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In mammals, histone 3 lysine 4 methylation (H3K4me) is mediated by six different lysine methyltransferases. Among these enzymes, SETD1B (SET domain containing 1b) has been linked to syndromic intellectual disability in human subjects, but its role in the mammalian postnatal brain has not been studied yet. Here, we employ mice deficient for Setd1b in excitatory neurons of the postnatal forebrain, and combine neuron‐specific ChIP‐seq and RNA‐seq approaches to elucidate its role in neuronal gene expression. We observe that Setd1b controls the expression of a set of genes with a broad H3K4me3 peak at their promoters, enriched for neuron‐specific genes linked to learning and memory function. Comparative analyses in mice with conditional deletion of Kmt2a and Kmt2b histone methyltransferases show that SETD1B plays a more pronounced and potent role in regulating such genes. Moreover, postnatal loss of Setd1b leads to severe learning impairment, suggesting that SETD1B‐dependent regulation of H3K4me levels in postnatal neurons is critical for cognitive function., Comparative analyses of conditional deletion of mammalian H3K4 methyltransferases show a selective role for Setd1b in expression of neuron‐specific genes important for cognitive function.

The generation of neocortical neurons from neural progenitor cells (NPCs) is primarily controlled by transcription factors binding to DNA in the context of chromatin. To understand the complex layer of regulation that orchestrates different NPC types from the same DNA sequence, epigenome maps with cell type resolution are required. Here, we present genomewide histone methylation maps for distinct neural cell populations in the developing mouse neocortex. Using different chromatin features, we identify potential novel regulators of cortical NPCs. Moreover, we identify extensive H3K27me3 changes between NPC subtypes coinciding with major developmental and cell biological transitions. Interestingly, we detect dynamic H3K27me3 changes on promoters of several crucial transcription factors, including the basal progenitor regulator Eomes. We use catalytically inactive Cas9 fused with the histone methyltransferase Ezh2 to edit H3K27me3 at the Eomes locus in vivo, which results in reduced Tbr2 expression and lower basal progenitor abundance, underscoring the relevance of dynamic H3K27me3 changes during neocortex development. Taken together, we provide a rich resource of neocortical histone methylation data and outline an approach to investigate its contribution to the regulation of selected genes during neocortical development.

Heterochromatin is marked by methylation of lysine 9 on histone H3 (H3K9me). A puzzling feature of H3K9me is that this modification localizes not only in promoters but also in internal regions (bodies) of silent transcription units. Despite its prevalence, the biological significance of gene‐body H3K9me remains enigmatic. Here we show that H3K9me‐associated removal of H3K4 monomethylation (H3K4me1) in gene bodies mediates transcriptional silencing. Mutations in an Arabidopsis H3K9 demethylase gene IBM1 induce ectopic H3K9me2 accumulation in gene bodies, with accompanying severe developmental defects. Through suppressor screening of the ibm1 ‐induced developmental defects, we identified the LDL2 gene, which encodes a homolog of conserved H3K4 demethylases. The ldl2 mutation suppressed the developmental defects, without suppressing the ibm1 ‐induced ectopic H3K9me2. The ectopic H3K9me2 mark directed removal of gene‐body H3K4me1 and caused transcriptional repression in an LDL2‐dependent manner. Furthermore, mutations of H3K9 methylases increased the level of H3K4me1 in the gene bodies of various transposable elements, and this H3K4me1 increase is a prerequisite for their transcriptional derepression. Our results uncover an unexpected role of gene‐body H3K9me2/H3K4me1 dynamics as a mediator of heterochromatin silencing and epigenome differentiation. ![][1] A genetic interaction between histone demethylases IBM1 and LDL2 reveals that H3K9 methylation in gene bodies induces transcriptional silencing by triggering the loss of H3K4 monomethylation. [1]: /embed/graphic-1.gif

In light of the increasing number of identified cancer‐driven gain‐of‐function (GOF) mutants of p53, it is important to define a common mechanism to systematically target several mutants, rather than developing strategies tailored to inhibit each mutant individually. Here, using RNA immunoprecipitation‐sequencing (RIP‐seq), we identified the Polycomb‐group histone methyltransferase EZH2 as a p53 mRNA‐binding protein. EZH2 bound to an internal ribosome entry site (IRES) in the 5′UTR of p53 mRNA and enhanced p53 protein translation in a methyltransferase‐independent manner. EZH2 augmented p53 GOF mutant‐mediated cancer growth and metastasis by increasing protein levels of mutant p53. EZH2 overexpression was associated with worsened outcome selectively in patients with p53‐mutated cancer. Depletion of EZH2 by antisense oligonucleotides inhibited p53 GOF mutant‐mediated cancer growth. Our findings reveal a non‐methyltransferase function of EZH2 that controls protein translation of p53 GOF mutants, inhibition of which causes synthetic lethality in cancer cells expressing p53 GOF mutants.

Post‐translational modification of proteins by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) is catalyzed by O‐GlcNAc transferases (OGTs). O‐GlcNAc modification of proteins regulates multiple important biological processes in metazoans. However, whether protein O‐GlcNAcylation is involved in epigenetic processes during plant development is largely unknown. Here, we show that loss of function of SECRET AGENT (SEC), an OGT in Arabidopsis, leads to an early flowering phenotype. This results from reduced histone H3 lysine 4 trimethylation (H3K4me3) of FLOWERING LOCUS C (FLC) locus, which encodes a key negative regulator of flowering. SEC activates ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1), a histone lysine methyltransferase (HKMT), through O‐GlcNAc modification to augment ATX1‐mediated H3K4me3 histone modification at FLC locus. SEC transfers an O‐GlcNAc group on Ser947 of ATX1, which resides in the SET domain, thereby activating ATX1. Taken together, these results uncover a novel post‐translational O‐GlcNAc modification‐mediated mechanism for regulation of HKMT activity and establish the function of O‐GlcNAc signaling in epigenetic processes in plants.

Host cell defense against an invading pathogen depends upon various multifactorial mechanisms, several of which remain undiscovered. Here, we report a novel defense mechanism against mycobacterial infection that utilizes the histone methyltransferase, SUV39H1. Normally, a part of the host chromatin, SUV39H1, was also found to be associated with the mycobacterial bacilli during infection. Its binding to bacilli was accompanied by trimethylation of the mycobacterial histone‐like protein, HupB, which in turn reduced the cell adhesion capability of the bacilli. Importantly, SUV39H1‐mediated methylation of HupB reduced the mycobacterial survival inside the host cell. This was also true in mice infection experiments. In addition, the ability of mycobacteria to form biofilms, a survival strategy of the bacteria dependent upon cell–cell adhesion, was dramatically reduced in the presence of SUV39H1. Thus, this novel defense mechanism against mycobacteria represents a surrogate function of the epigenetic modulator, SUV39H1, and operates by interfering with their cell–cell adhesion ability.

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.

Histone chaperones affect chromatin structure and gene expression through interaction with histones and RNA polymerase II (PolII). Here, we report that the histone chaperone Spt6 counteracts H3K27me3, an epigenetic mark deposited by the Polycomb Repressive Complex 2 (PRC2) and associated with transcriptional repression. By regulating proper engagement and function of the H3K27 demethylase KDM6A (UTX), Spt6 effectively promotes H3K27 demethylation, muscle gene expression, and cell differentiation. ChIP-Seq experiments reveal an extensive genome-wide overlap of Spt6, PolII, and KDM6A at transcribed regions that are devoid of H3K27me3. Mammalian cells and zebrafish embryos with reduced Spt6 display increased H3K27me3 and diminished expression of the master regulator MyoD, resulting in myogenic differentiation defects. As a confirmation for an antagonistic relationship between Spt6 and H3K27me3, inhibition of PRC2 permits MyoD re-expression in myogenic cells with reduced Spt6. Our data indicate that, through cooperation with PolII and KDM6A, Spt6 orchestrates removal of H3K27me3, thus controlling developmental gene expression and cell differentiation.

PPARγ promotes adipogenesis while Wnt proteins inhibit adipogenesis. However, the mechanisms that control expression of these positive and negative master regulators of adipogenesis remain incompletely understood. By genome-wide histone methylation profiling in preadipocytes, we find that among gene loci encoding adipogenesis regulators, histone methyltransferase (HMT) G9a-mediated repressive epigenetic mark H3K9me2 is selectively enriched on the entire PPARγ locus. H3K9me2 and G9a levels decrease during adipogenesis, which correlates inversely with induction of PPARγ. Removal of H3K9me2 by G9a deletion enhances chromatin opening and binding of the early adipogenic transcription factor C/EBPβ to PPARγ promoter, which promotes PPARγ expression. Interestingly, G9a represses PPARγ expression in an HMT activity-dependent manner but facilitates Wnt10a expression independent of its enzymatic activity. Consistently, deletion of G9a or inhibiting G9a HMT activity promotes adipogenesis. Finally, deletion of G9a in mouse adipose tissues increases adipogenic gene expression and tissue weight. Thus, by inhibiting PPARγ expression and facilitating Wnt10a expression, G9a represses adipogenesis.

The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.

Endogenous small interfering RNAs (endo-siRNAs) have been discovered in many organisms, including mammals. In C. elegans, depletion of germline-enriched endo-siRNAs found in complex with the CSR-1 Argonaute protein causes sterility and defects in chromosome segregation in early embryos. We discovered that knockdown of either csr-1, the RNA-dependent RNA polymerase (RdRP) ego-1, or the dicer-related helicase drh-3, leads to defects in histone mRNA processing, resulting in severe depletion of core histone proteins. The maturation of replication-dependent histone mRNAs, unlike that of other mRNAs, requires processing of their 3′UTRs through an endonucleolytic cleavage guided by the U7 snRNA, which is lacking in C. elegans. We found that CSR-1-bound antisense endo-siRNAs match histone mRNAs and mRNA precursors. Consistently, we demonstrate that CSR-1 directly binds to histone mRNA in an ego-1-dependent manner using biotinylated 2′-O-methyl RNA oligonucleotides. Moreover, we demonstrate that increasing the dosage of histone genes rescues the lethality associated with depletion of CSR-1 and EGO-1. These results support a positive and direct effect of RNAi on histone gene expression.

Setd8/PR-Set7/KMT5a-dependent mono-methylation of histone H4 at lysine 20 is essential for mitosis of cultured cells; yet, the functional roles of Setd8 in complex mammalian tissues are unknown. We use skin as a model system to explore how Setd8 may regulate cell division in vivo. Deletion of Setd8 in undifferentiated layers of the mouse epidermis impaired both proliferation and differentiation processes. Long-lived epidermal progenitor cells are lost in the absence of Setd8, leading to an irreversible loss of sebaceous glands and interfollicular epidermis. We show that Setd8 is a transcriptional target of c-Myc and an essential mediator of Myc-induced epidermal differentiation. Deletion of Setd8 in c-Myc-overexpressing skin blocks proliferation and differentiation and causes apoptosis. Increased apoptosis may be explained by our discovery that p63, an essential transcription factor for epidermal commitment is lost, while p53 is gained upon removal of Setd8. Both overexpression of p63 and deletion of p53 rescue Setd8-induced apoptosis. Thus, Setd8 is a crucial inhibitor of apoptosis in skin and its activity is essential for epidermal stem cell survival, proliferation and differentiation.

Genomic imprinting in mammals is controlled by DNA methylation imprints that are acquired in the gametes, at essential sequence elements called 'imprinting control regions' (ICRs). What signals paternal imprint acquisition in male germ cells remains unknown. To address this question, we explored histone methylation at ICRs in mouse primordial germ cells (PGCs). By 13.5 days post coitum (d.p.c.), H3 lysine-9 and H4 lysine-20 trimethylation are depleted from ICRs in male (and female) PGCs, indicating that these modifications do not signal subsequent imprint acquisition, which initiates at ∼15.5 d.p.c. Furthermore, during male PGC development, H3 lysine-4 trimethylation becomes biallelically enriched at 'maternal' ICRs, which are protected against DNA methylation, and whose promoters are active in the male germ cells. Remarkably, high transcriptional read-through is detected at the paternal ICRs H19-DMR and Ig-DMR at the time of imprint establishment, from one of the strands predominantly. Combined, our data evoke a model in which differential histone modification states linked to transcriptional events may signal the specificity of imprint acquisition during spermatogenesis.

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.

Transcription factors and epigenetic modulators are involved in the maintenance of self-renewal in embryonic stem (ES) cells. Here, we demonstrate the existence of a regulatory loop in ES cells between Sox2, an indispensable transcription factor for self-renewal, and embryonic ectoderm development (Eed), an epigenetic modulator regulating histone methylation. We found that Sox2 and Eed positively regulate each other's expression. Interestingly, Sox2 overexpression suppressed the induction of differentiation-associated genes in Eed-deficient ES cells without restoring histone methylation. This Sox2-mediated suppression was prevented by knockdown of the histone acetyltransferase (HAT), Tip60 or Elp3, and Sox2 stimulated expression of these HATs. Furthermore, forced expression of either HAT resulted in repression of differentiation-associated genes in Eed-deficient cells. These results suggest that Sox2 overcame the phenotype of Eed-deficient ES cells by promoting histone acetylation. We also found that knockout of Eed and knockdown of these HATs synergistically enhanced the upregulation of differentiation-associated genes in ES cells. Taken together, our results suggest that the Eed/Sox2 regulatory loop contributes to the maintenance of self-renewal in ES cells by controlling histone methylation and acetylation.

Post-translational modifications of the N-terminal histone tails, including lysine methylation, have key roles in regulation of chromatin and gene expression. A number of protein modules have been identified that recognize differentially modified histone tails and provide their proteins with the capacity to sense such modifications. Here, we identify the CW domain of plant and animal chromatin-related proteins as a novel module that recognizes different methylated states of lysine 4 on histone H3 (H3K4me). The solution structure of the CW domain of the Arabidopsis ASH1 HOMOLOG2 (ASHH2) histone methyltransferase provides insight into how different CW domains can distinguish different methylated histone tails. We provide evidence that ASHH2 is acting on H3K4me-marked genes, allowing for ASHH2-dependent H3K36 tri-methylation, which contributes to sustained expression of tissue-specific and developmentally regulated genes. This suggests that ASHH2 is a combined 'reader' and 'writer' of the histone code. We propose that different CW domains, dependent on their specificity for different H3K4 methylations, are important for epigenetic memory or participate in switching between permissive and repressive chromatin states.

Lsm1 forms part of a cytoplasmic protein complex, Lsm1-7-Pat1, involved in the degradation of mRNAs. Here, we show that Lsm1 has an important role in promoting genomic stability in Saccharomyces cerevisiae. Budding yeast cells lacking Lsm1 are defective in recovery from replication-fork stalling and show DNA damage sensitivity. Here, we identify histone mRNAs as substrates of the Lsm1-7-Pat1 complex in yeast, and show that abnormally high amounts of histones accumulate in lsm1Δ mutant cells. Importantly, we show that the excess of histones is responsible for the lsm1Δ replication-fork instability phenotype, since sensitivity of lsm1Δ cells to drugs that stall replication forks is significantly suppressed by a reduction in histone gene dosage. Our results demonstrate that improper histone stoichiometry leads to genomic instability and highlight the importance of regulating histone mRNA decay in the tight control of histone levels in yeast.

In Arabidopsis, the rapid-flowering summer-annual versus the vernalization-requiring winter-annual growth habit is determined by natural variation in FRIGIDA (FRI) and FLOWERING LOCUS C (FLC). However, the biochemical basis of how FRI confers a winter-annual habit remains elusive. Here, we show that FRI elevates FLC expression by enhancement of histone methyltransferase (HMT) activity. EARLY FLOWERING IN SHORT DAYS (EFS), which is essential for FRI function, is demonstrated to be a novel dual substrate (histone H3 lysine 4 (H3K4) and H3K36)-specific HMT. FRI is recruited into FLC chromatin through EFS and in turn enhances EFS activity and engages additional HMTs. At FLC, the HMT activity of EFS is balanced by the H3K4/H3K36- and H3K4-specific histone demethylase (HDM) activities of autonomous-pathway components, RELATIVE OF EARLY FLOWERING 6 and FLOWERING LOCUS D, respectively. Loss of HDM activity in summer annuals results in dominant HMT activity, leading to conversion to a winter-annual habit in the absence of FRI. Thus, our study provides a model of how growth habit is determined through the balance of the H3K4/H3K36-specific HMT and HDM activities.

RNA-directed modification of histones is essential for the maintenance of heterochromatin in higher eukaryotes. In plants, cytosine methylation is an additional factor regulating inactive chromatin, but the mechanisms regulating the coexistence of cytosine methylation and repressive histone modification remain obscure. In this study, we analysed the mechanism of gene silencing mediated by MORPHEUS' MOLECULE1 (MOM1) of Arabidopsis thaliana. Transcript profiling revealed that the majority of up-regulated loci in mom1 carry sequences related to transposons and homologous to the 24-nt siRNAs accumulated in wild-type plants that are the hallmarks of RNA-directed DNA methylation (RdDM). Analysis of a single-copy gene, SUPPRESSOR OF drm1 drm2 cmt3 (SDC), revealed that mom1 activates SDC with concomitant reduction of di-methylated histone H3 lysine 9 (H3K9me2) at the tandem repeats in the promoter region without changes in siRNA accumulation and cytosine methylation. The reduction of H3K9me2 is not observed in regions flanking the tandem repeats. The results suggest that MOM1 transduces RdDM signals to repressive histone modification in the core region of RdDM.

In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Although selective H3K9me is critical for maintaining genome integrity, mechanisms to exclude H3K9me from active genes remain largely unexplored. Here, we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation). Loss-of-function ibm1 mutation results in ectopic H3K9me and non-CG methylation in thousands of genes. The ibm1-induced genic H3K9me depends on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks--H3K9me and non-CG methylation. Notably, IBM1 enhances loss of H3K9me in transcriptionally de-repressed sequences. Furthermore, disruption of transcription in genes induces ectopic non-CG methylation, which mimics the loss of IBM1 function. We propose that active chromatin is stabilized by an autocatalytic loop of transcription and H3K9 demethylation. This process counteracts a similarly autocatalytic accumulation of silent epigenetic marks, H3K9me and non-CG methylation.

Linker histones are involved in the formation of higher-order chromatin structure and the regulation of specific genes, yet it remains unclear what their principal binding determinants are. We generated a genome-wide high-resolution binding map for linker histone H1 in Drosophila cells, using DamID. H1 binds at similar levels across much of the genome, both in classic euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites for active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes; rather, all regions with low binding of H1 show enrichment of the histone variant H3.3. Knockdown of H3.3 causes H1 levels to increase at these sites, with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1, providing a mechanism to keep diverse genomic sites in an open chromatin conformation.

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti-oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1-to-S phase transition, E2F1 associates through a short DHQY sequence with the cell-cycle regulator HCF-1 together with the mixed-lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF-1-binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF-1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF-1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF-1-binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF-1 association with E2F1 is a regulator of E2F1-induced apoptosis.

Recognition and repair of damaged DNA occurs within the context of chromatin. The key protein components of chromatin are histones, whose post-translational modifications control diverse chromatin functions. Here, we report our findings from a large-scale screen for DNA-damage-responsive histone modifications in human cells. We have identified specific phosphorylations and acetylations on histone H3 that decrease in response to DNA damage. Significantly, we find that DNA-damage-induced changes in H3S10p, H3S28p and H3.3S31p are a consequence of cell-cycle re-positioning rather than DNA damage per se. In contrast, H3K9Ac and H3K56Ac, a mark previously uncharacterized in human cells, are rapidly and reversibly reduced in response to DNA damage. Finally, we show that the histone acetyl-transferase GCN5/KAT2A acetylates H3K56 in vitro and in vivo. Collectively, our data indicate that though most histone modifications do not change appreciably after genotoxic stress, H3K9Ac and H3K56Ac are reduced in response to DNA damage in human cells.

Histone H3K9 methylation is required for DNA methylation and silencing of repetitive elements in plants and filamentous fungi. In mammalian cells however, deletion of the H3K9 histone methyltransferases (HMTases) Suv39h1 and Suv39h2 does not affect DNA methylation of the endogenous retrovirus murine leukaemia virus, indicating that H3K9 methylation is dispensable for DNA methylation of retrotransposons, or that a different HMTase is involved. We demonstrate that embryonic stem (ES) cells lacking the H3K9 HMTase G9a show a significant reduction in DNA methylation of retrotransposons, major satellite repeats and densely methylated CpG-rich promoters. Surprisingly, demethylated retrotransposons remain transcriptionally silent in G9a(-/-) cells, and show only a modest decrease in H3K9me2 and no decrease in H3K9me3 or HP1alpha binding, indicating that H3K9 methylation per se is not the relevant trigger for DNA methylation. Indeed, introduction of catalytically inactive G9a transgenes partially 'rescues' the DNA methylation defect observed in G9a(-/-) cells. Taken together, these observations reveal that H3K9me3 and HP1alpha recruitment to retrotransposons occurs independent of DNA methylation in ES cells and that G9a promotes DNA methylation independent of its HMTase activity.

Understanding the function of histone modifications across inducible genes in mammalian cells requires quantitative, comparative analysis of their fate during gene activation and identification of enzymes responsible. We produced high-resolution comparative maps of the distribution and dynamics of H3K4me3, H3K36me3, H3K79me2 and H3K9ac across c-fos and c-jun upon gene induction in murine fibroblasts. In unstimulated cells, continuous turnover of H3K9 acetylation occurs on all K4-trimethylated histone H3 tails; distribution of both modifications coincides across promoter and 5′ part of the coding region. In contrast, K36- and K79-methylated H3 tails, which are not dynamically acetylated, are restricted to the coding regions of these genes. Upon stimulation, transcription-dependent increases in H3K4 and H3K36 trimethylation are seen across coding regions, peaking at 5′ and 3′ ends, respectively. Addressing molecular mechanisms involved, we find that Huntingtin-interacting protein HYPB/Setd2 is responsible for virtually all global and transcription-dependent H3K36 trimethylation, but not H3K36-mono- or dimethylation, in these cells. These studies reveal four distinct layers of histone modification across inducible mammalian genes and show that HYPB/Setd2 is responsible for H3K36 trimethylation throughout the mouse nucleus.

Post-translational modifications of histones are involved in transcript initiation and elongation. Methylation of lysine 36 of histone H3 (H3K36me) resides promoter distal at transcribed regions in Saccharomyces cerevisiae and is thought to prevent spurious initiation through recruitment of histone-deacetylase activity. Here, we report surprising complexity in distribution, regulation and readout of H3K36me in Drosophila involving two histone methyltransferases (HMTases). Dimethylation of H3K36 peaks adjacent to promoters and requires dMes-4, whereas trimethylation accumulates toward the 3' end of genes and relies on dHypb. Reduction of H3K36me3 is lethal in Drosophila larvae and leads to elevated levels of acetylation, specifically at lysine 16 of histone H4 (H4K16ac). In contrast, reduction of both di- and trimethylation decreases lysine 16 acetylation. Thus di- and trimethylation of H3K36 have opposite effects on H4K16 acetylation, which we propose enable dynamic changes in chromatin compaction during transcript elongation.

Transcription by RNA polymerase II is accompanied by dynamic changes in chromatin, including the eviction/deposition of nucleosomes or the covalent modification of histone subunits. This study examined the role of the histone H3/H4 chaperones, Asf1 and HIR, in histone mobility during transcription, with particular focus on the histone exchange pathway, using a dual histone expression system. The results showed that the exchange of H3/H4 normally occurs during transcription by the histone chaperones. Both Asf1 and HIR are important for histone deposition but have a different effect on histone exchange. While Asf1 mediated incorporation of external H3/H4 and renewal of pre-existing histones, HIR opposed it. The balance of two opposing activities might be an important mechanism for determining current chromatin states.

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.

Gfi1b is a 37 kDa nuclear protein with six C2H2 zinc-finger domains that can silence transcription upon binding to specific target gene promoters. Here we show by using a chromatin immunoprecipitation and cloning protocol that Gfi1b also binds to gamma-satellite sequences that mainly occur in pericentric heterochromatin. Immuno-FISH experiments demonstrated that Gfi1b is localized at foci of pericentric heterochromatin identified by DAPI staining. Elevated levels of Gfi1b correlated with increased histone H3 lysine 9 dimethylation at sites neighboring gamma-satellite sequences but also at Gfi1b target gene promoters. In Gfi1b-deficient cells, however, a decrease of histone H3 lysine 9 trimethylation and a loss of heterochromatic structures was observed. Strikingly, we found that Gfi1b binds to both SUV39H1 and G9A histone methyl transferases, which provides a direct link between histone methylation and Gfi1b at heterochromatic and euchromatic sites. We propose that Gfi1b functions in heterochromatin formation and silencing of euchromatic transcription by recruiting histone methyl transferases to either gamma-satellite sequences or specific target gene promoters.

International audience; Transcriptional activity and structure of chromatin are correlated with patterns of covalent DNA and histone modification. Previous studies have revealed that high levels of histone H3 dimethylation at lysine 9 (H3K9me2), characteristic of transcriptionally silent heterochromatin in Arabidopsis, require hypermethylation of DNA at CpG sites. Here, we report that CpG hypermethylation characteristic of heterochromatin specifically prevented H3K27 trimethylation (H3K27me3). H3K27 mono- and dimethylation mark silent heterochromatin independently of DNA methylation. Upon loss of CpG methylation, there was target-specific enrichment of H3K27me3 in heterochromatin that correlated with transcriptional reactivation. Moreover, using the kyp mutant affected in H3K9me2, we showed that changes in H3K27me3 occurred independently of the levels of H3K9me2. Therefore, CpG methylation provides distinct and direct information for a specific subset of histone methylation marks. The observed independence of the regulation of H3K9 and H3K27 methylation by CpG methylation refines the recently proposed combinatorial histone code involving these two marks.

Histone phosphorylation influences transcription, chromosome condensation, DNA repair and apoptosis. Previously, we showed that histone H3 Ser10 phosphorylation (pSer10) by the yeast Snf1 kinase regulates INO1 gene activation in part via Gcn5/SAGA complex-mediated Lys14 acetylation (acLys14). How such chromatin modification patterns develop is largely unexplored. Here we examine the mechanisms surrounding pSer10 at INO1, and at GAL1, which herein is identified as a new regulatory target of Snf1/pSer10. Snf1 behaves as a classic coactivator in its recruitment by DNA-bound activators, and in its role in modifying histones and recruiting TATA-binding protein (TBP). However, one important difference in Snf1 function in vivo at these promoters is that SAGA recruitment at INO1 requires histone phosphorylation via Snf1, whereas at GAL1, SAGA recruitment is independent of histone phosphorylation. In addition, the GAL1 activator physically interacts with both Snf1 and SAGA, whereas the INO1 activator interacts only with Snf1. Thus, at INO1, pSer10's role in recruiting SAGA may substitute for recruitment by DNA-bound activator. Our results emphasize that histone modifications share general functions between promoters, but also acquire distinct roles tailored for promoter-specific requirements.

The chromodomain is a conserved motif that functions in the epigenetic control of gene expression. Here, we report the functional characterization of a chromodomain protein, Chp1, in the heterochromatin assembly in fission yeast. We show that Chp1 is a structural component of three heterochromatic regions-centromeres, the mating-type region, and telomeres-and that its localization in these regions is dependent on the histone methyltransferase Clr4. Although deletion of the chp1(+) gene causes centromere-specific decreases in Swi6 localization and histone H3-K9 methylation, we show that the role of Chp1 is not exclusive to the centromeres. We found that some methylation persists in native centromeric regions in the absence of Chp1, which is also true for the mating-type region and telomeres, and determined that Swi6 and Chp2 are critical to maintaining this residual methylation. We also show that Chp1 participates in the establishment of repressive chromatin in all three chromosomal regions. These results suggest that different heterochromatic regions share common structural properties, and that centromeric heterochromatin requires Chp1-mediated establishment steps differently than do other heterochromatic regions.

The Set1 protein of Saccharomyces cerevisiae is a histone methyltransferase (HMTase) acting on lysine 4 of histone H3. Inactivation of the SET1 gene in a diploid leads to a sporulation defect. We have studied various processes that take place during meiotic differentiation in set1delta diploid cells. The absence of Set1 leads to a delay of meiotic S-phase onset, which reflects a defect in DNA replication initiation. The timely induction of meiotic DNA replication does not require the Set1 HMTase activity, but depends on the SET domain. In addition, set1delta displays a severe impairment of the DNA double-strand break formation, which is not only the consequence of the replication delay. Transcriptional profiling experiments show that the induction of middle meiotic genes, but not of early meiotic genes, is affected by the loss of Set1. In contrast to meiotic replication, the transcriptional induction of the middle meiotic genes appears to depend on the methylation of H3-K4. Our results unveil multiple roles of Set1 in meiotic differentiation and distinguish between HMTase-dependent and -independent Set1 functions.

Nuclear receptor corepressors SMRT (silencing mediator of retinoid and thyroid receptors) and N-CoR (nuclear receptor corepressor) recruit histone deacetylase (HDAC) activity to targeted regions of chromatin. These corepressors contain a closely spaced pair of SANT motifs whose sequence and organization is highly conserved. The N-terminal SANT is a critical component of a deacetylase activation domain (DAD) that binds and activates HDAC3. Here, we show that the second SANT motif functions as part of a histone interaction domain (HID). The HID enhances repression by increasing the affinity of the DAD-HDAC3 enzyme for histone substrate. The two SANT motifs synergistically promote histone deacetylation and repression through unique functions. The HID contribution to repression is magnified by its ability to inhibit histone acetyltransferase enzyme activity. Remarkably, the SANT-containing HID preferentially binds to unacetylated histone tails. This implies that the SMRT HID participates in interpreting the histone code in a feed-forward mechanism that promotes and maintains histone deacetylation at genomic sites of SMRT recruitment.

Cytosine methylation is critical for correct development and genome stability in mammals and plants. In order to elucidate the factors that control genomic DNA methylation patterning, a genetic screen for mutations that disrupt methylation‐correlated silencing of the endogenous gene PAI2 was conducted in Arabidopsis . This screen yielded seven loss‐of‐function alleles in a SET domain protein with histone H3 Lys9 methyltransferase activity, SUVH4. The mutations conferred reduced cytosine methylation on PAI2 , especially in non‐CG sequence contexts, but did not affect methylation on another PAI locus carrying two genes arranged as an inverted repeat. Moreover, an unmethylated PAI2 gene could be methylated de novo in the suvh4 mutant background. These results suggest that SUVH4 is involved in maintenance but not establishment of methylation at particular genomic regions. In contrast, a heterochromatin protein 1 homolog, LHP1, had no effect on PAI methylation.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.

The induction of immediate-early (IE) genes, including proto-oncogenes c-fos and c-jun, correlates well with a nucleosomal response, the phosphorylation of histone H3 and HMG-14 mediated via extracellular signal regulated kinase or p38 MAP kinase cascades. Phosphorylation is targeted to a minute fraction of histone H3, which is also especially susceptible to hyperacetylation. Here, we provide direct evidence that phosphorylation and acetylation of histone H3 occur on the same histone H3 tail on nucleosomes associated with active IE gene chromatin. Chromatin immunoprecipitation (ChIP) assays were performed using antibodies that specifically recognize the doubly-modified phosphoacetylated form of histone H3. Analysis of the associated DNA shows that histone H3 on c-fos- and c-jun-associated nucleosomes becomes doubly-modified, the same H3 tails becoming both phosphorylated and acetylated, only upon gene activation. This study reveals potential complications of occlusion when using site-specific antibodies against modified histones, and shows also that phosphorylated H3 is more sensitive to trichostatin A (TSA)-induced hyperacetylation than non-phosphorylated H3. Because MAP kinase-mediated gene induction is implicated in controlling diverse biological processes, histone H3 phosphoacetylation is likely to be of widespread significance.

Histones are structural and functional components of the eukaryotic chromosome, and their function is essential for normal cell cycle progression. In this work, we describe the characterization of two Saccharomyces cerevisiae cold-sensitive histone H2A mutants. Both mutants contain single amino acid replacements of residues predicted to be on the surface of the nucleosome and in close contact with DNA. We show that these H2A mutations cause an increase-in-ploidy phenotype, an increased rate of chromosome loss, and a defect in traversing the G(2)-M phase of the cell cycle. Moreover, these H2A mutations show genetic interactions with mutations in genes encoding kinetochore components. Finally, chromatin analysis of these H2A mutants has revealed an altered centromeric chromatin structure. Taken together, these results strongly suggest that histone H2A is required for proper centromere-kinetochore function during chromosome segregation.

We have studied the role of core histone tails in the assembly of mitotic chromosomes using Xenopus egg extracts. Incubation of sperm nuclei in the extracts led to the formation of mitotic chromosomes, a process we found to be correlated with phosphorylation of the N–terminal tail of histone H3 at Ser10. When the extracts were supplemented with H1-depleted oligosomes, they were not able to assemble chromosomes. Selective elimination of oligosome histone tails by trypsin digestion resulted in a dramatic decrease in their ability to inhibit chromosome condensation. The chromosome assembly was also inhibited by each of the histone tails with differing efficiency. In addition, we found that nucleosomes were recruiting through the flexible histone tails some chromosome assembly factors, different from topoisomerase II and 13S condensin. These findings demonstrate that histone tails play an essential role in chromosome assembly. We also present evidence that the nucleosomes, through physical association, were able to deplete the extracts from the kinase phosphorylating histone H3 at Ser10, suggesting that this kinase could be important for chromosome condensation.

The chromatin elements targeted by the ATPdependent, Swi-Snf nucleosome-remodeling complex are unknown. To address this question, we generated mutations in yeast histone H2B that suppress phenotypes associated with the absence of Swi-Snf. Sin- (Swi-Snf-independent) mutations occur in residues involved in H2A-H2B dimer formation, dimer- tetramer association, and in the H2B N-terminus. The strongest and most pleiotropic Sin- mutation removed 20 amino acid residues from the H2B N-terminus. This mutation allowed active chromatin to be formed at the SUC2 locus in a snf5Delta mutant and resulted in hyperactivated levels of SUC2 mRNA under inducing conditions. Thus, the H2B N-terminus may be an important target of Swi-Snf in vivo. The GCN5 gene product, the catalytic subunit of several nuclear histone acetytransferase complexes that modify histone N-termini, was also found to act in conjunction with Swi-Snf. The phenotypes of double gcn5Deltasnf5Delta mutants suggest that histone acetylation may play both positive and negative roles in the activity of the Swi-Snf-remodeling factor.

The three-way connection between DNA methylation, gene activity and chromatin structure has been known for almost two decades. Nevertheless, the molecular link between methyl groups on the DNA and the positioning of nucleosomes to form an inactive chromatin configuration was missing. This review discusses recent experimental data that may, for the first time, shed light on this molecular link. MeCP2, which is a known methylcytosine-binding protein, has been shown to possess a transcriptional repressor domain (TRD) that binds the corepressor mSin3A. This corepressor protein constitutes the core of a multiprotein complex that includes histone deacetylases (HDAC1 and HDAC2). Transfection and injection experiments with methylated constructs have revealed that the silenced state of a methylated gene, which is associated with a deacetylated nucleosomal structure, could be relieved by the deacetylase inhibitor, trichostatin A. Thus, methylation plays a pivotal role in establishing and maintaining an inactive state of a gene by rendering the chromatin structure inaccessible to the transcription machinery.

Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly., Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast The demonstration that H3K14 deacetylation promotes recruitment of the Clr4 histone methyltransferase establishes a new function for the Sir2 deacetylase in de novo heterochromatin formation.

The molecular mechanisms responsible for the remodeling of entire somatic erythrocyte nuclei in Xenopus laevis egg cytoplasm have been examined. These transitions in chromosomal composition are associated with the capacity to activate new patterns of gene expression and the re-acquisition of replication competence. Somatic linker histone variants H1 and H1 (0) are released from chromatin in egg cytoplasm, whereas the oocyte-specific linker histone B4 and HMG1 are efficiently incorporated into remodeled chromatin. Histone H1 (0) is released from chromatin preferentially in comparison with histone H1. Core histones H2A and H4 in the somatic nucleus are phosphorylated during this remodeling process. These transitions recapitulate the chromosomal environment found within the nuclei of the early Xenopus embryo. Phosphorylation of somatic linker histone variants is demonstrated not to direct their release from chromatin, nor does direct competition with cytoplasmic stores of linker histone B4 determine their release. However, the molecular chaperone nucleoplasmin does have an important role in the selective removal of linker histones from somatic nuclei. For Xenopus erythrocyte nuclei, this disruption of chromatin structure leads to activation of the 5S rRNA genes. These results provide a molecular explanation for the remodeling of chromatin in Xenopus egg cytoplasm and indicate the capacity of molecular chaperones to disrupt a natural chromosomal environment, thereby facilitating transcription.

Cellular senescence is an intrinsic defense mechanism to various cellular stresses: while still metabolically active, senescent cells stop dividing and enter a proliferation arrest. Here, we identify DPY30, a member of all mammalian histone H3K4 histone methyltransferases (HMTases), as a key regulator of the proliferation potential of human primary cells. Following depletion of DPY30, cells show a severe proliferation defect and display a senescent phenotype, including a flattened and enlarged morphology, elevated level of reactive oxygen species (ROS), increased SA-β-galactosidase activity, and formation of senescence-associated heterochromatin foci (SAHFs). While DPY30 depletion leads to a reduced level of H3K4me3-marked active chromatin, we observed a concomitant activation of CDK inhibitors, including p16INK4a, independent of H3K4me3. ChIP experiments show that key regulators of cell-cycle progression, including ID proteins, are under direct control of DPY30. Because ID proteins are negative regulators of the transcription factors ETS1/2, depletion of DPY30 leads to the transcriptional activation of p16INK4a by ETS1/2 and thus to a senescent-like phenotype. Ectoptic re-introduction of ID protein expression can partially rescue the senescence-like phenotype induced by DPY30 depletion. Thus, our data indicate that DPY30 controls proliferation by regulating ID proteins expression, which in turn lead to senescence bypass.

Efficient supply of new histones during DNA replication is critical to restore chromatin organization and maintain genome function. The histone chaperone anti-silencing function 1 (Asf1) serves a key function in providing H3.1-H4 to CAF-1 for replication-coupled nucleosome assembly. We identify Codanin-1 as a novel interaction partner of Asf1 regulating S-phase histone supply. Mutations in Codanin-1 can cause congenital dyserythropoietic anaemia type I (CDAI), characterized by chromatin abnormalities in bone marrow erythroblasts. Codanin-1 is part of a cytosolic Asf1–H3.1-H4–Importin-4 complex and binds directly to Asf1 via a conserved B-domain, implying a mutually exclusive interaction with the chaperones CAF-1 and HIRA. Codanin-1 depletion accelerates the rate of DNA replication and increases the level of chromatin-bound Asf1, suggesting that Codanin-1 guards a limiting step in chromatin replication. Consistently, ectopic Codanin-1 expression arrests S-phase progression by sequestering Asf1 in the cytoplasm, blocking histone delivery. We propose that Codanin-1 acts as a negative regulator of Asf1 function in chromatin assembly. This function is compromised by two CDAI mutations that impair complex formation with Asf1, providing insight into the molecular basis for CDAI disease.

MDM2 is a key regulator of the p53 tumor suppressor acting primarily as an E3 ubiquitin ligase to promote its degradation. MDM2 also inhibits p53 transcriptional activity by recruiting histone deacetylase and corepressors to p53. Here, we show that immunopurified MDM2 complexes have significant histone H3-K9 methyltransferase activity. The histone methyltransferases SUV39H1 and EHMT1 bind specifically to MDM2 but not to its homolog MDMX. MDM2 mediates formation of p53-SUV39H1/EHMT1 complex capable of methylating H3-K9 in vitro and on p53 target promoters in vivo. Furthermore, MDM2 promotes EHMT1-mediated p53 methylation at K373. Knockdown of SUV39H1 and EHMT1 increases p53 activity during stress response without affecting p53 levels, whereas their overexpression inhibits p53 in an MDM2-dependent manner. The p53 activator ARF inhibits SUV39H1 and EHMT1 binding to MDM2 and reduces MDM2-associated methyltransferase activity. These results suggest that MDM2-dependent recruitment of methyltransferases is a novel mechanism of p53 regulation through methylation of both p53 itself and histone H3 at target promoters.

The function of histone modifications in initiating and regulating the chromosomal events of the meiotic prophase remains poorly understood. In Saccharomyces cerevisiae, we examined the genome-wide localization of histone H3 lysine 4 trimethylation (H3K4me3) along meiosis and its relationship to gene expression and position of the programmed double-strand breaks (DSBs) that initiate interhomologue recombination, essential to yield viable haploid gametes. We find that the level of H3K4me3 is constitutively higher close to DSB sites, independently of local gene expression levels. Without Set1, the H3K4 methylase, 84% of the DSB sites exhibit a severely reduced DSB frequency, the reduction being quantitatively correlated with the local level of H3K4me3 in wild-type cells. Further, we show that this differential histone mark is already established in vegetative cells, being higher in DSB-prone regions than in regions with no or little DSB. Taken together, our results demonstrate that H3K4me3 is a prominent and preexisting mark of active meiotic recombination initiation sites. Novel perspectives to dissect the various layers of the controls of meiotic DSB formation are discussed.

Cells respond to DNA double-strand breaks (DSBs) and uncapped telomeres by recruiting checkpoint and repair factors to the site of lesions. Single-stranded DNA (ssDNA) is an important intermediate in the repair of DSBs and is produced also at uncapped telomeres. Here, we provide evidence that binding of the checkpoint protein Rad9, through its Tudor domain, to methylated histone H3-K79 inhibits resection at DSBs and uncapped telomeres. Loss of DOT1 or mutations in RAD9 influence a Rad50-dependent nuclease, leading to more rapid accumulation of ssDNA, and faster activation of the critical checkpoint kinase, Mec1. Moreover, deletion of RAD9 or DOT1 partially bypasses the requirement for CDK1 in DSB resection. Interestingly, Dot1 contributes to checkpoint activation in response to low levels of telomere uncapping but is not essential with high levels of uncapping. We suggest that both Rad9 and histone H3 methylation allow transmission of the damage signal to checkpoint kinases, and keep resection of damaged DNA under control influencing, both positively and negatively, checkpoint cascades and contributing to a tightly controlled response to DNA damage.

Coactivator-associated arginine methyltransferase (CARM1) is a transcriptional coactivator that methylates Arg17 and Arg26 in histone H3. CARM1 contains a conserved protein arginine methyltransferase (PRMT) catalytic core flanked by unique pre- and post-core regions. The crystal structures of the CARM1 catalytic core in the apo and holo states reveal cofactor-dependent formation of a substrate-binding groove providing a specific access channel for arginine to the active site. The groove is supported by the first eight residues of the post-core region (C-extension), not present in other PRMTs. In vitro methylation assays show that the C-extension is essential for all histone H3 methylation activity, whereas the pre-core region is required for methylation of Arg26, but not Arg17. Kinetic analysis shows Arg17 methylation is potentiated by pre-acetylation of Lys18, and this is reflected in k(cat) rather than K(m). Together with the absence of specificity subsites in the structure, this suggests an electrostatic sensing mechanism for communicating the modification status of vicinal residues as part of the syntax of the 'histone code.'

The Bur1-Bur2 and Paf1 complexes function during transcription elongation and affect histone modifications. Here we describe new roles for Bur1-Bur2 and the Paf1 complex. We find that histone H3 K36 tri-methylation requires specific components of the Paf1 complex and that K36 tri-methylation is more strongly affected at the 5' ends of genes in paf1delta and bur2delta strains in parallel with increased acetylation of histones H3 and H4. Interestingly, the 5' increase in histone acetylation is independent of K36 methylation, and therefore is mechanistically distinct from the methylation-driven deacetylation that occurs at the 3' ends of genes. Finally, Bur1-Bur2 and the Paf1 complex have a second methylation-independent function, since bur2delta set2delta and paf1delta set2delta double mutants display enhanced histone acetylation at the 3' ends of genes and increased cryptic transcription initiation. These findings identify new functions for the Paf1 and Bur1-Bur2 complexes, provide evidence that histone modifications at the 5' and 3' ends of coding regions are regulated by distinct mechanisms, and reveal that the Bur1-Bur2 and Paf1 complexes repress cryptic transcription through a Set2-independent pathway.