REH and in H4K5cr-bu high B-ALL (Fig. 6D). It is worthy to recall that the common point between these two systems is the comparison between the state of H4K5 acylations.

Accordingly, the Gene Set Enrichment Pathway Analysis (GSEA) shows that, in both REH FASTKD1 ko cells and patients' H4K5cr-bu high B-ALL cells, the genes encoding for mitochondrial functions are significantly upregulated, confirming the presence of active mitochondria in these cells (Fig. S7 top panels). Other common features are shared by the expression signatures of both these cells, including high translational and proliferation/cell cycle related activities, and a significant depletion in hematopoietic stem cells genes (Fig. S7).

Discussion

Fatty acid -oxidation has recently been shown to be a major source of acetyl-CoA driving histone acetylation (McDonnel et al., 2016). Our present investigations reveal that mitochondrial activity and fatty acid -oxidation in particular, is also the main source of histone acylations. Interestingly, this conclusion is in full agreement with the recent finding identifying fatty acid -oxidation, which in yeast takes place in the peroxisomes, as a driver of histone crotonylation in S. cerevisiae, particularly during Yeast Metabolic Cycles (Gowans et al., 2019).

As demonstrated here, histone acylations preferentially occur, and hence concentrate, on regions with high levels of histone acetylations. The functional meaning of this observation is that the sum of a series of low abundance histone acylations and their local concentration on specific regions could make a significant contribution to non-acetyl histone modifications, in terms of stoichiometry and in terms of gene expression regulation, by competing with histone acetylation. This situation should have a direct effect on the stability of the interaction of bromodomains with chromatin. Indeed, structural studies have demonstrated why most of the bromodomains are unable to bind longer acyllysine modifications and how these modifications could attract other types of binder factors (Andrews et al., 2016b). Consequently, an increase in histone acylations/acetylation ratio, while destabilising chromatin binding by bromodomains, could in contrast stabilise the interaction of factors bearing YEATS and DPF domains, which could in turn enhance the displacement of bromodomain factors and their increased availability.

Another important concept developed here is that of a bromodomain factor "reservoir". The pool of BRD4 bound to the genome-wide bulk of acetylated nucleosomes could be considered as a reservoir of BRD4. As shown here, a general increase of >3C acylations on H4K5 leads to a global increase in the solubility of BRD4. This "mobile" fraction of BRD4 would be released from numerous genomic sites and concentrate on a limited number of "hyperdynamic" chromatin loci, such as active gene TSSs. This explains the increase in BRD4 binding observed upon FASTKD1 inactivation at highly active gene TSSs in our ChIP-seq mappings. This mechanism also makes BRD4 available for interactions with TSS-bound non-histone factors, including the acetylated cyclin T1 subunit of pTEF-b, in a bromodomain-dependent (Schroder et al., 2012) and independent (Lambert et al., 2019) manner.

We also took advantage of the quantitative measurements of H4K5bu and H4K5cr in B-ALL samples to investigate the relationship between gene expression and the level of H4K5 acylations.

The correlation between the level of expression of the mitochondrial gene ND2 and the level of H4K5bu-cr in these patients' samples suggests that our observation made in REH cells can be extended to patients' B-ALL blast cells, where high mitochondrial transcriptional activity is also associated with an increased H4K5 acylation / acetylation ratio.

Based on all these data we propose that the activation of mitochondrial activity leads to an increase in the H4K5 acylation (butyrylation, crotonylation, etc..), loosening the binding of the genome-wide bulk of acetylated nucleosomes by BRD4 (BRD4 reservoir), making it available for recruitment and binding at active gene regulatory sites (Fig. 7). In the frame of ALL, we also propose that a decrease of mitochondrial activity in blasts due to aberrant FASTKD1 expression or for any other reasons, could favour a gene expression pattern associated with aggressive forms of this pathology (Wang et al., 2015).

Overall, this work highlights several important concepts in the biology of chromatin and transcription. We propose that there is a systematic co-existence of histone acetylation and histone acylations at chromatin dynamic spots. With respect to bromodomain factors' function, histone acylations should be considered collectively and not individually. Namely, in terms of stoichiometry relative to acetylation, >3C histone acylations, including butyrylation, crotonylation, etc., are permanently exchanged and hence should be considered as one functional entity. Histone acylations occur and concentrate on regions of high histone acetylation. The ratio of histone acyl/acetyl is a critical functional parameter, which is tuned by upstream cell metabolic reactions. Mitochondrial activity and -oxidation in particular, are important drivers of histone acylations. Histone acyl/acetyl ratio fine tunes the availability of BRD4 for recruitment at its sites of action and could represent a general mean of controlling bromodomain-containing factors availability and function. See Figure S1 for information on the generation of REH knock-down and knock-out cells.

See Figure S2 for the mitochondrial targeting of FASTKD1 (related to Fig. 1B) A-Histone extracts from wild-type and FASTKD1 ko cells were analysed by label free mass spectrometry method. The relative abundance of the indicated identified site-specific modifications was determined and expressed as the ratio FASTKD1 ko to wild-type. The corresponding core histone peptides were used to normalize the considered modified peptides. Please note that K8bu and K12bu containing peptides could not be distinguished following these analyses. Consequently, the quantification regarding these two modification sites were plotted as H4K8bu-K12bu.

B-Total extracts from wild-type and FASTKD1 ko cells were used to detect the indicated H4K5 and H4K8 modifications as well as actin in two parallel immunoblots using the corresponding specific antibodies as indicated. The specificities of these antibodies were confirmed by dot blot analyses on peptides bearing the target H4K5 acylations as well as unrelated acylations (data file S1).

C-Wild-type and FASTKD1 ko cells were treated with Rotenone (0.5 M) for one or two hours. Protein extracts were prepared, and the corresponding immunoblots were probed as in B.

D-Wild-type and FASTKD1 ko cells were treated with Triacsin (3 M) for 16 hours and protein extracts were prepared and analysed as in B.

E-Wild-type and FASTKD1 ko cells were treated with ND-630 (100 nM) for 6 hours, extracts were prepared and analysed as in B.

F-Wild-type REH cells were treated with the indicated concentrations of octanoate for 24 hours and the indicated histone PTMs were analysed as in B.

G-Wild-type and FASTKD1 ko cells were treated with Ranolazine (0.5 mM) for 6 hours and protein extracts were analysed as in B.

H-The scheme represents the metabolic pathways and the key enzymes targeted by the indicated inhibitors in this series of experiments (D to G). FAO stands for Fatty Acids Oxidation.

See Figure S3 for accumulation of H4K5 acetylation and various acylations in FASTKD1 wildtype and ko cells after octanoate treatment (related to Fig. 2F). See Tables S1,S2 and S3 for details on the unbiased determination of histone H3 and H4 propionylation and butyrylation (related to Fig. 2A). See Table S4 for the Characteristics of B-ALL patients samples.

Figure 4: H4K5 acyl/acetyl ratio controls the dynamics of BRD4-chromatin interaction

A-Total wild-type REH cell extracts were used in pull-down experiments with the indicated peptides. After pull-down the peptide-bound proteins were recovered in SDS-PAGE loading buffer, submitted to PAGE and BRD4 was visualized using the corresponding antibody.

B-High salt extracts (total BRD4) from wild-type and FASTKD1 ko cells were prepared and a peptide pull-down experiment was performed using unmodified H4 tail peptide or the corresponding acetylated peptide.

C-Wild-type and FASTKD1 ko cells were lysed in a buffer containing the indicated concentrations of NaCl. After centrifugation, the corresponding supernatants and pellets (salt extracts and nuclei, respectively) were analysed by immunoblotting for the presence of BRD4.

Actin was also visualized as a loading control for the soluble extracts and H3 for the nuclei fraction (upper panels).

Wild-type and FASTKD1 ko cells were treated with the indicated concentrations of JQ1 for 3 hours, cells were lysed, and the nuclei pelleted by centrifugation. The soluble extracts and the nuclei fractions ("soluble" and "nuclei", respectively) were analysed as above (lower panels). See Figure S4A for the selective action of CobB on H4K5 acetylation compared to butyrylation and crotonylation in COS-7 cells (related to Fig. 4D).

D-

See Figure S4B for the effect of CobB expression in COS-7 cells on BRD4 -chromatin interaction stability (related to Fig. 4E). For all TSS, the mean normalized read counts/TSS were calculated for each genotype (wildtype, ko-1 and ko-2) and each experiment H4K5cr, H4K5bu and H4K5ac. Considering each genotype, the TSS regions were then plotted according to their mean normalized read counts for H4K5cr (upper panels) or H4K5bu (lower panels) ChIP-seq (y-axis) as a function of their mean normalized read counts for H4K5ac ChIP-seq (x-axis). In order to evaluate the relationship between the measured parameters, the non-acetyl acylation (H4K5cr or H4K5bu)

and the acetylation of H4 lysine 5 (H4K5ac), in wild-type cell and in the two FASTKD1 ko cells, a linear regression model (grey lines) and an exponential regression model (red lines) were fitted.

B-

The two gene groups defined in A were respectively used as genesets for GSEA plots to test for their enrichment/depletion in the JQ1-treated REH cells. These plots show a significant depletion of genes up or down regulated in FASTKD1 ko cells. Both these genes' categories are therefore JQ1 sensitive.

C-

D-

The two gene groups defined in C were respectively used as genesets for GSEA plots to test for their enrichment/depletion in the FASTKD1 ko REH cells. These plots show significant depletion or enrichment of genes respectively down or up regulated in FASTKD1 ko cells suggesting that the transcriptional effect of high acylation in B-ALL blasts is similar to the effect of FASTKD1 ko -induced high acylation in REH cells.

See Figure S6 showing differential expression between B-ALL blasts with high compared to low acylation levels.

See Figure S7 for the GSEA of the transcriptomic signatures of REH cells (FASTKD1 ko versus wild-type, or JQ1 treated versus untreated) and B-ALL with high versus low H4K5cr/bu levels. acylation/acetylation ratio. Bromodomain containing factors, such as BRD4, lose their tight binding to the genome-wide acetylated nucleosomes (reservoir pool) and therefore become available to be redistributed from this reservoir pool towards specific and localized genome regions including active genes' TSSs (functional pool).

Key resource table

RESOURCE AVAILABILITY Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Saadi Khochbin (saadi.khochbin@univ-grenoble-alpes.fr).

Materials Availability

Plasmids and cell lines generated in this study are available upon request. • This study does not report original code.

Data and Code Availability

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell lines and cell cultures

Human lymphoblastic leukemia cell line REH (Female, CVCL_1650) was obtained from National Collection of Authenticated Cell Cultures and was authenticated by STR analysis prior to use. REH and the derivative cells were maintained in RPMI-1640 (Gibco) supplemented with 10% FBS (Dominique DUTSCHER), 4mM L-glutamine (Gibco) and 1% Penicillin-Streptomycin (Gibco). COS-7 (Male, CVCL_0224) were purchased from ATCC, and were cultured in DMEM (Low glucose, 1g/L, Gibco) supplemented with 10% FBS, 4mM Lglutamine and 1% Penicillin-Streptomycin. All cells were incubated at 37°C with 5% CO2.

CRISPR/Cas9 mediated knockout cell line REH cells were co-introduced with lenti-Cas9 and lenti-sgRNAs plasmids (targeting sequences indicated in Key Resource Table ) using the lentivirus infection approach described in the method details. 3 days after infection, positive cells with sgRNAs were enriched using 1.0μg/ml puromycin (Sigma-Aldrich, Cat# P8833). Genotypes of the resultant cell populations were analysed by PCR amplification (primers shown in Table S5) and sanger sequencing. Successful genome-editing of cell populations was documented by multi-spikes in the sequence map of PCR products. After being seeded into 96-well plates for 2-3 weeks, single cell clones were obtained and genotypes of each allele of single cell clones was analysed through sanger sequencing following TA cloning of PCR products. Knockout clones with frameshift indels within the exons were used for further experiments.

B-ALL patients' bone marrow samples

All the patients (n = 31) enrolled in this study were newly diagnosed in Ruijin hospital (See Table S4). Bone marrow aspiration was conducted at diagnosis. Mononuclear cells were enriched from BM samples by density gradient centrifugation with LymphoPrep™ Solution (Axis Shield PoC, Cat#1114547) and were stored at -80°C as dry pellets for further experimental analysis. This study was approved by the ethical board of Shanghai Institute of Hematology. All patients and their guardians were provided with informed consent for sample collection and research in agreement with the Declaration of Helsinki.

METHOD DETAILS

Drug treatment and sample preparations

For sodium octanoate (Sigma-Aldrich, Cat#C5038) treatment, 5 x10 5 /ml REH cells were seeded into 6-well plates, treated with or without indicated concentrations of sodium octanoate for 24 hours. For other compounds, REH wild-type, ko-1 and ko-2 cells were seeded at the density of 7.5 x10 5 /ml in 6-well plates. Treatment strategy was as following: 100 nM ND-630 (MedChemExpress, Cat#HY-16901) for 6 hours, 0.5 μM Rotenone (Sigma-Aldrich, Cat#R8875) for 1 and 2 hours respectively, 0.5 mM Ranolazine (Sigma-Aldrich, Cat#R6152) for 6 hours, 3 μM Triacsin C (Sigma-Aldrich, Cat#T4540) for 16 hours. After exposition to the different compounds, these cells were lysed with 8 M urea and subjected to sonication (Biorupter, High, 30s ON/OFF, total 10min) to obtain total protein. Protein solutions were quantified using Bradford assay (Bio-rad) before being added with SDS-PAGE loading buffer for western blot analysis. For JQ1 treatment, REH wild-type cells were exposed to 0.1 μM or 0.5 μM JQ1 or DMSO as control respectively for 24 hours. 5x10 6 cells were then harvested and yielded to RNA extraction with 500 μl TRIzol reagent (Invitrogen, Cat#15596018) according to the provider's protocol. Precipitated RNA was dissolved in DEPC-treated water and used for RNAsequencing.

For salt elution experiments around 1x107 REH wild-type, ko-1 and ko-2 or 4x106 COS-7 control and CobB-ha expressing cells were harvested, washed twice with ice cold PBS, and split into equal aliquots. Each aliquot of cells was lysed in LSDB lysis buffer (50mM HEPES pH7.0, 3mM MgCl2, 20% glycerol, 0.1% NP-40, 1mM DTT, 1xprotease cocktail inhibitors) containing different concentrations of salts and 10mM sodium butyrate for 30 min on ice. For REH cells, the KCl concentrations of 100, 150, 200 or 250 mM were respectively used and for COS-7 cells, the KCl concentrations of 100, 150, 200 mM KCl were respectively added to the lysis buffer. After centrifugation at 12000g 4°C for 10min, the supernatants were saved to detect soluble BRD4, while the nuclei pellets were subjected to SDS-PAGE loading buffer to detect chromatin tightly bound BRD4. For JQ1 treatment assay correlated with Figure 4C, lower panel, 1x10 6 /ml REH wild-type, ko-1 and ko-2 cells were exposed to 0.1 μM, 0.5 μM or 5 μM JQ1 or DMSO as control in 6-well plates. After 3 hours, cells were collected and lysed in LSDB lysis buffer with 100 mM KCl and 10 mM sodium butyrate for 30 min on ice. After centrifugation at 12000g for 10 min at 4°C, supernatants were collected to detect soluble BRD4, while the nuclei pellets were subjected to SDS-PAGE loading buffer to detect chromatin tightly bound BRD4.

Plasmids, shRNA and sgRNA FASTKD1 (NM_024622) was chemically synthesized and was cloned into LeGO-iG2 (Addegene #27341) vector to generate LeGO-FASTKD1-3x Flag construct. Co1491-IRES-RFP modified from pSicoR PGK (Addgene #12084) was kindly provided by Corinne Albiges-Rizo. Co1491-NLS-CobB-HA was subcloned from CMV-NLS-CobB, which was kindly provided by Heinz Neumann (Spinck et al., 2020). shRNAs and sgRNAs were designed using GPP Web Portal(https://portals.broadinstitute.org) or referring to GeCKO v2 library (Sanjana et al., 2014) with the corresponding targeting sequences indicated in the Key Resource Table, and were cloned into pLVX-shRNA1 (Clontech, Cat#632177) and LentiGuide-Puro plasmid (Addgene plasmid#52963) respectively. All plasmids were confirmed by sanger sequencing before being used in this study.

Lentivirus production and infection 5μg lentiviral transfer and package plasmids were co-transfected into 293T cells in each well of 6-well plates using lipofectamine 2000 (Invitrogen, Cat#11668019) according to the manufacturer's instructions. For pLVX-shRNA1, lentiCas9-blast, lentiGuide-Puro and the derivative plasmids, second generation packaging plasmids (psPAX2 and pMD2G) were used for generating viral particles, and an additional pRSV-Rev plasmid was used for transferring LeGO-iG2 and Co1491-IRES-RFP backbone as well as the subcloned plasmids. Virus supernatants were collected at 24 and 48 hours after plasmid transfection and precleared with 0.45 μm filter (Millipore). Amicon Ultra-15 fiters (Millipore) were used to concentrate virus supernatants by centrifugation at 4000 rpm 4°C for 30 min. Concentrated virus was diluted with complete growth medium and immediately used to infect the cells of interests. Infection was performed in 12-well plates, with each well containing 1ml diluted virus medium and 2x10 5 REH cells, or at 20% confluency in the case of COS-7 cells. 24 hours after infection, virus medium was washed out and replaced with fresh culture medium. Cells were then maintained regularly before further selection. For shRNA knockdown, positive cells were enriched 3 days after virus infection by 1.0 μg/ml puromycin treatment and were maintained for an average of 1 week before experimental analysis. For CobB and FASTKD1 re-expressing cells, RFP and GFP positive cells were respectively sorted with FacsAria IIu-cell sorter (BD Biosciences) 1-2 weeks after infection.

Histone preparation

Histone samples were prepared by acid extraction protocol as previously described with minor modifications (Buchou et al., 2017). In brief, 1x10 7 REH wild-type, ko-1 and ko-2 cells, or 1x10 7 COS-7 control and CobB expressing cells were lysed in 1 ml lysis buffer (0.06% NP-40, 10 mM HEPES pH 7.0, 10 mM KCl, 1.5 mM MgCl2, 0.34 M sucrose, 1xprotease cocktail inhibitor) with 10 mM sodium butyrate for 10 min on ice. Cell nuclei were pelleted by centrifugation at 250g 4°C for 5 min, and subjected to histones extraction using 0.2 M H2SO4 for 16 hours at 4°C. After centrifugation at 16000 g for 10 min at 4°C, solubilized histones were collected and then precipitated by adding TCA drop by drop to the final volume of 20%. Histone pellets were then washed once with cold acetone + 0.1%HCl, twice with cold acetone and were dried completely in air.

MS quantification of histone acylations

5 μg of each histone sample from REH wild-type, ko-1 and ko-2 cells were separated by SDS-PAGE and each spliced histone band was in-gel digested with trypsin. The tryptic peptides were analyzed by Orbitrap Fusion following an EASY-nLC 1000 HPLC system (Thermo Fisher Scientific, San Jose, CA). Mass spectrometry data were analyzed by Mascot software (version 2.3.01, Matrix Science Ltd., London, UK) against an in-house human histone sequence database (83 sequences; 13,870 residues) generated from the UniProt database (updated on 01/27/2015). All identified MS/MS spectra were manually verified. Peptides containing modifications were manually quantified using the Qual Browser (version 3.0.63, Thermo Fisher Scientific, San Jose, CA) by the area under the curve (AUC) of the extracted precursor ion of each peptide. Acylated histone peptides were normalized to the corresponding histone peptides. The relative abundance of indicated histone acylations in two ko clones versus wild-type cells was plotted in the column chart.

Peptide pull-down assay

Peptides pull-down assay was performed using a protocol described previously (Goudarzi et al., 2016). Briefly, peptides (H4 /H4K5acK8ac /H4K5buK8bu /H4K5acK8bu /H4K5buK8ac /H4K5bu /H4K8bu) were first bound to beads by incubating equal amount of each type of peptide with Streptavidin Sepharose beads (GE Healthcare, Cat#17-5113-01) in PBS supplemented with 100ng/ml TSA for 20min. Bound beads were then washed with PBS and LSDB lysis buffer (50 mM HEPES pH 7.0, 3 mM MgCl2, 20% glycerol, 0.1% NP-40, 1 mM DTT, 1xprotease cocktail inhibitors) with 25mM KCl and 10mM sodium butyrate. Around 1x10 7 FASTKD1 ko or wild-type cells were harvested and lysed in 500 μl LSDB lysis buffer with 500 mM KCl and 10 mM sodium butyrate for 20 min on ice. After centrifugation at 12000g for 10 min at 4 °C, protein supernatants were collected and diluted with LSDB lysis buffer with 10 mM sodium butyrate to achieve the salt concentration of 250 mM. A small volume of diluted protein supernatants was saved for input, and the rest was split into equal aliquots, each incubated with the corresponding conjugated beads for 2 hours at 4 °C. Beads then were pelleted and washed twice with LSDB lysis buffer with 250mM KCl and 10 mM sodium butyrate and once with PBS with 10 mM sodium butyrate. After incubating with 1x SDS-PAGE loading buffer at 100°C for 5min, pulled-down complexes were eluted from beads and preserved for western blotting analysis.

Western blotting

Western blotting with SDS-PAGE were carried out according to standard procedures using the antibodies listed in Key Resource Table . After adding ECL substrates (Bio-rad), revelation was performed with Chemidoc (Bio-rad) or Vilber Chemiluminescence system (Vilber). The dilutions of antibodies used in this study are as follows: anti-BRD4 (Bethyl Lab, 1:2000), anti-H4K5bu (PTM biolabs, 1:1000), anti-H4K8bu (PTM biolabs, 1:1000), anti-H4K5cr (PTM biolabs, 1:1000), anti-H4K8cr (PTM biolabs, 1:1000), anti-H4K5ac (PTM biolabs, 1:1000), anti-H4K8ac (PTM biolabs, 1:1000), anti-H4K5bhb (PTM biolabs, 1:1000), anti-H4K5lac (PTM biolabs, 1:1000), anti-HA (Abcam, 1:2000), anti-H4 (Abcam 1:1000), Anti-β-Actin (Sigma-Aldrich, 1:5000), Goat anti Rabbit IgG(H + L)-HRP (Bio-rad, 1:5000), Goat anti-Mouse IgG (H + L)-HRP (Bio-rad, 1:10000).

Relative quantification of histone acylations with ELISA Indirect ELISA was set up according to a protocol described previously (Dai et al., 2011). Total protein was obtained from 5x10 6 mononuclear cells of each patient in parallel with RNA extraction using AllPrep DNA/RNA/Protein Mini Kit (Qiagen) following the manufacturer's instructions. In the case of COS-7 control and CobB expressing cells, histones prepared through acid extraction protocol were used for ELISA assay. These precipitated proteins were dissolved in a small volume of 8 M urea and quantified by Bradford reagent (Bio-rad). Protein solution was diluted with PBS and coated into triplicate wells of 96-well flat-bottom plates (Nunc-Immuno products, Thermo Fisher) at 4°C for 16 hours. The amount of protein being coated was optimized according to the histone marks being detected and the protein samples being used.

In the case of total protein of B-ALL patients' samples, the protein amount for each well was as follows: 2.5μg for H4K5cr and H4K5bu detection, 0.0125 μg for H3 detection. In the case of protein from COS-7 control and CobB expressing cells, the amount of protein for each well was as follows: 0.15μg for H4K5bu and H4K5ac detection, 0.3 μg for H4K5cr detection, 0.015 μg for H4 detection. Coated plates were then blocked with 5% BSA for 1 hour at room temperature and sequentially incubated with primary antibodies accordingly and secondary antibodies. Dilutions of the antibodies used in this assay were: H4K5cr (PTM biolabs, 1:500), H4K5bu (PTM biolabs, 1:500), H3 (Abcam,1:5000), H4K5ac (Abcam, 1:2000), H4 (Abcam, 1:1000), HRP linked Goat anti Rabbit IgG (Bio-rad or CST, 1:4000). After the addition of TMB substrate (Abcam, Cat#ab171523) and acid stop solution (Abcam, Cat#ab171529), the signal intensity was recorded at OD 450 nm. Relative quantity of histone marks was calculated based on the value of signal intensity.

Immunofluorescence

Empty vector and FASTKD1-Flag expressing cells were treated with 250 nM Mitotracker Red (Invitrogen, Cat#M7512) dye for 20 min at 37 ºC in CO2 incubator. Approximately 8x10 4 cells were then collected, washed once with PBS and spined onto glass slide at 800 rpm for 5 min. Fixation was performed using 4% paraformaldehyde for 15 min, followed by permeabilization with 0.3% Triton-X100 for 10 min at room temperature. After being blocked with 10% BSA for 1 hour at room temperature, slides were sequentially incubated with anti-Flag antibody (Sigma-Aldrich, 1:500 dilution) and anti-Mouse IgG DyLight 405 (Invitrogen, 1:500 dilution). Fluorescent images were captured by confocal laser scanning microscope (TCS SP8, Leica) under a 63x 1.40 numeric aperture oil-immersion lens and were processed with ImageJ.

Fluorescence recovery after photobleaching (FRAP) 2x10 5 COS-7 control and CobB expressing cells were seeded in 1-well Chambered Coverglass (LAB-TEK brand products, Thermo Fisher) respectively the day before being transfected with 1μg BRD4-NUT-GFP plasmid using lipofectamine 2000 (Invitrogen). 24 hours after transfection, FRAP was performed on 8 independent cells of each group using fluorescent microscope (LSM710 NLO-LIVE7-Confocor3, Zeiss) equipped with a 488 nm laser and a LP505 filter. A circular region on GFP foci was bleached for 1.592 seconds and the recovery of fluorescence was recorded each second for a duration of 75 seconds after photobleaching by software ZEN lite. Immunofluorescence photos of BRD4-NUT-GFP foci from control and CobB expressing cells were captured before FRAP.

For the data analysis and plotting, fluorescence intensity was normalized and rescaled to a reference axis from 0 to 1. 8 datasets from each cell group were individually fitted using the single exponential model: 𝐼(𝑡) = 𝛼(1 -2 -𝑡 𝜅 ) . The 8 biological replicates giving the best fitting statistics were used to calculate the average half-life (t1/2) of fluorescence recovery (κ) and average mobile fractions (α). Values of mean ± 2 SEM are shown in the figure. FRAP curve was plotted on the mean of the 8 biological replicates ± 2 SEM at each time point.

Metabolic assay

Metabolic assay was performed according to the Seahorse XF Cell Mito Stress Test user manuals. Briefly, 1 × 10 5 REH wild-type, ko-1 and ko-2 cells were resuspended in 180 μl assay medium formulated as pH 7.4 ± 0.1 bicarbonate-free Seahorse XF base medium (Agilent, Cat#103334-100), with 10 mM glucose (Gibco), 1 mM sodium pyruvate (Gibco) and 4 mM Lglutamine (Gibco). Resuspended cells were then seeded in XF96 cell culture plates (Agilent, Cat#102601-100), which had previously been coated with 0.1% gelatin (Sigma-Aldrich, Cat#G1393) for at least 1 hour at room temperature. The seeded plates were then incubated at 37 °C for 1 hour in a non-CO2 incubator before being loaded onto Seahorse XFe96 Analyzer (Agilent). Oxygen consumption rate (OCR) was measured at basal level as well as after injection of 1 µM oligomycin A (Sigma-Aldrich, Cat#75351), 1 µM FCCP (Sigma-Aldrich, Cat#C2920), 0.5 µM rotenone (Sigma-Aldrich, Cat#R8875) and 1 µM antimycin A (Sigma-Aldrich, Cat#A8674). OCR values (pmol/min) were normalized to cell number. Mean ± SEM based on 5 replicates were plotted in the figure .   RT-qPCR 5x10 6 cells from FASTKD1 wild-type and two ko clones, or from shCtl and two shRNAs knockdown cell lines were collected and lysed in 500 μl TRIzol reagent (Invitrogen, Cat#15596018). Total RNA was obtained by phenol-chloroform extraction and isopropanol precipitation using standard procedure. For B-ALL patients' samples, total RNA was extracted from 5x10 6 mononuclear cells of each patient using AllPrep DNA/RNA/Protein Mini Kit (Qiagen, Cat# 80004) following the manufacturer's instructions. cDNA was produced from 1μg RNA with superscript III transcriptase (Invitrogen, Cat#18080-051) using random hexamers according to the manufacturer's instructions. Quantitative PCR (qPCR) was performed with SYBR green reagent (Yeasen, Cat#11202ES08) on ViiA 7(The Applied Biosystem) using primers described in Table S5. CPT1A and ND2 with patients' samples were calculated via -dCt against GAPDH. The correlation between CPT1A and ND2 was analysed with Spearman correlation. Mean values of triplicates were plotted with scatterplot using GraphPad Prism 5. Fold changes of gene expression levels were calculated via 2^-ddCt and are represented by mean ± SEM based on at least three independent experiments. Statistical differences between wild-type and two ko, or between shCtl and shRNAs groups were calculated with Fisher's Least Significant Difference (LSD) post one-way ANOVA test. * p<0.05, ** p<0.01, ***p<0.001.

RNA-seq

Three independent RNA extractions from FASTKD1 wild-type and two ko clones (ko-1, ko-2), or from control and two knockdown cell lines (sh-1, sh-2), or from solvent and JQ1 treated wild-type cells, or from 25 B-ALL patients' bone marrow samples were sequenced. For each sample, 1 µg RNA samples (RIN = 10) were used for libraries preparations with TruSeq Stranded Total RNA (RiboZero Human/Mouse/Rat) Library Prep (Illumina, Cat#RS-122-2201) according to manufacturer's instructions. Each library was quantified on Qubit with Qubit® dsDNA HS Assay Kit (Life Technologies) and the size distribution was examined on the Fragment Analyzer with High Sensitivity NGS Fragment Analysis kit (Agilent). Libraries prepared from solvent and JQ1 treated cells samples were sequenced on Illumina NS500 (PE75) at the TGML Platform of Aix-Marseille Université, and those prepared from FASTKD1 control and knockdown, wild-type and knockout cells samples, and B-ALL patients' samples were sequenced on Hiseq 4000 (PE150) platform in Novagene company. The sequenced reads were aligned from raw sequence fastq data using STAR v2.5.2b software on UCSC hg19 reference genome. The aligned reads were normalized, and log transformed using the R bioconductor package DEseq2 (http://bioconductor.org/packages/3.12/bioc/html/DESeq2.html). The standardized and normalized read counts of mitochondrial genes were used to generate a heatmap in Figure 1A. Supervised transcriptomic analyses were performed to identify genes significantly up-and down regulated between two conditions using thresholds of Student t-test p-value <0.05 and fold change absolute value of 1.5.

ChIP-seq

ChIP assays for H4K5cr, H4K5ac and BRD4 were carried out as previously described with minor modifications (Buchou et al., 2017, Barral et al. 2017). Around 5 -10 x10 7 REH wildtype, ko-1 and ko-2 cells were harvested and lysed in 1.5 ml lysis buffer (0.05% Triton-X 100, 15 mM Tris-HCl pH 7.4, 60 mM KCl, 15 mM NaCl, 0.34 M sucrose, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 0.65 mM spermidine, 1x protease cocktail inhibitors) with 10 mM sodium butyrate and incubated for 5 minutes at 4°C. Cell nuclei were pelleted by centrifugation at 250 g for 5 min at 4°C and resuspended in MNase buffer (10 mM Tris-HCl pH 7.5, 10 mM KCl, 2 mM CaCl2) with10 mM sodium butyrate. Then the cell nuclei solution was subjected to micrococcal nuclease S7 (Sigma-Aldrich, Cat#10107921001) digestion (5U MNase per 100μg nuclei) at 37°C for 20 min to obtain mononucleosomes. Small aliquots of mononucleosomes solutions were collected for input and used to check the efficiency of digestion before immunoprecipitation.

Immunoprecipitations were carried out as follows: 5 μg anti-H4K5cr (PTM biolabs, Cat#PTM-521), anti-H4K5bu (PTM biolabs, Cat#PTM-310), anti-H4K5ac (abcam, Cat#ab51997), anti-BRD4 (Bethyl lab, Cat#A301-985A100) antibodies were coupled with 50 µl Dynabeads protein G (Thermo Fisher, Cat#10007D) respectively according to the manufacturer's instructions. Digested mononucleosomes solutions were diluted with LSDB500 buffer (50 mM HEPES pH 7.0, 3mM MgCl2, 500 mM KCl, 20% glycerol, protease cocktail inhibitors) with 10 mM sodium butyrate to achieve the final KCl concentration of 350 mM. For each reaction, around 100 μg chromatin was incubated with antibody-coupled beads at 4°C for 16 hours. Immunoprecipitated beads were then washed three times with LSDB350 buffer (50 mM HEPES pH 7.0, 3 mM MgCl2, 350 mM KCl, 20% glycerol, protease cocktail inhibitors) with 10 mM sodium butyrate and one time with elution buffer (10 mM Tris-HCl pH 8.5, 1 mM EDTA). ChIP samples were eluted from beads with elution buffer containing 1%SDS at 65°C for 15 min and were purified by phenol-chloroform extraction and ethanol precipitation in parallel with input samples. For sequencing, ChIP libraries were prepared using MicroPlex Library Preparation Kit v2 (Diagenode) according to manufacturer's instructions. Each library was quantified on Qubit with Qubit® dsDNA HS Assay Kit (Life Technologies) and size distribution was examined on the Fragment Analyzer with High Sensitivity NGS Fragment Analysis kit (Agilent). ChIP libraries against H4K5cr, H4K5ac and BRD4 (1 st experiment) were sequenced on a Highoutput flow cell (400M clusters) using the NextSeq® 500/550 High Output v2.5 150 cycles kit (Illumina), in paired-end 75/75nt mode, according to manufacturer's instructions at the TGML Platform of Aix-Marseille University. ChIP libraries against H4K5bu and BRD4 (2 nd experiment) were sequenced using the NovaSeq 6000 in PE150 mode according to manufacturer's instructions at the Novogene company.

Raw fastq files were processed by 5 prime trimming, keeping 30 bp-length fragments, using `fastx_trimmer` (with options -l 30 -Q33). The trimmed fastq files were aligned on the USCS human hg38 genome using the Bowtie2 aligner (Langmead and Salzberg, 2012), with options -end-to-end, -no-mixed, -no-discordant. The aligned reads were filtered according to alignment quality (mapping quality score > 30) and normalized using bamCoverage by Reads Per Kilobase per Million mapped reads (RPKM) for the anti K5ac, K5cr and Kbu ChIP-seq and scaling factors for the anti-BRD4 ChIP-seq. For the anti-BRD4 ChIP-seq, scaling factors were computed according to the Spike in free method (Jin et al., 2020). Accordingly, for the first experiment, the respective scaling factors 4. 25, 3.58 and 5.81 were applied when calculating the BRD4 ChIP-seq read coverage in wildtype, ko1 and ko2 cells. For the second experiment the corresponding scaling factors of 4. 85, 4.6 and 5.72 were respectively applied. The aligned read counts were converted into a 10 bp bin matrix of the signal 2Kb upstream and downstream genes TSS, using computeMatrix (from the package deepTools2, Ramírez et al., 2016), heatmaps and profiles were generated using the respective deepTools2 packages, plotHeatmap and plotProfile.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis was performed with SPSS v20. Details of the number and type of replication for each experiment were described in method details or figure legends where appropriate. Each dot represents one individual in patients' experiment. Data were presented as median with interquartile range for non-normally distributed populations of patients' samples or mean ± 2 standard error of the mean (SEM) for FRAP experiment. All other data were presented as mean ± SEM. When comparing normally distributed two groups, statistical significance was determined by Student t-test. When comparing multiple groups, Fisher's LSD post one-way ANOVA test was used to determine the significance between groups. When comparing nonnormally distributed data, Mann-Whitney U test was used to determine the significance, and Spearman correlation was used to determine the correlation. p < 0.05 were considered statistically significant. Statistics are *p < 0.05, **p < 0.01, ***p < 0.001. ko REH cells histone samples were calculated by the peak area of the corresponding peaks. The PTM with more than 1.5 times variations are highlighted (see Table S2 for details).

Table S2: Relative quantitative analysis of site-specific histone H3 and H4 propionylation and butyrylation in wild-type and two FASTKD1 ko clones (related to Figure 2A). Detailed quantitative analyses of site-specific histone propionylation and butyrylation used to generate Table S1 is shown.

Table S4: Characteristics of B-ALL patients samples (related to Figure 3, 6C&D, S6, S7).

Table S5: Primers used in this study (related to STAR Methods).

  Puthier from Aix Marseille Université to support the tremendous work of sequencing. I would like to thank Yingming Zhao and Zhongyi Cheng, who provided the important antibodies in my work. I thank Minjia Tan and Lulu Pan from Shanghai Institute of Materia Medica for helping with the work by mass spectrometry, I thank Heinz Neumann and Martin Spinck from Max-Planck-Institute of Molecular Physiology to support the work of CobB.

Figure 1 .Figure 3 .

 13 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 4 .Figure 5 .Figure 6 .

 456 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 7 .Figure 8 .Figure 9 .

 789 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 11 .

 11 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 13 .

 13 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 14 .

 14 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 15 .

 15 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 17 .

 17 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 18 .

 18 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 19 .

 19 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 20 .

 20 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 21 .

 21 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 22 .

 22 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 23 .

 23 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 24 .

 24 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 31 .

 31 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 32 .

 32 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 33 .

 33 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 35 .

 35 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 36 .

 36 Figure 1. Hierarchical organization of chromatin. ...................................................................Figure 2. Chromatin Packaging. ............................................................................................... Figure 3. Nucleosome core particle structure. ..........................................................................Figure 4. Sequence preference of nucleosome. ........................................................................ Figure 5. Nucleosome positioning. .......................................................................................... Figure 6. Gene transcription. ....................................................................................................Figure 7. Histone N-terminal tail modifications. .....................................................................Figure 8. Writers and erasers of lysine methylation. ................................................................Figure 9. Distribution of histone lysine methylation and their association with gene transcription. ........................................................................................................................................... Figure 10. HDACs. ................................................................................................................... Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. ................................ Figure 12. Bromodomain structure. .........................................................................................Figure 13. Structure of YEATS domain binding H3K9ac. .......................................................Figure 14. Histone non-acetyl acylations. ................................................................................Figure 15. Other histone modifications. ................................................................................... Figure 16. Combinatorial readout of histone PTMs. ................................................................Figure 17. One carbon metabolism. .........................................................................................Figure 18. Production and utilization of acetyl-CoA. ..............................................................Figure 19. β-oxidation. .............................................................................................................Figure 20. Acyl-CoAs metabolism ...........................................................................................Figure 21. C/T genes contribute to various pro-tumoral processes. .........................................Figure 22. Identification of six gene signature in ALL. ...........................................................Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts. .................Figure 24. FASTKD1 is associated with poor prognosis. ........................................................ Figure 25. mtDNA. ................................................................................................................... Figure 26. OXPHOS complexes. ........................................................................................... Figure 27. Domains of FASTK family proteins. .................................................................... Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. ............ Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. ................................................................ Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. ..........................Figure 31. Mitochondrial activity drives histone modifications. ...........................................Figure 32. β-oxidation drives histone acylations. ..................................................................Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients. ................................................................................................................... Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu. ..................Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin. ................Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. ......................................................................................................................................... Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. .........................................................................................................

Figure 38 .

 38 Figure 38. H4K5cr/ac ratio confers to BRD4 genomic re-distribution. ................................. 123 Figure 39. Genomic redistribution of BRD4 regulates specific sets of genes in REH cells and ALL patients. ................................................................................................................... 126

  Chromatin is formed of DNA bound to histones. It has long been known that chromatin displays two global states : euchromatin which is associated with active or potentially active genomic regions, and heterochromatin, which is generally associated with the repressed genome.

Figure 2 .

 2 Figure 2. Chromatin Packaging.

Figure 3 .

 3 Figure 3. Nucleosome core particle structure. A. Histone fold domains of H3. Core histones have conserved motifs of histone fold, namely α1-L1-α2-L2-α3 from N terminus to C terminus. B. Nucleosome core particle (NCP) is composed of two copies of each H2A, H2B, H3 and H4. Disc view (left) and gyre view (right) of NCP show the crucial domains and flexible tails of core histones

Figure 4 .

 4 Figure 4. Sequence preference of nucleosome.

Figure 5 .

 5 Figure 5. Nucleosome positioning.

  recognizes and interacts with promoters, and produces nascent RNA at transcription start sites (TSS). Recognition of RNA polymerases is aided by transcription factors (TFs), including general transcriptional factors (GTFs) and associated activators or coactivators. TFIID is the first GTF that interacts with DNA, followed by sequential recruitment of TFIIA, TFIIB, TFIIF, TFIIE, and TFIIH. All the recruited GTFs together with RNA Pol II, form the pre-initiation complexes (PICs) at promoter regions. The PICs are in closed form when DNA is still in a double helix state, before XBP, the translocase subunit of TFIIH, hydrolyzes ATP, through which, unwinds dsDNA and pushes DNA strand towards the catalytic center of RNA polymerase to facilitate the synthesis of nascent RNAs. After which, RNA Pol II is released from associated GTFs and promoters (termed as promoter escape) and RNA synthesis is hence elongated. In many cases, RNA Pol II pauses after transcribing around 30-50 nucleotides downstream of TSS, which is known as promoter-proximal pause. The paused Pol II is stabilized by 5,6dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) sensitivity-inducing factor (DSIF) and negative elongation factor (NELF), which consequently, inhibits early processive transcription elongation. Release of paused RNA Pol II requires NELF, DSIF and carboxy terminal domain (CTD) of Pol II. CTD is phosphorylated by kinase CDK9, which is a subunit of positive transcription elongation factor b (P-TEFb).

Figure 6 .

 6 Figure 6. Gene transcription.

Figure 7 .

 7 Figure 7. Histone N-terminal tail modifications. Histone tails are subject to a variety of modifications. P: Phosphorylation. Ac: Acetylation. Me: Methylation. Adapted from 26 .

Figure 8 .

 8 Figure 8. Writers and erasers of lysine methylation. The graph summarizes the known KMTs (left) and KDMs (right) families with their subcellular localization. The graph also shows the potential inhibitors and their clinical status relating methylation enzymes. From 33 .

Figure 9 .

 9 Figure 9. Distribution of histone lysine methylation and their association with gene transcription.

  and IIb (HDAC6, 10); Class III HDACs are known as sirtuin (SIRT) family proteins, which are homologous to yeast Sir2 and comprise of sirtuin1-7; Class IV HDACs has only one member HDAC11, which is closely related to HDACs of class I and II. In general, HDACs members perform catalysis of acetyl groups removal in an either zinc ion (Zn 2+ )-dependent (e.g., HDAC I, II, IV) or nicotinamide adenine dinucleotide (NAD+) dependent mechanism (e.g., SIRT family proteins) and they do not seem to have much preference for specific acetylated sites in vitro. However, different classes of HDACs tend to have distinctive roles in vivo due to their specific subcellular localization.

Figure 10 .

 10 Figure 10. HDACs. HDACs are classified into four families, Class I-IV. Class II can be further divided into IIa and IIb subclasses. The members of each family, their domains, subcellular localization are shown in the figure. DAC: Deacetylase

  Sirtuin family share NAD+ dependent deacetylase domains but have distinct flanking C and N terminal parts, facilitating their different subcellular locations, enzymatic activities and substrates specificities. SIRT1 and 2 shuttle between cytoplasm and nucleus, SIRT3-5 reside in mitochondrion, while SIRT 6 and 7 are located in nucleus and nucleolus respectively. This family utilize acylated lysine and NAD+ as substrates and catalyze the production of unacylated lysine, 2'-O-acyl-ADP-ribose and nicotinamide (NAM). Recent studies uncovered that these family of proteins can also perform desuccinylation, deglutarylation, demalonylation etc., and can act as ADP-ribosyl-transferases and lipoamidases as well (Figure11)[START_REF] Kumar | For Certain, SIRT4 Activities! Trends[END_REF] .

Figure 11 .

 11 Figure 11. Sirtuins are protein deacetylase and ADP-ribosyltransferase. A shows the NAD+ dependent deacetylase catalysis by Sirtuin. B shows the ADP ribosylation activity performed by Sirtuin. From 80 .

Figure 12 .

 12 Figure 12. Bromodomain structure. A. (PDB:1N72) shows the structure of bromodomain from p300/CBP. BrD comprises of four helix bundles (αA, αB, αC, αD), and two loops (ZA and BC). B and C (PDB:1E6I) show bromodomain from GCN5 binding with H4K16ac peptide. C shows hydrogen bonds formed by amide group of bromodomain and carbonyl oxygen group of acetyllysine. Red sphere: oxygen atom; Blue sphere: nitrogen atom; orange rods: histone peptide chain.

Figure 13 .

 13 Figure 13. Structure of YEATS domain binding H3K9ac. A. (PDB: 4TMP) The figure shows the structure of YEATS from AF9 binding with H3K9ac peptide. YEATS comprises of 8 antiparallel β-strands and 2 short α-helices. B The figure shows the interaction between H3K9ac and YEATS structural module.

Figure 14 .

 14 Figure 14. Histone non-acetyl acylations.

Figure 15 .

 15 Figure 15. Other histone modifications. A Glycation. B ADP-ribosylation. C Serotonylation. D Citrullination.

  , and eicosapentaenoate (20:5)) onto proteins via thioester (S-), amide (N-) and ester (O-) bonds depending on the residues they associate with. Using unbiased proteomic analysis, Wilson and colleagues have discovered H3 variants to be substrates of S-fatty acylation 191 . Besides, Zou and colleagues reported that histone H4ser47 is subjected to O-palmitoylation catalyzed by Acyl-CoA:Lysophosphatidylcholine Acyltransferase I (LPCAT1) in response to exogenous calcium ions. H4 O-palmitoylation is associated with RNA Pol II activation and increased gene transcription 192 . In addition to histones, other chromatin interacting proteins can also undergo fatty acylation, which in turn might participate in chromatin structure and gene transcription regulation.

Figure 16 . 2 .

 162 Figure 16. Combinatorial readout of histone PTMs. The binding specificity of readers protein to histone marks can be influenced by the adjacent histone marks. For example, certain reader domains can recognize multiple PTMs simultaneously (A). Combined reader domains can recognize different marks on cis-histone (B), different histone proteins of cis-nucleosome (C, left) or of transnucleosomes (C, right). Moreover, reader proteins can form complexes with other proteins, including additional readers in vivo. Different subunits in the complexes therefore could exert multiple functions as a whole (D). From 194 .

  molecules and intracellular regulatory pathways by deacetylating histone and non-histone substrates. Hagelkruys and colleagues have reviewed studies of HDACs and cancer in 2011, where they highlighted various aspects of HDACs activities in cancer biology, predominantly including cell proliferation and cell cycle, apoptosis, differentiation, DNA damage response, metastasis, angiogenesis, autophagy, etc. 382 .

  HDACsInhibition of HDACs promotes cell apoptosis by directly activating intrinsic and extrinsic pathways (e.g., caspase-8, 10 expression)398, 399 , or by sensitizing cells to apoptosis via downregulating antiapoptotic proteins (e.g., cFLIP, Bcl2, survivin, XIAP, Mcl-1 etc)371, 400-403 [494-496].SIRTsInhibition of SIRT1/2 inhibition was found to activate caspase-3, -8 and -9 391 . Ectopic overexpression of SIRT6 could upregulate Bcl-2 expression and decrease cleaved caspase-3 and Bax 404 . Knocking out SIRT4 confer to 5-FU chemoresistance via inhibiting cell can bind with HDAC1, 2 and 3 and recruit them to promoters of AML1 targeted genes in acute leukemia 406 .HDAC3PML-RARα could recruit HDAC3 as component of nuclear receptor corepressor (NCoR) complexes and repress gene expression program associated with hematopoietic differentiation 407 .

  anti-tumor functions by bringing about changes on specific oncogenes/tumor suppressors or oncogenic programs513 . For instance, DNMTi azacitidine and decitabine causes robust demethylation of specific tumor suppressor gene promoters such CDKN2B, or global genespecific demethylation concomitant with genome-wide DNA hypomethylation[514][515][516] . HDACi treatment resulted in differential expression of many oncoproteins (e.g., PI3-kinase catalytic subunit PI3KCB) and tumor suppressors (e.g., programmed cell death protein 4 PDCD4) in malignant B-and T-cell lines 517 . In addition, HDACi was reported to directly increase the acetylated c-Myc at K323 and hence regulating c-Myc mediated oncogenic program 518 .Epigenetic drugs (HDACi, KMTi and DNMTi) have also been documented to impact cell differentiation, metabolic reprogramming, cell immunity etc. For instance, Combination of LSD1/HDAC inhibitors increase H3K27ac and H3K4me1 and control differentiationassociated genes expression in diffuse intrinsic pontine glioma519 . Treatment with DNMTi/HDACi was also reported to downregulate IRF4 and MYC and induce a gene expression profile of mature plasma cells in myeloma cells 520 . In the case of immune response, DNMTi treatment was reported to upregulate the expression of tumor associated antigens (e.g., surface marker of MHC, cancer testis antigens, etc.). In addition, DNMTi combined with HDACi was reported to induce interferon-α/β based transcription program and enhanced antigen presentation, accompanied by altered host immunity, therefore contributing to tumor immune cell invasion of non-small-cell lung cancers 521 . Epigenetic drugs could also facilitate metabolic reprogramming and therefore make the cancer cells sensitive to the corresponding treatment. For example, in glioblastoma models, HDACi treatment triggered metabolic reprogramming of cells with impaired glycolysis and enhanced oxidative phosphorylation, by suppressing c-Myc and increasing transcriptional drivers of fatty acid oxidation, PGC1α and PPARD 522 . Other regulators targeted by epigenetic drugs that are involved in the cell cycle, proliferation, apoptosis, angiogenesis etc. had been discussed in section 2.1.2 and are reviewed

(

  hCys) and adenosine by SAH hydrolase (SAHH). Homocysteine is then remethylated and converted back to methionine by vitamin B12-dependent methionine synthase (MS) using 5methyl-tetrahydrofolate (5-mTHF, MTHF) as methyl group donor or alternatively, in the case of liver, by betaine homocysteine methyltransferase (BHMT) with the production of dimethylglycine (DMG).Methionine cycle is fueled by the folate cycle. 5-mTHF in folate cycle donates its one-carbon methyl group to homocysteine. After transferring the methyl group, 5-mTHF is converted to THF. THF is then used to produce 5,10-meTHF by receiving methyl group from serine, which subsequently is converted to glycine by serine hydroxymethyltransferase (SHMT). This reaction that converts serine to glycine is reversible. The last step to complete folate cycle is to convert 5,10-meTHF back to 5-mTHF via NADPH-dependent catalysis acted by 5,10 methylenetetrahydrofolate reductase (MTHFR).

Figure 17 .

 17 Figure 17. One carbon metabolism. The methionine cycle (red, middle) can provide methyl group in the form of SAM to fuel biochemical reactions, such as histone methylation (yellow, right). The methyl group in the methionine cycle is provided by the folate cycle (blue, left). The main donor of the above one-carbon unit is serine. SAM: S-adenosylmethionine; MAT: Methionine adenosyltransferase; SAH: S-adenosyl homocysteine; SAHH: SAH hydrolase; hCys: homocysteine; MS: methionine synthase; MET: methionine; THF: tetrahydrofolate; 5-mTHF: 5-methyl-tetrahydrofolate; BHMT: betaine homocysteine methyltransferase; 5;10-meTHF: 5;10; methylenetetrahydrofolate. From 544 .

Figure 18 . 3 . 2 . 2

 18322 Figure 18. Production and utilization of acetyl-CoA. Acetyl-CoA can be produced in mitochondrion, cytosol and nucleus. Acetyl-CoA produced in mitochondrion can be exported to cytosol in the form of citrate, where it is converted back to acetyl-CoA under the reaction of ACLY. In addition, ACSS2 can generate acetyl-CoA from acetate in cytosol and in nucleus. PDC can be translocated to nucleus, where it produces acetyl-CoA from pyryvate. ACLY: Acetyl-CoA lyase; ACSS2: Acetyl-CoA synthase; PDC: Pyruvate dehydrogenase complex. From 555 .

  metabolism. In addition to protein acetylation, intracellular acetyl-CoA can also be consumed in TCA cycle or serve as building blocks for lipid biosynthesis, therefore perturbation of mitochondrial respiration and lipid synthesis might in turn impact protein acetylation by competing with the usage of acetyl-CoA. Inhibition of acetyl-CoA carboxylase expression, which catalyzes the first and rate-limiting step of fatty acid synthesis, results in increased acetyl-CoA available for histone acetyltransferases and enhances histone acetylation566, 567 . Yucel and colleagues reported that compared to proliferating muscle stem cell (MuSCs), quiescent and differentiated MuSCs have elevated utilization of glucose and acetyl-CoA for mitochondrial respiration, and therefore have decreased acetylation 568 . Conversely, using mitochondrial DNA mutated cell model, and inhibition of mitochondrial translation, Kopinski and colleagues identified that decrease of mitochondrially derived acetyl-CoA causes a reduction in histone H4 acetylation569 .The signaling between metabolic state and histone acetylation via acetyl-CoA can rewire the transcriptional program in response to nutrient availability. For example, in adipocyte, expression of glucose transporter gene GLUT4 is dependent on glucose and ACLY, which fuels acetyl-CoA production and histone acetylation at Slc2a (encoding GLUT4) locus, suggesting a positive feedback between glucose metabolism and gene expression 570 . Under hypoxic stress, acetyl-CoA produced from acetate induces H3 hyperacetylation at the promoter regions of ACACA and FASN genes and therefore enhances lipid synthesis 571 .Of note, evidences began to flourish uncovering that acetyl-CoA concentration alterations can impact specific sets of genes and therefore could contribute to cell fate and cell functioning 560 . For example, toll-like receptor activation induces glycolytic flux and ACLY activation, which enhances acetyl-CoA production and histone acetylation, thereby facilitating the transcription of LPS (lipopolysaccharide) -inducible gene sets 572 . During the muscle regeneration process, glucose metabolism program determines histone acetylation profile, which dominates stem cell and cell-cycle genes transcription and cell functioning 568 . In glioblastoma cells, ACSS2 is phosphorylated and translocated into nucleus, where it binds with transcription factor EB (TFEB) and enhances lysosomal and autophagy-related gene expression via recycling acetate and providing acetyl-CoA to TFEB targeted gene promoters 573 . The above examples demonstrate the crucial role of cellular metabolism in determining cellular phenotypes and point to epigenetic metabolites as potential targets for therapeutic usage. One key question arises as how the level of acetyl-CoA impacts histone acetylation at specific loci and regulates specific sets of genes. Campbell and Wellen summarized two possibilities that explain this issue 560 : 1) spatiotemporal regulation of acetyl-CoA production (i.e., spatiotemporal control); and 2) transcription factor ability to respond to acetyl-CoA concentrations.

  factors mediating specific sets of genes expression in response to acetyl-CoA concentration alteration Increasing evidence indicate that acetyl-CoA abundance can impact gene transcription via the regulation of transcription factors. Firstly, transcription factors per se can be acetylated, which might consequently impact its subcellular location, protein stability and trans-activating capacity. For example, elevated acetyl-CoA levels resulted from ACC1 inhibition induces Smad2 acetylation, which then translocates into the nucleus and activates Snail and Slug mediated epithelial-mesenchymal transition program and promotes cancer invasion 567 . Similarly, in chondrocyte, inhibition of ACLY reduces acetylation of p65, NF-κB and H3K27, which attenuate IL-1β induced gene transcription. Besides, under these conditions SOX9 acetylation is reduced, promoting its nuclear localization and increasing the mRNA level of aggrecan and Col2a1 581 . Secondly, transcription factors can be regulated by acetyl-CoA levels through an indirect mechanism. For example, a change of acetyl-CoA abundance regulates H3K27ac at specific loci correlated with integrin signaling and cell adhesion. This pathway participates to the modulation of calcium signalings, which trigger nuclear translocation of the transcription factor NFAT1 (nuclear factor of activated T cells), where it mediates acetyl-CoA dependent genes transcription 582 .

Fatty

  acid uptake is coupled with CoA esterification by acyl-CoA synthetases (ACSs) known as lipid activation. This reaction consumes energy leading to ATP breakdown to AMP and pyrophosphate(PPi). ACSs present several members, who have specificity for different lipids depending on the carbon-chain of lipids, namely, long chain specific acyl-CoA synthetases (ACSL), medium chain acyl-CoA synthetases (ACSM), and short chain acyl-CoA synthetases (ACSS). ACSL is associated with outer membrane of mitochondria, peroxisomes and endoplasmic reticulum, and ACSM are located in mitochondrial matrix while ACSS can be found both in mitochondrial matrix or cytosol.In mammals, both peroxisome and mitochondria ensure lipid oxidation but with separate enzymatic systems and have distinct features. Some types of lipids such as very long chain fatty acids (VLCFA), branched-chain fatty acids, bile acid intermediates and long chain dicarboxylic acids are poorly taken up by mitochondrion and are mainly degraded in peroxisome. However, peroxisome β-oxidation only generates shortened fatty acids (i.e., medium chain acyl-CoA, C6-8), while mitochondrion can proceed complete oxidation of lipids 584 .Long chain fatty acyl-CoA cannot penetrate mitochondrial membrane and must be assisted by carnitine-acylcarnitine shuttle system. The acyl group of fatty acyl-CoA is transferred to carnitine at the outer membrane catalyzed by carnitine palmitoyl-transferase I (CPT I), acyl-carnitine can then cross the membrane through carnitine translocation catalyzed by carnitineacyl-cartnine translocase (CACT). Acyl-carnitine is then converted back to acyl-CoA and carnitine at the inner membrane by carnitine palmitoyl-transferase II (CPT II). In contrast, medium (C6-12) and short chain (C4-6) can directly enter mitochondrial and are then activated to the corresponding acyl-CoAs in mitochondrial matrix.β-oxidation process proceeds as repetitive cycles of four reactions, namely 1) dehydrogenation, which introduced double bond between α and β carbon, 2) hydration which add water molecules and breaks the carbon double bond, 3) dehydrogenation, which dehydrogenate 3-hydroxyacyl-CoA to 3-ketoacyl-CoA, and 4) thiolation which cleaves the bond between α and β carbon in the presence of CoA, producing one acetyl-CoA and a shortened acyl-CoA (Figure19). Acetyl-CoA produced here can then enter TCA cycle for complete oxidation. These reactions are catalyzed by acyl-CoA dehydrogenase, enoyl-CoA hydratase, 3 (β)-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase (acyl-CoA acetyltransferase) respectively. In mammals, the first step of β-oxidation is catalyzed by several dehydrogenase, with (very) long chain acyl-CoA dehydrogenase ((V)LCAD) catalyzing C12-24, medium chain acyl-CoA dehydrogenase (MCAD) catalyzing C6-12, and short chain acyl-CoA dehydrogenase (SCAD) catalyzing C4-6. The last 3 steps of LCFAs and MCFAs are mainly catalyzed by mitochondrial trifunctional proteins (MTP, or TFP abbreviated), while SCFAs are catalyzed by ECHS, HADH and KAT respectively 585 .

Figure 19 .

 19 Figure 19. β-oxidation. β-oxidation spiral contains dehydrogenation, hydration, dehydrogenation and thiolation. Through each round of β-oxidation, a acetyl-CoA and a shortened acyl-CoA (-2C) are produced.

  Ketogenesis occurs in the mitochondria of liver cells triggered by glucose depletion conditions such as fasting. The reaction starts with two molecules of acetyl-CoA being converted to acetoacetyl-CoA by thiolase, which is then modified by an additional acetyl-CoA molecule and generates hydroxylmethylglutrayl-CoA (HMG-CoA) by HMG-CoA synthase. HMG-CoA lyase then cleaves HMG-CoA and produces acetoacetate. Acetoacetate either makes acetone through non-enzymatic decarboxylation or D (R)-β-hydroxybutyrate catalyzed by βhydroxybutyrate dehydrogenase.

Figure 20 .

 20 Figure 20. Acyl-CoAs metabolism Acyl-CoAs can be produced and consumed in various catabolic and anabolic pathways including β-oxidation, glutaminolysis, glycolysis, ketogenesis and amino acid catabolism. The various pathways involved and the key corresponding metabolites are indicated in the figure and are highlighted in different color. β-oxidation is labeled in red, ketogenesis is labeled in orange, cholesterol and fatty acid synthesis are labeled in magenta, TCA cycle in blue, amino acid metabolism in cyan, glycolysis in olive green. From 597 .

  In mammals, nicotinamide adenine dinucleotide (NAD) is synthesized from tryptophan or vitamin B3 precursors, which are provided by diet. Tryptophan is degraded through kynurenine pathway, which produces quinolinic acid and then nicotinic acid mononucleotide (NAMN).Vitamin B3 includes nicotinamide (NAM, NM), nicotinic acid (NA), nicotinamide riboside (NR) and nicotinic acid riboside (NAR), which can either generate NAMN (e.g., NA is used) or nicotinamide mononucleoside (NMN) (e.g., NR, NAM as sources). NAMN and NMN are then converted to their corresponding dinucleotide (NAAD or NAD+) by NMN adenylyltransferases (NMNAT), of which NAAD is amidated to NAD+ by NAD synthetase (NADS)598 . NAD exists as NAD+ and the reduced state NADH in cells which senses intracellular metabolic state.NAD is a cofactor for SIRT family deacetylase. During deacetylation reaction, acetyl group cleaved from acetyl-lysine is transferred to ADP-ribose of NAD, generating O-acetyl-ADP ribose and unmodified lysine (See Figure11). Therefore, NAD homeostasis can regulate histone acetylation in a SIRT-dependent manner. For example, knocking down NMNAT1 results in decreased intracellular NAD and increased levels of histone acetylation in retina 599 .Similarly, expression of nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the generation of NMN from NAM was shown to increase NAD levels and activate SIRT1, which consequently reduces histone acetylation and represses histone gene expression 599 .During the transition from quiescence to proliferation of muscle stem cell, decreased intracellular NAD level resulting from metabolism reprogramming leads to reduced SIRT1 activity, which promotes H4K16ac and transcriptional activation of muscle genes 600 .NAD also serves as co-substrates for ADP-ribosylation. ADP-ribose transferases use NAD and transfer single or several ADP-ribosyl moieties from NAD to acceptor proteins including histones, generating protein ADP-ribosylation (See Figure11). Adding NAD to cell nuclei increased tremendously protein ADP-ribosylation 601 . Nuclear NAD+ synthase NMNAT-1 can promote histone H2B ADP-ribosylation via synthesizing NAD and directing poly (ADP-ribose) polymerase (PARP)-1 catalytic activity, which subsequently inhibits phosphorylation of adjacent residue and precludes adipocyte differentiation 129 .

Figure 22 .

 22 Figure 22. Identification of six gene signature in ALL.

Figure 23 .

 23 Figure 23. Six genes' signature stratify prognosis in two childhood ALL cohorts.AK022211, FASTKD1 and STARD4 whose expression is associated with poor outcome are designated as negative genes. CAMSAP1, PCGF6 and SH3RF3, whose expression is associated with good outcome are designated as positive genes. Tumors expressing at least one of negative genes and less than 2 of the positive genes are defined as P3 group, while the rest are defined as P1&P2 group. This six genes-based algorithm stratifies patients' prognosis in GSE11877 (N=207) and GSE7440 (N=59) cohorts. Compared to P3 group (red line), P1&P2 group of patients (grey line) present a better outcome. From 711 .

Figure 24 .

 24 Figure 24. FASTKD1 is associated with poor prognosis. FASTKD1 predicts outcome in two cohorts (GSE11877 N=207, GSE7440 N=59): expression of FASTKD1 (black line) is associated with poorer prognosis. HR: Hazard ratio. From 711 .

Figure 25 .

 25 Figure 25. mtDNA. mtDNA is double strand circular DNA that encodes 13 mRNAs: ND1-4, ND4L, ND5-6, CO I-III, CYTB, ATP6/8. This figure also shows the important structures of mtDNA. For instance, the D-loop is located at non-coding region (NCR). More details about D-loop are described in 714 . From 714 .

  mtDNA and nuclear genome (nDNA). Complex I (also known as NADH dehydrogenase) is the largest enzyme of electron transport chain (ETC), it oxidizes NADH to NAD+ and transfers two electrons each cycle via flavin mononucleotide (FMN), iron-sulphur (Fe-S) clusters to ubiquinone (UbQ), namely NADH→FMN→Fe-S→Q, and produces ubiquinol (QH2). Complex I consists of ~44 subunits, of them ~37 subunits come from nDNA, while the other 7 subunits (ND1-6, ND4L) are encoded by mtDNA. Complex II (also known as succinate dehydrogenase, SDH or succinate-coenzyme Q reductase) forms the second entrance door for electrons. In complex II electrons transfer from succinate via FAD, Fe-S to UbQ, namely succinate→FAD→Fe-S→Q and produce ubiquinol and fumarate. Complex II is composed of four subunits, SDHA, SDHB, SDHC, SDHD, which are all encoded by nDNA. Complex III (known as cytochrome C reductase) transfers electrons from ubiquinol (QH2) to cytochrome C. Complex III is a symmetric dimer, with each monomer composed of ~11 subunits, among them cytB is encoded by the mtDNA, while the rest of ~10 subunits are encoded by nDNA. Complex IV (known as cytochrome C oxidase) receives electrons from cytochrome C and reduces oxygen to water molecule. Complex IV contains 13 subunits, among them COX1-3 are encoded by mtDNA, while the other ~10 are encoded by the nDNA. Except complex II, all respiratory complexes pump protons across inner mitochondrial membrane. The created proton gradient drives the rotation of F1F0 ATP synthase (known as complex V), thereby, generating ATP. ATP synthase is formed by ~14 subunits, two of them (ATP6, ATP8) are encoded by mtDNA (Figure 26) 715 .

Figure 26 .

 26 Figure 26. OXPHOS complexes. OXPHOS complexes are composed of Complex I-V. Each Complex contains multiple subunits encoded by either mtDNA or nDNA. Complex I, III and IV pump proton across IMM. The resultant gradient is then used to generate ATP by complex V (also known as ATP synthase). OMM: Outer mitochondrial membrane; IMM: Inner mitochondrial membrane; coQ: Coenzyme Q, ubiquinone ; CytC: Cytochrome C. From 716 .

Figure 27 .

 27 Figure 27. Domains of FASTK family proteins.FASTK family encompasses five members, FASTK, FASTKD1-5. They share MLS (brown), FAST_1 (green), FAST_2 (red) and RAP (navy) domains. MLS (MTS), mitochondrial localization signal; LC, low complexity; FAST_1/FAST_2, FAST kinase like domains 1/2; RAP, putative RNA-binding domain particularly abundant in Apicomplexans.

RS4; 11 ,

 11 and NALM6 cell lines, which is shown in Figure 28B. Two pieces of single guide RNAs targeting exon 4 and exon 7 respectively were used separately to generate knockout cell lines. After co-transfection of lenti-cas9 and lentiguide RNAs, multiplex cell populations were firstly genotyped with PCR amplification to check if gene-editing occurred. Multiple spikes were observed around sgRNA targeting sequence in sanger sequence map, indicating that a certain percentage of cells have been edited (Figure 28C, left). Genotype of both alleles of single clone was determined by PCR amplification followed by TA cloning. Knockout clones showing frame-shift indels were nominated as ko-1, ko-2 (Figure 28C, right) and were used for further experiments.

Figure 28 .

 28 Figure 28. Exogenous expression and depletion of FASTKD1 in B-ALL cell lines. A. Overexpression of FASTKD1 in REH and NALM6 cells. Flag tag is at the C terminus of FASTKD1 protein. Antiflag antibody was used to probe the FASTKD1-flag protein (left panels). Relative FASTKD1 mRNA level was determined using RT-qPCR. Compared to the corresponding control cells (LeGO-EV), the expression of FASTKD1

Figure 29 .

 29 Figure 29. Deletion or overexpression of FASTKD1 do not impair cell proliferation, cell cycle or chemotherapy responses in ALL cell lines. A. Cell cycle analysis of knockdown cells in RS4;11, NALM6 and REH knockout cells. Cells were harvested and fixated with 70% ethanol. After RNase treatment and PI staining, fluorescence signal was collected with flow cytometry and analysed with Flowjo. B. Cell cycle analysis of FASTKD1 overexpressing REH (upper panel) and NALM6 (lower panel) cell lines. Compared with control (LeGO-EV) cells, the FASTKD1 expressing cells (LeGO-FAKD1) do not show significant changes in cell cycle.C. Cell number of FASTKD1 control (LeGO-EV) and overexpressing (LeGO-FAKD1) cells was counted for 10 days.

Figure 30 .F.

 30 Figure 30. FASTKD1 is a negative regulator of mitochondrial respiration. A. Immunofluorescence to detect the localization of FASTKD1-flag in overexpressing cell line. Anti-Flag signal overlaps Mitotracker dye. Scale bar = 50μM. B. Heatmap of mitochondrial transcriptomic data of FASTKD1 knockdown (sh-1, sh-2) and knockout (ko-1, ko-2) cell lines (REH). Data shown are from three independent RNA extractions. C. Relative level of ND2 and ND3 in FASTKD1 knockdown (left) and knockout (right); GAPDH was used as internal

Figure 31 .

 31 Figure 31. Mitochondrial activity drives histone modifications. A. Histones acid extracts from WT and two knockout cell lines were subjected to HPLC/MS/MS to determine different H3 and H4 PTMs indicated in the figure. Column represents the relative amount in KO versus WT. B. Western blot of the indicated histone PTMs using total cell protein extracts from WT and two ko cell lines. Histone acetylation (ac), butyrylation (bu), crotonylation (cr), β-hydroxybutyrylation (bhb), lactylation (lac), and succinylation were probed with indicated site-specific anti-PTM antibodies.C. WT and knockout cells (ko-1, ko-2) were exposed to 0.5μM rotenone for the indicated time. Total protein was used for probing H4K5bu and H4K5ac.

2

 2 Fatty acid metabolism (β-oxidation) is a major driver of histone acetylation-acylationAcyl-CoAs are predominantly involved in lipid metabolism, including FA biosynthesis, degradation.

Figure 32 .

 32 Figure 32. β-oxidation drives histone acylations. A. Treatment strategy targeting fatty acid metabolism. ACC, Acetyl-CoA carboxylase; ACSL, long chain acyl-CoA ligase; CPT, carnitine palmitoyltransferase.

  ), implicating the correlation of mitochondrial activity and β-oxidation in patients. Besides, the positive correlation between D F E mitochondrial activity and histone acylation (H4K5cr/bu), and between CPT1A and H4K5cr/bu were determined in B-ALL patients (Figure 33C, D).

Figure 33 .

 33 Figure 33. Mitochondrial activity is associated with β-oxidation and histone acylation in B-ALL patients.

Figure 34 .

 34 Figure 34. The binding of BRD4 to acetylated peptides are perturbed by K5bu.

Figure 35 .

 35 Figure 35. Acyl/acetyl ratio determines the BRD4 binding affinity to chromatin.

  Western blot of soluble BRD4 dissociated by JQ1. WT and two ko cell lines were pre-exposed to JQ1

Figure 36 .

 36 Figure 36. Acyl/acetyl ratio determines BRD4-chromatin binding dynamics in COS-7 cells. A. COS-7 cells stably expressing CobB were used in this experiment. Left panels showed the levels of histones acetylation and butyrylation in control and CobB expressing cells. Left panel shows the expression of the exogenous CobB-HA. B. H4K5ac, H4K5bu, H4K5cr levels were determined between control and CobB-expressing COS-7 cells. CobB expressing cells displayed a significant decreased H4K5ac but mildly decreased H4K5bu and H4K5cr. Median and interquartile range from 5 datasets are plotted in the boxplot and the Whiskers represent the top and bottom quartiles. Mean values were indicated as "+".

Figure 37 .

 37 Figure 37. H4K5cr is enriched on the highly acetylated TSS regions and is associated with active transcription. A. Heatmap of H4K5cr and H4K5ac distribution across genome from WT cells. According to the level of H4K5ac/cr ratio, genes are divided into 5 groups, as shown in the figure (left panel). Those which are covered by high H4K5cr/ac marks are most highly transcribed (right panel). B. Total level of H4K5ac and H4K5cr on gene TSSs. As shown in the figure, most gene TSSs which are covered with low to median level of H4K5ac are also covered with modest level of H4K5cr, as shown by the linear correlation between H4K5ac and H4K5cr in the figure. However, genes that are covered by very high level of H4K5ac are covered with extremely high level of H4K5cr, as shown by the 3 rd degree polynomial correlation between H4K5ac and H4K5cr. In ko-1 and ko-2 cells, the polynomial correlation is even more obvious than the control cells, as indicated by the red arrows in the figure.

Figure 38 .

 38 Figure 38. H4K5cr/ac ratio confers to BRD4 genomic re-distribution.

Figure 39 .

 39 Figure 39. Genomic redistribution of BRD4 regulates specific sets of genes in REH cells and ALL patients.Volcano plot demonstrating the differential gene expression signature between FASTKD1 KO (A) and control, and between B-ALL blasts with high H4K5 acylations (crotonylation and/or butyrylation, n = 15) and B-ALL blasts with low acylations (n = 10) (C). Y axis: -log10 (p-value); x-axis: log (ratio of normalized expression values between ko and WT cells) (A), log (ratio of normalized expression values between B-ALL with high and low acylation levels) (C). The genes down-regulated or up-regulated with a fold change >1.5 and a Student t-test p-value < 0.05 are respectively represented in blue and red.

DISCUSSION 1 .

 1 Analysis (GSEA) plots representative of genesets enriched or depleted in the transcriptomic signature of KO versus WT cells (left panels), of JQ1 treated versus untreated (middle panels) REH cells and B-ALL with high versus low H4K5cr/bu levels (right panel). NES = normalized enrichment score; Pval = nominal p-value, a p-value of 0 indicates a p-value < 1/2000 (since our analysis was performed with 2000 permutations); FDR = False discovery rate adjusted for gene set size and multiple hypotheses testing. Oncogenic role of FASTKD1 in ALL An increasing body of evidence established that reactivation of C/T genes initiates additional cell signaling required for cell transformation and tumor aggressiveness 702 . Based on this theory, my colleagues previously established specific data mining strategies to identify ectopically activated C/T genes, which are correlated with prognosis in ALL. Among the identified genes the expression of the single gene FASTKD1 predicts inferior outcome 711 . My PhD thesis project aimed to unravel the oncogenic role of this gene in ALL.

Figure 1 :

 1 Figure 1: FASTKD1 controls mitochondrial genome expression and mitochondrial activity.

Figure 2 :

 2 Figure 2: FASTKD1 gene inactivation, mitochondrial activation and -oxidation lead to an increase of H4K5K8 acylations.

Figure 3 :

 3 Figure 3: -oxidation and mitochondrial activity correlate with histone acylations in B-ALL tumour cells

  Protein extracts were prepared from COS-7 cells stably expressing the Ha-tagged bacterial deacetylase CobB or from control cells and used to visualize of CobB-ha and actin (upper panel), or H4 and its indicated modified forms using cell extracts (middle panel). 1X to 3X indicate increasing amounts for extracts loadings: one-fold to three-folds. The relative amounts of H4, H4K5ac, H4K5bu and H4K5cr were measured by ELISA in COS-7 cells or in cells expressing CobB-ha in biological triplicates and were repeated for at least three times and the values presented in the lower panel (see also Fig.S4). E-COS-7 cells described in "D", expressing CobB-ha or not, were transfected with a GFP-BRD4-NUT expression vector, and for each condition, 8 GFP-BRD4-NUT foci (one in each of 8 different cells) were bleached and the recovery of GFP fluorescence (FRAP) was monitored as a function of time. The 16 datasets corresponding to each FRAP experiment were individually fitted allowing for the determination of the half-life (t1/2) of fluorescence recovery and the mobile fractions (values indicated below the figure). The mean ± 2 SEM are shown for each time point. Scale bar represents 10μm.

Figure 5 :

 5 Figure 5: Characterization of H4K5cr-bu/H4K5ac ratio and BDR4 redistribution between wild-type and FASTKD1 ko cells

B

  and C-Anti-BRD4 ChIP-seqs were performed on the MNase digested chromatin of wildtype and FASTKD1 ko cell lines. The heatmap (B) and profiles (C) show BRD4 ChIP-seq normalized read counts in wild-type as well as in FASTKD1 ko REH cells over all gene TSS centred regions (TSSs +/-2000 bp). The TSS were ranked as a function of their transcriptional activity in wild-type REH cells and grouped into quartiles, Q1, Q2, Q3 and Q4, the first quartile (Q1: 75-100%) corresponding to the 25% genes with highest expression and the fourth quartile to the lowest 25% (Q4: 0-25%); (B) These gene groups are shown on the heatmap from top to bottom: highest (Q1: 75-100%) to lowest (Q4: 0-25%) gene expression levels; (C) The profiles corresponding to the different quartile groups are shown in different colours as indicated.

Figure 6 :

 6 Figure 6: Mitochondrial activity drives H4K5 acylation and directs BRD4dependent gene expression in REH cell line and in B-ALL patients blasts A-Volcano plot illustrating the differential gene expression signature between FASTKD1 ko (ko-1 and ko-2) and wild-type REH cells. Y axis: -log10 (p-value); x-axis: log (ratio of normalized expression values between ko and wild-type cells). The genes down-regulated or up-regulated with a fold change >1.5 and a Student t-test p-value < 0.05 are respectively represented in blue and red.

  Volcano plot illustrating the differential gene expression signature between B-ALL blasts with high H4K5 acylations (crotonylation and/or butyrylation, n = 15) and B-ALL blasts with low acylations (n = 10). Y axis: -log10 (p-value); x-axis: log (ratio of normalized expression values between B-ALL with high and low acylation levels). The genes down-regulated or upregulated with an absolute fold change >1.5 and a Student t-test p-value < 0.05 are respectively represented in blue and red.

Figure 7 :

 7 Figure 7: Control of BRD4 reservoir by mitochondrial activity and cell metabolism: a working model Enhanced mitochondrial activity leads to a global increase of histone H4K5

  Script for bamCoverage normalizing with RPKM: bamCoverage -b myfile_notrim.srt.bam --extendReads --binSize 4 --minMappingQuality 30 --normalizeUsing RPKM -o myfile_notrim.bw Script for bamCoverage with scaling factors: bamCoverage -b my_file.bam --extendReads --numberOfProcessors 4 --binSize 4 --minMappingQuality 30 --normalizeUsing None --scaleFactor my_value -o my_file.bw

FRAP

  CobB (t 1/2 : 6.26+/-0.33; mobile fraction : 0.84+/-0.05 BRD4-NUT (t 1/2 : 8.94+/-0.73; mobile fraction : 0.76+/-0.02 Relative quantitative analysis of site-specific histone H3 and H4 propionylation and butyrylation in wild-type and two FASTKD1 ko clones (related to Figure2A). All the identified peptides with Mascot score above 20 were manually verified. The relative ratios of abundance of histone peptides in wild type and FASTKD1

Table 1 .

 1 Mammalian HATs members and their histone acetylation targets. ............................

Table 2 .

 2 Common HMTs and their regulatory contexts in cancer. ...........................................

Table 3 .

 3 Common KDMs and their implications in cancer. .....................................................

Table 4 .

 4 Common HATs and their implications in cancer. .......................................................

Table 5 .

 5 Histone deacetylases regulate various hallmarks of cancer. .......................................

Table 6 .

 6 Epigenetic drugs . .......................................................................................................

Table 7 .

 7 Key resources table..................................................... Error! Bookmark not defined.

Table 1 . Mammalian HATs members and their histone acetylation targets. Members Acronyms In vitro substrates In vivo complexes In vivo substrates Functions

 1

	GNATs family
	HAT1	KAT1	H4K5, H4K12	HAT-B complex		Newly synthesized histone H4	Histone deposition and chromatin assembly 49, 50
	KAT2A	GCN5	H3K14, H4K8,	2MDa SAGA-like complex	Core histones, with preference	Global acetylation across the genome and targeted histone
	KAT2B	PCAF	H4K16	(STAGA / TFTC / PCAF),	for H3 (at K9)		acetylation at specific loci 51 .
				700kDa (ATAC)
	MYST family
	KAT5	TIP60	H2A, H3 and H4 NuA4 complex		Catalyze nucleosomal H2A, H4,	Chromatin structure alterations, DNA damage response, cell
							and non-histone substrates,	cycle progression and transcriptional regulation, act as
							preference for H2A (at K5) 52 .	coactivators of corresponding factors (e.g., acetylating ATM and
	KAT8	MOF,	/	MSL	complex,	NSL	MSL	complex	specifically	p53) 53, 54 .
		MYST1		complex			acetylates nucleosomal H4 at
							K16.
							NSL complex broadly acetylates
							K5, K8 and K16 on nucleosomal
							H4
	KAT6A	MYST3,	H3 and H4	MOZ/MORF complexes	Histone H3 at K9, K14 and K23	Acts more on locus-specific regions, might impact global
		MOZ		(contain ING5, MEAF6 and				H3K23ac
				BRPF1/2/3 subunits)

Table 2 . Common HMTs and their regulatory contexts in cancer.

 2

	Name	Histone targets	Types of aberrations	Cancer types	Roles in cancer
	EZH2	H3K27me	gain-of-function	mutation	Melanoma,	lymphoid	Generally associated with accelerated transformation and
			(Y641 mutations 211-213 , A677	malignancies 212, 215	progression, can be tumor-suppressive in certain types of
			mutaion 214 ), loss-of-function		cancer 216, 217 .
			mutation,	amplification,
			dysregulation 207-209
	MLL/KMT2 family	H3K4me	Translocates with more than	Acute leukemias	Oncogenic in hematological malignancies through activating
			70 partner genes (e.g., AF4,		transcriptional factors that control hematopoietic stem cell
			AF9, AF10, ENL family and p-		program, including Hoxa9 and Meis1 218-220 .
			TEFb)
			Nonsense	or	frameshift	Colorectal	cancer,	KMT2A/B cooperate with genuine oncogenes in
			mutations 221			glioblastoma,	melanoma,	transcription regulation 222, 223 .
							pancreatic	carcinoma,
							hepatocellular carcinoma
	NSD family	H3K36me	NSD1 mutation, NUP98-NSD1	Human papillomavirus (HPV)-	Loss-of-function NSD1 mutations confer to decreased risk of
			fusion				negative head and neck	death in HNSCC 224 . NSD1 expression is associated with
							squamous cell carcinoma	cancer metastasis in various solid tumors 225-227 .
							(HNSCC), laryngeal, prostate,	NUP98-NSD1 binds gene elements adjacent to HoxA7/9 228
							pancreatic ductal tumors,	and predicts poor outcome in AML 229-231 .
							acute myeloid leukemia
			NSD2	mutation	(E1099K,	Acute lymphoblastic leukemia	Contribute to abnormal gene expression programs
			activating mutation)		(ALL), mantle cell lymphoma,	associated with oncogenesis and cancer aggressiveness 233,
							chronic	lymphocytic	234 .
							leukemia 232	Correlated with clonal evoluation and drug resistance in
								ALL 235, 236 .

KMT2C/D/F might be either oncogenic or tumor suppressive in solid tumors

221

.

Associated with DNA damage responses (e.g., DNA mismatch repair, double strand break repair etc.)

[248][249][250]

, RNA splicing machineries

[251][252][253]

. Required for cancer proliferation, DNA damage response respectively

[254][255][256]

.

In general, SETD2 has tumor-suppressive potentials across various cancer entities, whose inactivating mutations are associated with chemotherapy resistance 257 . leukemia Promote cancer survival and tumorigenesis via repressing specific genes (e.g., hypoxic response associated genes), or activating oncogenes (e.g., mTOR), or activating metabolic pathways (e.g., serine-glycine biosynthetic pathway, iron metabolism) in various cancer cells

[266][267][268][269]

. Cooperates with transcriptional factor YY1 to prevent tumor progression in leukemic cells 270 . G9a depletion was shown to exhibit distinct roles as repressive in short-term tumor initiation but confers to more aggressive behaviors in long-term progression in skin tumors 271 .

Table 5 . Histone deacetylases regulate various hallmarks of cancer.

 5

	Hallmark		HDACs members	Signaling networks
	capabilities
	Proliferation and	HDAC1 and HDAC2	Inhibition of HDAC1 and HDAC2 leads to increased p21, p57 and p27, causing cell cycle arrest at G1 phase 383, 384 .
	cell	cycle	HDAC3, 4, 5	Inhibits p21 385-387 . HDAC4 was also reported to act as corepressor on cyclin D1 promoter 388 . HDAC3 can induce G2/M arrest via
	progression			impaired Aurora B activity 389
			HDAC6	Deacetylate α-tubulin and impairing BCL3 translocation and cyclin D1 expression 77 .
			HDAC10	cyclin A2 expression and regulate G2/M progression 390 .

SIRT1 and SIRT2

Inhibition of SIRT1 and SIRT2 induces decreased expression of cyclin D1/E, CDK2/4/6 and leads to G0/G1 arrest in colorectal cancer cells 391 . SIRT2deacetylates tubulin and regulates heterochromatin structure

[392][393][394]

  120,[464][465][466] . Indeed, acetylation readers, predominantly including BET family members, have been extensively studied and exploited as therapeutic targets in various cancers.

	a global hypoacetylation and silencing of differentiation program in BRD4-NUT midline
	carcinoma 210, 470, 471 .
	Most studies on BRD4 in cancers, prominently in hematological malignancies are focusing
	on its role in regulating of MYC in various cancers. Targeting BRD4 via bromodomain
	inhibitors or protein degradation, selectively down-regulates MYC and MYC-dependent target
	genes, prompting cell-cycle arrest and repressing cell proliferation in multiple myeloma,
	Burkitt's lymphoma, acute myeloid leukemia, colorectal cancer, gastric cancer, bladder cancer
	etc. 472-476 . Interestingly, BRD4 was shown to directly phosphorylate MYC at Thr58, leading to
	MYC protein ubiquitination and degradation 477 . In addition to MYC, BRD4 also regulates other
	key oncogenic pathways. For example, BET inhibition disrupts the key oncogenic driver,
	Forkhead box protein M1 (FoxM1) and its down-stream pathway and leads to antitumor effect
	in ovarian cancer 478 . In prostate cancer, BRD4 could promote the expression of HOXB13, which
	is a lineage-specific transcription factor and maintains pro-proliferative network in castration
	resistance cells 479 . Besides, genomic profiling identified that BRD4 binds to cell type-specific
	enhancer regions and can activate STAT5 pathway in AML cells 480 .
	However, these properties of BRD4 were not reported by any other group and should be taken
	into account with caution. Functionally, BRD4 is involved in cell identity determination via
	accumulating on hyper-acetylated regions (both promoters and enhancers) as well as by
	regulating lineage specific transcription factors during embryonic development and cell
	differentiation. While in transformed cells, oncogenic role of BRD4 is represented by its impact
	on 1) the expression of key oncogenic factors; 2) the expression of cell identity program.
	Oncogenic role of BRD4 was firstly investigated in NUT midline cancer. BRD4 gene could
	be translocated to NUT gene, generating a driver oncoprotein BRD4-NUT that blocks

BET BET family proteins comprise of BRDT, BRD2, BRD3 and BRD4. Of them BRDT is exclusively present in the male geminal tissue, while the other three members are ubiquitously expressed in all tissues. BET family members share two tandem BrDs and C-terminal extraterminal (ET) domain. BrDs of BET specifically bind acetylated histones, while the C terminal extended domain is involved in recruiting chromatin regulator factors. BRD4 was firstly characterized as a scaffold for transcription machineries, predominantly in association with elongation factors P-TEFb. It therefore promotes RNA Pol II -dependent transcription. Furthermore, BRD4 might have an intrinsic kinase activity, which can directly phosphorylates CTD Ser2 of RNA Pol II 467 . Besides, BRD4 is found as a cofactor of Mediator complexes, which integrates information from transcription factors and coactivators at enhancer regions to promoters and RNA polymerases, therefore regulates transcription initiation 468 . BRD4 was also shown to possess intrinsic acetyltransferase activity. It can acetylate H3K122 and contributes to nucleosome eviction and chromatin decompaction, therefore promoting transcription 469 .

differentiation. Mechanistically, BRD4-NUT forms discrete nuclear foci that are enriched with p300 and acetylated histones dependent the BrDs of BRD4, which binds acetylated histones, and AD1 domain of NUT, which binds and activates p300. The BRD4-NUT chromatin foci sequester p300, which becomes depleted from the differentiation-associated genes, leading to

Table 6 .

 6 Epigenetic drugs .

	Compound	Target	Cancer type	Phase
	Azacitidine	DNMT	MDS	EMA and FDA
	Decitabine	DNMT	AML, MDS	EMA and FDA
	Guadecitabine	DNMT	AML	Phase III
	Belinostat	HDAC class I and II	Peripheral T cell lymphoma	FDA
	Panobinostate	HDAC class I, II and	Multiple myeloma	FDA
		IV
	Romidepsin	HDAC class I	Cutaneous T cell lymphoma	FDA

Abbrevations: AML: acute myeloid leukemia; MDS: myeodysplasic syndrome; MM:multiple myeloma; NHL: non-Hodgkin lymphoma; R/R relapsed/refractory; DLBCL: difuse large B cell lymphoma; MCL: mantle cell lymphoma; FL: follicular lymphoma; tFL: transformed FL; NSCLC: non small cell lung cancer; NMC: NUT-midline carcinoma; CRPC: castration-resistant prostate cancer; IDH: isocitrate dehydrogenase; PMBL: primary mediastinal large B-cell lymphoma.

manuscript submitted 1.1 Manuscript text Metabolically controlled histone H4K5 acylation/acetylation ratio drives BRD4 genomic distribution

  These are in accord with less aggressive tumors, and a relatively favorable outcome in this group of patients.Mengqing Gao 1, 2, 3 + , Jin Wang 1, 3 + , Sophie Rousseaux 2, 3 , Minjia Tan 4 , Lulu Pan 4 , Lijun Peng 1, 3 , Sisi Wang1, 3 , Wenqian Xu1, 3 , Jiayi Ren 1, 3 , Yuanfang Liu 1 , Martin Spinck 5 , SophieBarral 2, 3 , TaoWang 2, 3 , Florent Chuffart 2, 3 , Ekaterina Bourova-Flin 2, 3 , Denis Puthier 6 , Sandrine Curtet 2, 3 , Lisa Bargier 6 , Zhongyi Cheng 7 , Heinz Neumann 5 , Jian Li 8 , Yingming Zhao 9 , Jian-QingMi 1,

	ANNEXES
	1. A
	Collectively, our work highlights the importance of these relatively low abundant histone
	marks in regulating gene transcription. More specifically, these acylations preferentially
	regulate highly transcribed genes where in combination with acetylation, they tune BRD4
	availability across various genomic regions. Therefore, our work also proposes a new
	hypothesis on the functional importance of histone PTM combination, in addition to their
	individual role.

  The ChIP-seq data and RNA-seq data generated during this study haven been deposited at GEO and are publicly available as of the date of publication. Accession numbers are listed in the key resources table. Original western blot images have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the key resources table. Microscopy data reported in this paper will be shared by the lead contact upon request.

•

ACKNOWLEDGEMENTS

This program was supported by Fondation ARC program N° RF20190208471, by ANR Episperm4 program and by the "Université Grenoble Alpes" ANR-15-IDEX-02 LIFE and SYMER programs, by INCa and IreSP, by Plan Cancer Pitcher and from MSD Avenir ERICAN programs. JM's lab is supported by National Natural Science Foundation of China (81670147), clinical research plan of Shanghai Hospital Development Center (16CR3008A), International Cooperation Projects of Shanghai Science and Technology Committee (15410710200) and Innovation Program of Shanghai Municipal Education Commission (8201001096). MG follows a Shanghai Jiao Tong University -Grenoble Alpes University joint PhD program of Cai Yuanpei -Campus de France supported by China Scholarship Council. This work was supported by the University of Chicago, Nancy and Leonard Florsheim family fund (YZ), NIH grants GM135504, DK118266 (YZ). MT's group acknowledges funding from "the Natural Science Foundation of China (91753203)". The TGML Platform is supported by grants from Inserm, GIS IBiSA, Aix-Marseille Université, and ANR-10-INBS-0009-10. NH and SM's work was funded by the Max-Planck Institute of Molecular Physiology.

AUTHORS' CONTRIBUTIONS

MG generated model cell lines and performed most of the experiments. MT performed H3 and H4 PTMs measurements. LB prepared all the libraries and performed the sequencing under the supervision of DP. SR performed and supervised all the bio-informatic analyses involving also FC and EFB. MS and HN produced the CobB expression vector and discussed the results. SC performed the FRAP experiments and analysed the data with the help of FC. SB performed anti-H4K5ac-cr ChIP experiments and TW, SW, WX helped with the RT-qPCR experiments.

ZC produced and characterized the anti-histone PTM antibodies. JL helped with and discussed about the statistical analysis. JW recruited the B-ALL patients and took care of bio-bank setup and the formalities including the acquisition of the ethic committee approval. LP, JR, YL helped with the collection and disposition of patients' samples. JQM supervised the clinical part of the experiments and co-supervised MG work. YZ supervised the work on histone PTM and provided access to antibodies and helped with data interpretation. SK designed and coordinated the whole project and wrote the manuscript. All the authors read and commented the manuscript.

DECLARATION OF INTERESTS

All the authors declare no conflict of interest except ZC and YZ. Y.Z. is a founder, board member, advisor to, and inventor on patents licensed to PTM Biolabs Inc (Hangzhou, China and Chicago, IL) and Maponos Therapeutics Inc. (Chicago, IL). Zhongyi Cheng is an employee and equity holder of PTM BioLabs Inc.

Supplementary data

Supplementary Figure S1: Generation of stable FASTKD1 knock-down and knock out REH cell lines (related to Figure 1A).

A-Viral expression vectors expressing two independent anti-FASTKD1 shRNAs or an empty vector were established and used to generate RNA-seq data (Fig. 1A). The raw reads corresponding to the FASTKD1 gene were aligned on FASTKD1 gene exons and shown on the same diagram.

B-

The CRISPR/Cas9 system was used to knock-out FASTKD1 gene in REH cells using two different guide RNAs.

The genomic DNAs of a pool of cells after CRISPR/Cas9 genome modification were sequenced around the targeted regions (indicated as multi-clones). Limit dilution of the two resulting multi-clone cells allowed for the isolation of single cell clones called ko-1 and ko-2. The sequence of the two isolated FASTKD1 clones and the corresponding sequence alterations are shown.

C-The level of

B.

The gene group defined in A was used as a geneset for a GSEA plot to test for its enrichment/depletion in JQ1 treated REH cells. This plot shows a significant depletion of this group of genes, visualizing their down regulation in JQ1 treated cells, which demonstrates that their expression is BRD4 dependent. Table S3: Relative quantitative analysis of site-specific histone H3 and H4 propionylation and butyrylation in wild-type and two FASTKD1 ko clones (related to Figure 2A).

Supplementary

Normalization information (upper Table ): values of the precursor ion AUC used to normalize the amount of each histone in label-free quantification.

Coverage information (lower Table ): values of histone coverage for each experiment.