Histone H3


Table of contents

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression H3 lysine 4-specific methylation

MLL, the human homolog of Drosophila trithorax, maintains Hox gene expression in mammalian embryos and is rearranged in human leukemias resulting in Hox gene deregulation. How MLL or MLL fusion proteins regulate gene expression remains obscure. MLL regulates target Hox gene expression through direct binding to promoter sequences. The MLL SET domain is a histone H3 lysine 4-specific methyltransferase whose activity is stimulated with acetylated H3 peptides. This methylase activity is associated with Hox gene activation and H3 (Lys4) methylation at cis-regulatory sequences in vivo. A leukemogenic MLL fusion protein that activates Hox expression had no effect on histone methylation, suggesting a distinct mechanism for gene regulation by MLL and MLL fusion proteins (Milne, 2002).

How MLL regulates Hox gene expression is poorly understood. The domain structure of MLL is complex, making it difficult to unravel the key components of MLL function. Domains that may have a role in MLL function include the AT hooks, which bind DNA, a region homologous to DNA methyl transferases (DNMT), the cysteine-rich PHD domain, and a highly conserved SET domain. The SET domain is found in many proteins now demonstrated to mediate lysine-directed histone methylation. These findings suggest a possible role for MLL in chromatin remodeling mediated by histone methylation. However, early studies of this domain in MLL did not reveal evidence of enzymatic activity, leaving its function enigmatic. Furthermore, rearrangements of MLL that occur in leukemia consistently delete the PHD and SET domains and replace these sequences with one of over 30 different translocation partners that in general share little sequence homology (Milne, 2002).

Progress in understanding the mechanistic role of MLL in maintenance and gene regulation has also been slowed by a lack of known target binding sites for mammalian PcG or trxG homologs. To address these issues, attention was focused on how MLL regulates transcription of Hox c8. This target was chosen because it is tightly regulated by MLL and because it is the only Hox gene in which the sequences required for the correct initiation and maintenance of expression have been extensively mapped in vivo. Hox c8 is upregulated by MLL, supporting a transcriptional activating role for MLL. MLL binds directly to proximal promoter sequences but not to other regions of the Hox c8 locus, including the 5' and 3' enhancer sequences, suggesting that MLL-dependent regulatory elements in mammalian Hox genes are organized differently from those in Drosophila. The Hox c8 promoter is necessary and sufficient for MLL responsiveness and, along with the 5' enhancer, exhibits differential histone acetylation and H3 (Lys4) methylation in Mll+/+ as compared to Mll-/- cells. Reexpression of MLL in null cells results in methylation of H3 (Lys4) at the Hox c8 5' enhancer and promoter as well as at other Hox gene promoters. H3 (Lys4) methylation is dependent on an intact MLL SET domain and this methyltransferase activity is stimulated by H3 peptides that are acetylated at Lys9 or Lys14. Collectively, these experiments underscore the importance of a concerted series of histone and DNA modifications in the regulation and maintenance of target genes during mammalian development and provide a framework for comparing mechanisms of epigenetic forms of gene regulation by MLL and MLL fusion proteins (Milne, 2002).

ALL-1 is a member of the human trithorax/Polycomb gene family and is also involved in acute leukemia. ALL-1 is present within a stable, very large multiprotein supercomplex composed of ~29 proteins. The majority of the latter are components of the human transcription complexes TFIID (including TBP), SWI/SNF, NuRD, hSNF2H, and Sin3A. Other components are involved in RNA processing or in histone methylation. The complex remodels, acetylates, deacetylates, and methylates nucleosomes and/or free histones. The complex's H3-K4 methylation activity is conferred by the ALL-1 SET domain. Chromatin immunoprecipitations show that ALL-1 and other complex components examined are bound at the promoter of an active ALL-1-dependent Hox a9 gene. In parallel, H3-K4 is methylated, and histones H3 and H4 are acetylated at this promoter (Nakamura, 2002).

Strikingly, most ALL-1-associated proteins can be classified into well-known complexes involved in transcription. Of these, the SWI/SNF(BRM) and NuRD complexes and the hSNF2H protein are ATP-dependent chromatin remodelers: Sin3A and NuRD are histone deacetylases; two human homologs of components of the yeast Set1 complex (but not the Set1 protein) are involved in H3-K4 methylation, and TFIID acts in promoter recognition and in mediating activator responsiveness. The identification of TFIID components, including TBP, within the ALL-1 supercomplex is one of the most significant observations of this work. This finding indicates a direct connection between ALL-1 and the general transcription machinery. Several TFIID proteins have been identified as components of the Drosophila Polycomb multiprotein complex. Considering the known functions of the other complexes included within the ALL-1 supercomplex, SWI/SNF and hSNF2H may act both as activators and repressors, but Sin3A and NuRD complexes have been generally associated with transcriptional silencing. Since ALL-1 is an activator, the inclusion of these last two complexes within the ALL-1 supercomplex is surprising. However, HDAC1, a component of both Sin3A and NuRD complexes, has been found bound to active promoters of some Drosophila genes, including the trithorax-regulated Abd-B. Further, histone deacetylation might be required to enable H3-K4 methylation. Also, deacetylation might be applied to modulate the level of histone acetylation conferred by the ALL-1 complex and/or by acetyltransferases transiently associated with ALL-1. Moreover, the deacetylating complexes might target transcription factors regulated by acetylation. Finally, the inclusion within the ALL-1 supercomplex of CPSF and Symplekin involved in polyadenylation and of p116 associated with splicing provides support for the notion of direct connection between the promoter of a gene and how its transcript is processed (Nakamura, 2002and references therein).

A major finding in this work is that the ALL-1 SET domain methylates H3-K4. Previous attempts to show methyltransferase activity of ALL-1 SET were unsuccessful, probably due to the low activity of this domain (at least 10-fold lower than the activity of SUV39H1 SET, which methylates H3-K9). Presently, two other proteins have been implicated in H3-K4 methylation. Human Set7/Set9 possess this intrinsic enzymatic activity conferred by the SET domain. A Saccharomyces cerevisiae complex containing the Set1 protein methylates H3-K4, and mutation analysis of the gene implicates it directly in that histone modification in yeast. Whereas human SET7/SET9 activates transcription, yeast Set1 represses transcription of ribosomal DNA. Nevertheless, diverse findings correlate H3-K4 methylation with an active state of transcription. Thus, this modification is specifically associated with transcriptionally active macronuclei but not with inactive micronuclei in Tetrahymena. Also, immunofluorescence studies of human female chromosomes show that H3-K4 methylation accumulates at transcribed regions of autosomes but is largely excluded from the inactive X chromosome. Moreover, ChIP experiments at the mating type locus of fission yeast have shown that, while histone H3-K4 methylation is localized to actively transcribed regions, H3-K9 methylation is detected in silent heterochromatin. Similar results have been observed in ChIP analysis of the beta-globin locus during erythropoiesis (Nakamura, 2002 and references therein).

Basic helix-loop-helix (bHLH) transcription factors such as atonal homolog 5 (ATH5) and neurogenin 2 (NGN2) determine crucial events in retinogenesis. Using chromatin immunoprecipitation, their interactions with target promoters have been demonstrated to undergo dynamic changes as development proceeds in the chick embryo. Chick ATH5 associates with its own promoter and with the promoter of the ß3 nicotinic receptor specifically in retinal ganglion cells and their precursors. NGN2 binds to the ATH5 promoter in retina but not in optic tectum, suggesting that interactions between bHLH factors and chromatin are highly tissue specific. The transcriptional activations of both promoters correlate with dimethylation of lysine 4 on histone H3. Inactivation of the ATH5 promoter in differentiated neurons is accompanied by replication-independent chromatin de-methylation. This report is one of the first demonstrations of correlation between gene expression, binding of transcription factors and chromatin modification in a developing neural tissue (Skowronska-Krawczyk, 2004).

To determine whether the correlation between promoter activity and histone H3-K4 dimethylation is a general phenomenon, it was of interest to analyze the methylation patterns of genes expressed both in the retina and in the optic tectum. NeuroM and NeuroD are good candidates for this study as they are dynamically expressed in both tissues. In the optic tectum, the transient expression of NeuroM peaks at E6, at a time when the various cell classes exit from the mitotic cycle. In the retina, NeuroM expression obeys the same principle as in the optic tectum; however, expression does not stop at the end of neurogenesis but persists in mature bipolar and horizontal cells. In the optic tectum and retina, NeuroD has a later onset than NeuroM. In early (E4-6) retina, expression of NeuroD is detected in precursor cells and may correlate at later stages with the differentiation of photoreceptors and amacrine cells. In the optic tectum, NeuroD expression is detected at around E6 and increases slowly during development of the tissue. Immunoprecipitation of chromatin from retina and optic tectum was performed using an anti dimethylated H3-K4 antibody and correlations in both tissues were observed between histone dimethylation and the known expression patterns of NeuroM and NeuroD. In retina, methylation of the NeuroM promoter is detected at E3 and reaches its highest level at E9. It remains high in the developed retina, in accordance with the sustained NeuroM mRNA expression seen in this tissue. In the optic tectum, the transient expression of this gene is at a much lower level than in the retina and no significant enhancement of methylation was detected. This could reflect the fact that the fraction of tectal cells that express NeuroM is too small to be detected in the assay, or it may suggest different histone modification requirements for brief versus continuous expression of the gene. The level of methylation of NeuroD promoter sequences remained very low during retina and optic tectum development, but was strongly enhanced in the developed retina and optic tectum. This delayed methylation of the NeuroD promoter is congruent with the late onset of NeuroD expression in both tissues. Incidentally, the ability to detect H3 methylation at the NeuroD promoter in both retina and optic tectum demonstrates that the paucity in optic tectum methylation observed for other promoters is physiologically relevant and not due to a tissue-specific bias in chromatin quality (Skowronska-Krawczyk, 2004).

Best known as epigenetic repressors of developmental Hox gene transcription, Polycomb complexes alter chromatin structure by means of post-translational modification of histone tails. Depending on the cellular context, Polycomb complexes of diverse composition and function exhibit cooperative interaction or hierarchical interdependency at target loci. The present study interrogated the genetic, biochemical and molecular interaction of BMI1 [Drosophila homologs Psc and Su(z)2] and EED (Drosophila homolog; Esc), pivotal constituents of heterologous Polycomb complexes, in the regulation of vertebral identity during mouse development. Despite a significant overlap in dosage-sensitive homeotic phenotypes and co-repression of a similar set of Hox genes, genetic analysis implicated eed and Bmi1 in parallel pathways, which converge at the level of Hox gene regulation. Whereas EED and BMI1 formed separate biochemical entities with EzH2 and Ring1B, respectively, in mid-gestation embryos, YY1 engaged in both Polycomb complexes. Strikingly, methylated lysine 27 of histone H3 (H3-K27), a mediator of Polycomb complex recruitment to target genes, stably associated with the EED complex during the maintenance phase of Hox gene repression. Juxtaposed EED and BMI1 complexes, along with YY1 and methylated H3-K27, were detected in upstream regulatory regions of Hoxc8 and Hoxa5. The combined data suggest a model wherein epigenetic and genetic elements cooperatively recruit and retain juxtaposed Polycomb complexes in mammalian Hox gene clusters toward co-regulation of vertebral identity (Kim, 2006).

At least two PcG complexes with diverse composition and function in chromatin remodeling have been identified in mammals. The Polycomb repressive complex 1 (PRC1) involves the paralogous PcG proteins BMI1/MEL18, M33/PC2, RAE28, and RING1A. Evidence for PRC1-mediated chromatin modification derived from ubiquitylation at lysine 119 of histone H2A (H2A-K119). A second PcG complex, PRC2, encompasses EED, the histone methyltransferase EZH2, the zinc finger protein SUZ12, the histone-binding proteins RBAP46/RBAP48, and the histone deacetylase HDAC1. Several EED isoforms, generated by alternate translation start site usage of eed mRNA, differentially engage in PRC2-related complexes (PRC2/3/4), targeting the histone methyltransferase activity of EZH2 to H3-K27 or H1-K26. PcG complexes bind to cis-acting Polycomb response elements (PREs), which encompass several hundred base pairs and are necessary and sufficient for PcG-mediated repression of target genes. Whereas the function of several PREs has been delineated in Drosophila, similar elements await characterization in mammals (Kim, 2006 and references therein).

An antibody raised against residues 123-140 of the EED amino terminus precipitated three distinct isoforms of approximately 50 and 75 kDA from E12.5 trunk, representing three of the four EED isoforms previously reported in 293 cells. In addition to EZH2 and YY1, dimethylated H3-K27 co-immunoprecipitated with EED. Immunoprecipitation identified three BMI1 isoforms of approximately 39-41 kDA. BMI1 was found in a complex with RING1B, but not dimethylated H3-K27. Similar to the EED complex, the BMI1 complex also contained YY1. It should be emphasized that all (co-)immunoprecipitating bands were detected by at least two antibodies against different epitopes. Strikingly, while dimethylated H3-K27 engaged in the EED complex, trimethylated H3-K27 did not appear to associate with either the EED or the BMI1 complex. Importantly, reciprocal co-immunoprecipitation detected EED and BMI1 in separate protein complexes (Kim, 2006).

Ectopic expression in mutant embryos revealed Hoxc8 and Hoxa5 as downstream targets of EED and BMI1 function. ChIP detected EED and BMI1 binding immediately upstream of the Hoxc8 transcribed region near putative promoter elements. The binding sites could not be separated, indicating close proximity of the complexes. EED and BMI1 binding also clustered within a small fragment 1.5 kb upstream of the Hoxc8 transcription start site, suggesting long-range juxtaposition of heterologous PcG complexes. Similar to EED and BMI1, YY1 localized to both regions. In support of YY1 binding to Hox regulatory regions, inspection of the mouse genome sequence revealed clusters of putative YY1 binding sites in both regions a and b, including TGTCCATTAG and CCCCCATTCC (region a), as well as ACACCATGGC, TTTCCATTAG and TCCCCATAAA (region b). CCAT represents the core of the YY1 consensus binding site, while flanking sequences exhibited significant tolerance for multiple nucleotides. EED, BMI1 and YY1 also co-localized approximately 1.5 kb upstream of the transcription start site of Hoxa5. In addition to PcG binding, ChIP detected trimethylated H3-K27 throughout the regulatory regions of Hoxc8 and Hoxa5. Furthermore, dimethylated H3-K27 localized to region b of Hoxc8 (Kim, 2006).

Spatial regulation of EED and BMI1 binding to Hox regulatory regions was evident from ChIP analysis of dissected anterior and posterior regions of E12.5 trunk. In agreement with transcriptional silencing of Hoxc8 and Hoxa5, EED and BMI1 binding was detected upstream of these loci in anterior regions of the trunk. By contrast, EED and BMI1 binding was absent from posterior regions of the trunk, where Hoxc8 and Hoxa5 are transcribed. These findings implicate PcG complexes in Hox gene repression in anterior regions of the AP axis (Kim, 2006).

The combined interpretation of the co-immunoprecipitation and ChiP results indicates that trimethylated H3-K27 did not form a complex with EED or BMI1, despite co-localization of the three proteins in Hox regulatory regions. By contrast, co-immunoprecipitation demonstrated physical association of the EED complex with dimethylated H3-K27. In aggregate, the results support a model in which EED- and BMI1-containing chromatin remodeling complexes exist as separate, but juxtaposed, biochemical entities at Hox target loci (Kim, 2006).

Automethylation activities within the Mixed Lineage Leukemia-1 (MLL1) core complex reveal evidence supporting a 'two-active site' model for multiple histone H3 lysine 4 methylation

The Mixed Lineage Leukemia-1 (MLL1) core complex predominantly catalyzes mono- and dimethylation of histone H3 at lysine 4 (H3K4) and is frequently altered in aggressive acute leukemias. The molecular mechanisms that account for conversion of mono- to dimethyl H3K4 (H3K4me1,2) are not well understood. This paper reports that the SET domains from human MLL1 and Drosophila Trithorax undergo robust intramolecular automethylation reactions at an evolutionarily conserved cysteine residue in the active site, which is inhibited by unmodified histone H3. The location of the automethylation in the SET-I sub-domain indicates that the MLL1 SET domain possesses significantly more conformational plasticity in solution than suggested by its crystal structure. It is also reported that MLL1 methylates Ash2L in the absence of histone H3, but only when assembled within a complex including WDR5 and RbBP5, suggesting a restraint for the architectural arrangement of subunits within the complex. Using MLL1 and Ash2L automethylation reactions as probes for histone binding, it was observed that both automethylation reactions are significantly inhibited by stoichiometric amounts of unmethylated histone H3, but not by histones previously mono-, di- or trimethylated at H3K4. These results suggest that the H3K4me1 intermediate does not significantly bind to the MLL1 SET domain during the dimethylation reaction. Consistent with this hypothesis, it was demonstrated that the MLL1 core complex assembled with a catalytically inactive SET domain variant preferentially catalyzes H3K4 dimethylation using the H3K4me1 substrate. Taken together, these results are consistent with a 'two-active site' model for multiple H3K4 methylation by the MLL1 core complex (Patel, 2013).

Chd1 chromodomain links histone H3 methylation with SAGA- and SLIK-dependent acetylation

The specific post-translational modifications to histones influence many nuclear processes including gene regulation, DNA repair and replication. Recent studies have identified effector proteins that recognize patterns of histone modification and transduce their function in downstream processes. For example, histone acetyltransferases (HATs) have been shown to participate in many essential cellular processes, particularly those associated with activation of transcription. Yeast SAGA (Spt-Ada-Gcn5 acetyltransferase) and SLIK (SAGA-like) are two highly homologous and conserved multi-subunit HAT complexes, which preferentially acetylate histones H3 and H2B and deubiquitinate histone H2B. This study identifies the chromatin remodelling protein Chd1 (chromo-ATPase/helicase-DNA binding domain 1; see Drosophila Chd1) as a component of SAGA and SLIK. These findings indicate that one of the two chromodomains of Chd1 specifically interacts with the methylated lysine 4 mark on histone H3 that is associated with transcriptional activity. Furthermore, the SLIK complex shows enhanced acetylation of a methylated substrate and this activity is dependent upon a functional methyl-binding chromodomain, both in vitro and in vivo. This study identifies the first chromodomain that recognizes methylated histone H3 (Lys 4) and possibly identifies a larger subfamily of chromodomain proteins with similar recognition properties (Pray-Grant, 2005).

The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex

The MLL family of histone methyltransferases maintains active chromatin domains by methylating histone H3 on lysine 4 (H3K4). How MLL complexes recognize specific chromatin domains in a temporal and tissue-specific manner remains unclear. This study shows that the DNA-binding protein PAX2 promotes assembly of an H3K4 methyltransferase complex through the ubiquitously expressed nuclear factor PTIP (pax transcription activation domain interacting protein). PTIP copurifies with ALR, MLL3, and other components of a histone methyltransferase complex. PTIP promotes assembly of the ALR complex and H3K4 methylation at a PAX2-binding DNA element. Without PTIP, Pax2 binds to this element but does not assemble the ALR complex. Embryonic lethal ptip-null mutants and conditional mutants both show reduced levels of methylated H3K4. Thus, PTIP bridges DNA-binding developmental regulators to histone methyltransferase-dependent epigenetic regulation (Patel, 2007).

Double chromodomains cooperate to recognize the methylated histone H3 tail

Chromodomains are modules implicated in the recognition of lysine-methylated histone tails and nucleic acids. CHD (for chromo-ATPase/helicase-DNA-binding) proteins regulate ATP-dependent nucleosome assembly and mobilization through their conserved double chromodomains and SWI2/SNF2 helicase/ATPase domain. The Drosophila CHD1 localizes to the interbands and puffs of the polytene chromosomes, which are classic sites of transcriptional activity (Stokes, 1996). Other CHD isoforms (CHD3/4 or Mi-2) are important for nucleosome remodelling in histone deacetylase complexes. Deletion of chromodomains impairs nucleosome binding and remodelling by CHD proteins. This study describes the structure of the tandem arrangement of the human CHD1 chromodomains, and its interactions with histone tails. Unlike HP1 and Polycomb proteins that use single chromodomains to bind to their respective methylated histone H3 tails, the two chromodomains of CHD1 cooperate to interact with one methylated H3 tail. The human CHD1 double chromodomains target the lysine 4-methylated histone H3 tail (H3K4me), a hallmark of active chromatin. Methylammonium recognition involves two aromatic residues, not the three-residue aromatic cage used by chromodomains of HP1 and Polycomb proteins. Furthermore, unique inserts within chromodomain 1 of CHD1 block the expected site of H3 tail binding seen in HP1 and Polycomb, instead directing H3 binding to a groove at the inter-chromodomain junction (Flanagan, 2005).

SET protein complex in vertebrates methylates H3

The abundant and chromatin-associated protein HCF-1 is a critical player in mammalian cell proliferation as well as herpes simplex virus (HSV) transcription. Separate regions of HCF-1 critical for its role in cell proliferation associate with the Sin3 histone deacetylase (HDAC) and a previously uncharacterized human trithorax-related Set1/Ash2 histone methyltransferase (HMT). The Set1/Ash2 HMT methylates histone H3 at Lys 4 (K4), but not if the neighboring K9 residue is already methylated. HCF-1 tethers the Sin3 and Set1/Ash2 transcriptional regulatory complexes together even though they are generally associated with opposite transcriptional outcomes: repression and activation of transcription, respectively. Nevertheless, this tethering is context-dependent because the transcriptional activator VP16 selectively binds HCF-1 associated with the Set1/Ash2 HMT complex in the absence of the Sin3 HDAC complex. These results suggest that HCF-1 can broadly regulate transcription, both positively and negatively, through selective modulation of chromatin structure (Wysocka, 2003).

Dimethylation of H3K4 by Set1 recruits the Set3 histone deacetylase complex to 5' transcribed regions

Cotranscriptional histone methylations by Set1 and Set2 have been shown to affect histone acetylation at promoters and 3' regions of genes, respectively. While histone H3K4 trimethylation (H3K4me3) is thought to promote nucleosome acetylation and remodeling near promoters, this study shows that H3K4 dimethylation (H3K4me2) by Set1 leads to reduced histone acetylation levels near 5' ends of genes. H3K4me2 recruits the Set3 complex via the Set3 PHD finger, localizing the Hos2 and Hst1 subunits to deacetylate histones in 5' transcribed regions. Cells lacking the Set1-Set3 complex pathway are sensitive to mycophenolic acid and have reduced polymerase levels at a Set3 target gene, suggesting a positive role in transcription. It is proposed that Set1 establishes two distinct chromatin zones on genes: H3K4me3 leads to high levels of acetylation and low nucleosome density at promoters, while H3K4me2 just downstream recruits the Set3 complex to suppress nucleosome acetylation and remodeling (T. Kim, 2009).

Feedback control of Set1 protein levels is important for proper H3K4 methylation patterns

Methylation of histone H3 lysine 4 by the Set1 subunit of COMPASS correlates with active transcription. This study shows that Set1 levels are regulated in yeast by protein degradation in response to multiple signals. Set1 levels are greatly reduced when COMPASS recruitment to genes, H3K4 methylation, or transcription is blocked. The degradation sequences map to N-terminal regions that overlap a previously identified autoinhibitory domain, as well as the catalytic domain. Truncation mutants of Set1 that cause under- or overexpression produce abnormal H3K4 methylation patterns on transcribed genes. Surprisingly, SAGA-dependent genes are more strongly affected than TFIID-dependent genes, reflecting differences in their chromatin dynamics. It is proposed that careful tuning of Set1 levels by regulated degradation is critical for the establishment and maintenance of proper H3K4 methylation patterns (Soares, 2014).

Histone H3 lysine 4 monomethylation modulates long-range chromatin interactions at enhancers
Long-range chromatin interactions between enhancers and promoters are essential for transcription of many developmentally controlled genes in mammals and other metazoans. Currently, the exact mechanisms that connect distal enhancers to their specific target promoters remain to be fully elucidated. This study shows that the enhancer-specific histone H3 lysine 4 monomethylation (H3K4me1) and the histone methyltransferases MLL3 and MLL4 (MLL3/4; see Drosophila Trithorax) play an active role in this process. This study demonstrates that in differentiating mouse embryonic stem cells, MLL3/4-dependent deposition of H3K4me1 at enhancers correlates with increased levels of chromatin interactions, whereas loss of this histone modification leads to reduced levels of chromatin interactions and defects in gene activation during differentiation. H3K4me1 facilitates recruitment of the Cohesin complex, a known regulator of chromatin organization, to chromatin in vitro and in vivo, providing a potential mechanism for MLL3/4 to promote chromatin interactions between enhancers and promoters. Taken together, these results support a role for MLL3/4-dependent H3K4me1 in orchestrating long-range chromatin interactions at enhancers in mammalian cells (Yan, 2018).

Mammalian ASH1L is a histone methyltransferase that occupies the transcribed region of active genes

Histone lysine methylation regulates genomic functions, including gene transcription. Previous reports found various degrees of methylation at H3K4, H3K9, and H4K20 within the transcribed region of active mammalian genes. To identify the enzymes responsible for placing these modifications, ASH1L, the mammalian homolog of the Drosophila Trithorax group (TrxG) protein Ash1, was examined. Drosophila Ash1 has been reported to methylate H3K4, H3K9, and H4K20 at its target sites. This study demonstrates that mammalian ASH1L associates with the transcribed region of all active genes examined, including Hox genes. The distribution of ASH1L in transcribed chromatin strongly resembles that of methylated H3K4 but not that of H3K9 or H4K20. Accordingly, the SET domain of ASH1L methylates H3K4 in vitro, and knockdown of ASH1L expression reduced H3K4 trimethylation at HoxA10 in vivo. Notably, prior methylation at H3K9 reduced ASH1L-mediated methylation at H3K4, suggesting cross-regulation among these marks. Drosophila ash1 and trithorax interact genetically, and the mammalian TrxG protein MLL1 and ASH1L display highly similar distributions and substrate specificities. However, by using MLL null cell lines it was found that their recruitments occur independently of each other. Collectively, these data suggest that ASH1L occupies most, if not all, active genes and methylates histone H3 in a nonredundant fashion at a subset of genes (Gregory, 2007).

Ash1l methylates Lys36 of histone H3 independently of transcriptional elongation to counteract polycomb silencing

Molecular mechanisms for the establishment of transcriptional memory are poorly understood. 5,6-dichloro-1-D-ribofuranosyl-benzimidazole (DRB) is a P-TEFb kinase inhibitor that artificially induces the poised RNA polymerase II (RNAPII), thereby manifesting intermediate steps for the establishment of transcriptional activation. In this study, using genetics and DRB, it was shown that mammalian Absent, small, or homeotic discs 1-like (Ash1l), a member of the trithorax group proteins, methylates Lys36 of histone H3 to promote the establishment of Hox gene expression by counteracting Polycomb silencing. Importantly, it was found that Ash1l-dependent Lys36 di-, tri-methylation of histone H3 in a coding region and exclusion of Polycomb group proteins occur independently of transcriptional elongation in embryonic stem (ES) cells, although both were previously thought to be consequences of transcription. Genome-wide analyses of histone H3 Lys36 methylation under DRB treatment have suggested that binding of the retinoic acid receptor (RAR) to a certain genomic region promotes trimethylation in the RAR-associated gene independent of its ongoing transcription. Moreover, DRB treatment unveils a parallel response between Lys36 methylation of histone H3 and occupancy of either Tip60 or Mof in a region-dependent manner. It was also found that Brg1 is another key player involved in the response. These results uncover a novel regulatory cascade orchestrated by Ash1l with RAR and provide insights into mechanisms underlying the establishment of the transcriptional activation that counteracts Polycomb silencing (Miyazaki, 2013).

Arginine methylation at histone H3R2 controls deposition of H3K4 trimethylation

Modifications on histones control important biological processes through their effects on chromatin structure. Methylation at lysine 4 on histone H3 (H3K4) is found at the 5' end of active genes and contributes to transcriptional activation by recruiting chromatin-remodelling enzymes. An adjacent arginine residue (H3R2) is also known to be asymmetrically dimethylated (H3R2me2a) in mammalian cells, but its location within genes and its function in transcription are unknown. This study shows that H3R2 is also methylated in budding yeast (Saccharomyces cerevisiae), and by using an antibody specific for H3R2me2a in a chromatin immunoprecipitation-on-chip analysis the distribution of this modification has been determined on the entire yeast genome. H3R2me2a is enriched throughout all heterochromatic loci and inactive euchromatic genes and is present at the 3' end of moderately transcribed genes. In all cases the pattern of H3R2 methylation is mutually exclusive with the trimethyl form of H3K4 (H3K4me3). Methylation at H3R2 abrogates the trimethylation of H3K4 by the Set1 methyltransferase. The specific effect on H3K4me3 results from the occlusion of Spp1, a Set1 methyltransferase subunit necessary for trimethylation. Thus, the inability of Spp1 to recognize H3 methylated at R2 prevents Set1 from trimethylating H3K4. These results provide the first mechanistic insight into the function of arginine methylation on chromatin (Kirmizis, 2007).

These findings place methylation at H3R2 and H3K4 in the same pathway and support a role of H3R2me2a as a negative regulator of H3K4 trimethylation. A model is presented of how H3R2me2a may function during the transition from a repressed to a transcriptionally active state on a gene. Global analysis shows that when a gene is inactive, H3R2me2a is present throughout the promoter and coding region (step 0). Methylation of H3R2 in yeast is likely to be catalysed by a previously unknown and as yet unidentified methyltransferase, because combinatorial deletion of the three known arginine methyltransferases (Rmt1, Rmt2 and Hsl7) does not affect the degree of this modification. At this silent stage (step 0) very little, if any, methylation of H3K4 takes place. During activation, the presence of methylated H3R2 does not inhibit Set1p from monomethylating or dimethylating H3K4 (step 1). However, for trimethylation of H3K4 to take place, methylation at H3R2 has to be removed (step 2). The clearing of methylation at H3R2 must be mediated either by histone replacement or by the action of an as yet unidentified arginine demethylase. Once a region becomes devoid of H3R2 methylation, the Spp1 protein can recognize H3K4me2 by its PHD domain. This binding probably extends the time of interaction between the Set1 complex and its substrate, thus promoting the trimethylation of H3K4 by Set1p (step 3). Spp1 then associates with H3K4me3 (step 4), possibly to protect this methyl state from the action of the H3K4me3 demethylase Jhd2. At the same time, Spp1 may protect H3R2 from methylation; structural studies have shown that this arginine residue is absolutely required for the association of the Spp1 PHD finger with methylated H3K4. Together these data indicate that arginine methylation at H3R2 may influence transcription by regulating the H3K4 trimethylation capacity of the Set1 methyltransferase (Kirmizis, 2007).

Methylation of histone H3R2 by PRMT6 and H3K4 by an MLL complex are mutually exclusive

Eukaryotic genomes are organized into active (euchromatic) and inactive (heterochromatic) chromatin domains. Post-translational modifications of histones (or 'marks') are key in defining these functional states, particularly in promoter regions. Mutual regulatory interactions between these marks -- and the enzymes that catalyse them -- contribute to the shaping of this epigenetic landscape, in a manner that remains to be fully elucidated. Asymmetric di-methylation of histone H3 arginine 2 (H3R2me2a) counter-correlates with di- and tri-methylation of H3 lysine 4 (H3K4me2, H3K4me3) on human promoters. This study shows that the arginine methyltransferase PRMT6 catalyses H3R2 di-methylation in vitro and controls global levels of H3R2me2a in vivo. H3R2 methylation by PRMT6 is prevented by the presence of H3K4me3 on the H3 tail. Conversely, the H3R2me2a mark prevents methylation of H3K4 as well as binding to the H3 tail by an ASH2/WDR5/MLL-family methyltransferase complex. Chromatin immunoprecipitation showed that H3R2me2a is distributed within the body and at the 3' end of human genes, regardless of their transcriptional state, whereas it is selectively and locally depleted from active promoters, coincident with the presence of H3K4me3. Hence, the mutual antagonism between H3R2 and H3K4 methylation, together with the association of MLL-family complexes with the basal transcription machinery, may contribute to the localized patterns of H3K4 tri-methylation characteristic of transcriptionally poised or active promoters in mammalian genomes (Guccione, 2007).

RAD6-Mediated transcription-coupled H2B ubiquitylation directly stimulates H3K4 methylation in human cells

H2B ubiquitylation has been implicated in active transcription but is not well understood in mammalian cells. Beyond earlier identification of hBRE1 as the E3 ligase for H2B ubiquitylation in human cells, this study now shows (1) that hRAD6 serves as the cognate E2-conjugating enzyme; (2) that hRAD6, through direct interaction with hPAF-bound hBRE1, is recruited to transcribed genes and ubiquitylates chromatinized H2B at lysine 120; (3) that hPAF-mediated transcription is required for efficient H2B ubiquitylation as a result of hPAF-dependent recruitment of hBRE1-hRAD6 to the Pol II transcription machinery; (4) that H2B ubiquitylation per se does not affect the level of hPAF-, SII-, and p300-dependent transcription and likely functions downstream; and (5) that H2B ubiquitylation directly stimulates hSET1-dependent H3K4 di- and trimethylation. These studies establish the natural H2B ubiquitylation factors in human cells and also detail the mechanistic basis for H2B ubiquitylation and function during transcription (J. Kim, 2009).

The PAF complex requirement for both H2B ubiquitylation and downstream H3K4 and H3K79 methylation has been well-documented by yeast genetics. However, there has been no clear biochemical evidence that explains the direct role of the PAF complex in H2B ubiquitylation. In the present study, chromatin-templated ubiquitylation and transcription assays with defined factors have revealed distinct PAF complex functions in H2B ubiquitylation. (1) The PAF complex can recruit H2B ubiquitylation factors to chromatin through direct interactions with both the BRE1 complex and Pol II. (2) The PAF complex couples transcription and efficient H2B ubiquitylation through its intrinsic chromatin transcription enabling activity rather than through a direct stimulation of RAD6-BRE1 activity. It is plausible that the PAF complex-enhanced passage of Pol II through nucleosomes allows H2B ubiquitylation factors (recruited by the PAF complex) easier access to the H2B ubiquitylation site. The fact that H2B ubiquitylation is further enhanced by SII, which synergistically increases transcription with the hPAF complex, strengthens the claim that efficient H2B ubiquitylation is coupled to transcription. This relationship (hRAD6->hBRE1 complex->hPAF complex->Pol II) nicely fits the yeast genetic data wherein yBre1 deletion completely abrogates yRad6 association with the entire body of Pol II-dependent genes and wherein deletion of the PAF complex subunit yRtf1 leads to dissociation of yRad6 from coding regions. Along with these observations, the data contrast with a previous report that hUbcH6, rather than the hBRE1 complex, interacts with the hPAF complex to physically link the H2B ubiquitylation machinery to Pol II and that the hPAF complex enhances hUbcH6-mediated H2B ubiquitylation in the absence of ongoing transcription (J. Kim, 2009).

Cell-based assays have shown that H2B ubiquitylation is required for proper activation of several inducible genes and, conversely, that a number of factors implicated in transcription initiation and elongation are required for H2B ubiquitylation. An intriguing question raised by these results is whether H2B ubiquitylation itself stimulates transcription or whether transcription facilitates H2B ubiquitylation for purpose of a subsequent ubiquitylated H2B function. Relevant to this issue, transcription-coupled chromatin ubiquitylation assays with biochemically defined factors clearly show that ongoing transcription is required for efficient H2B ubiquitylation. Of note, the overall level of H2B ubiquitylation in this assay (about 15%) is markedly higher than the level (<1%) observed with oligonucleosome substrates in the absence of transcription. Moreover, since only a small portion of the chromatin template is transcribed in vitro, the level of H2B ubiquitylation may be much greater than 15% in the transcribed region. In addition, the demonstration of low-level transcription-independent H2B ubiquitylation with natural HeLa cell-derived oligonucleosomes, but not with recombinant chromatin, raises the possibility that natural transcriptionally active chromatins (with associated histone modifications) may serve as preferential substrates for H2B ubiquitylation in vitro. The demonstration of a transcription requirement for H2B ubiquitylation also provides a plausible explanation for why H2B ubiquitylation-dependent H3K4 methylation marks recent transcription (J. Kim, 2009).

Strikingly, transcription assays with a recombinant, fully H2B-ubiquitylated chromatin template demonstrate that H2B ubiquitylation per se has no demonstrable effect on the level of hPAF complex- and SII-enhanced transcription mediated by p53 in conjunction with p300. This contrasts with a previously reported observation that hUbcH6-mediated H2B ubiquitylation directly stimulates FACT-dependent histone displacement and transcription elongation in vitro. Although no any effect of FACT, in the presence or absence of RAD6-dependent H2B ubiquitylation, was seen on the overall level of transcription, this may reflect in part the utilization of NAP1, rather than FACT, as a histone chaperone in these assays. The results are consistent with studies that showed an in vivo role for H2B ubiquitylation in the efficient reassembly of nucleosomes after Pol II passage, thereby repressing cryptic transcription initiation, but no role in nucleosome disassembly was found during Pol II passage. Hence, H2B ubiquitylation was proposed to function in the wake of elongating Pol II rather than by directly stimulating Pol II elongation. Nontheless, and of note, the current inability to see an effect of robust H2B ubiquitylation on the overall level of transcription or on transcription-related events may relate to the use of a defined transcription system that lacks downstream factors that act in conjunction with ubH2B (see below). Thus, the results overall indicate that H2B ubiquitylation does not directly affect the function of the transcriptional machinery, but that it is a consequence of transcription that is important for events following passage of Pol II (J. Kim, 2009).

Current observations suggest several possibilities for a positive role for H2B ubiquitylation in transcription-related events. First, ubH2B may provide a binding platform for a factor(s) that is responsible for downstream events such as histone modification and chromatin remodeling during transcription elongation. Second, and potentially related to the first possibility, H2B ubiquitylation-dependent H3 methylation may affect transcription. In this regard, H2B ubiquitylation was shown to directly stimulate both hDOT1L-mediated H3K79 methylation and hSET1 complex-mediated H3K4 di- and trimethylation. In relation to transcription, H3K4 trimethyl marks are known to be recognized, for example, by PHD fingers in factors affecting chromatin remodeling or histone modifications. Third, H2B ubiquitylation, followed by deubiquitylation, may also be required for promoter-proximal transcription events on certain genes, and these requirements could be imposed by other unknown factors. The defined transcription-H2B ubiquitylation system described in this study should prove critical for a further characterization of transcription-related factors that are dependent upon H2B ubiquitylation (J. Kim, 2009).

A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling

Lysine methylation of histones is recognized as an important component of an epigenetic indexing system demarcating transcriptionally active and inactive chromatin domains. Trimethylation of histone H3 lysine 4 (H3K4me3) marks transcription start sites of virtually all active genes. The WD40-repeat protein WDR5 is important for global levels of H3K4me3 and control of HOX gene expression. A PHD finger of nucleosome remodelling factor (NURF), an ISWI-containing ATP-dependent chromatin-remodelling complex, mediates a direct preferential association with H3K4me3 tails. Depletion of H3K4me3 causes partial release of the NURF subunit, BPTF (bromodomain and PHD finger transcription factor), from chromatin and defective recruitment of the associated ATPase, SNF2L (also known as ISWI and SMARCA1), to the HOXC8 promoter. Loss of BPTF in Xenopus embryos mimics WDR5 loss-of-function phenotypes, and compromises spatial control of Hox gene expression. These results strongly suggest that WDR5 and NURF function in a common biological pathway in vivo, and that NURF-mediated ATP-dependent chromatin remodelling is directly coupled to H3K4 trimethylation to maintain Hox gene expression patterns during development. This study identifies a previously unknown function for the PHD finger as a highly specialized methyl-lysine-binding domain (Wysocka, 2005).

Mono-, di- and tri-methylated states of particular histone lysine residues are selectively found in different regions of chromatin, thereby implying specialized biological functions for these marks ranging from heterochromatin formation to X-chromosome inactivation and transcriptional regulation. A major challenge in chromatin biology has centered on efforts to define the connection between specific methylation states and distinct biological read-outs impacting on function. For example, histone H3 trimethylated at lysine 4 (H3K4me3) is associated with transcription start sites of active genes, but the molecular 'effectors' involved in specific recognition of H3K4me3 tails remain poorly understood. This study demonstrates the molecular basis for specific recognition of H3(1-15)K4me3 (residues 1-15 of histone H3 trimethylated at K4) by a PHD finger of human BPTF (bromodomain and PHD domain transcription factor), the largest subunit of the ATP-dependent chromatin-remodelling complex, NURF (nucleosome remodelling factor). Crystallographic and NMR structures of the bromodomain-proximal PHD finger of BPTF in free and H3(1-15)K4me3-bound states is reported. H3(1-15)K4me3 interacts through anti-parallel beta-sheet formation on the surface of the PHD finger, with the long side chains of arginine 2 (R2) and K4me3 fitting snugly in adjacent pre-formed surface pockets, and bracketing an invariant tryptophan. The observed stapling role by non-adjacent R2 and K4me3 provides a molecular explanation for H3K4me3 site specificity. Binding studies establish that the BPTF PHD finger exhibits a modest preference for K4me3- over K4me2-containing H3 peptides, and discriminates against monomethylated and unmodified counterparts. Furthermore, key specificity-determining residues were identified from binding studies of H3(1-15)K4me3 with PHD finger point mutants. These findings call attention to the PHD finger as a previously uncharacterized chromatin-binding module found in a large number of chromatin-associated proteins (H. Li, 2006).

Subnuclear segregation of genes and core promoter factors in myogenesis: Recognition of H3K4Me3 by the TAF3 PHD finger is required for the sequestration of TAF3 to the nuclear interior

Recent findings implicate alternate core promoter recognition complexes in regulating cellular differentiation. This study reports a spatial segregation of the alternative core factor TAF3, but not canonical TFIID subunits, away from the nuclear periphery, where the key myogenic gene MyoD is preferentially localized in myoblasts. This segregation is correlated with the differential occupancy of TAF3 versus TFIID at the MyoD promoter. Loss of this segregation by modulating either the intranuclear location of the MyoD gene or TAF3 protein leads to altered TAF3 occupancy at the MyoD promoter. Intriguingly, in differentiated myotubes, the MyoD gene is repositioned to the nuclear interior, where TAF3 resides. The specific high-affinity recognition of H3K4Me3 by the TAF3 PHD finger appears to be required for the sequestration of TAF3 to the nuclear interior. It is suggested that intranuclear sequestration of core transcription components and their target genes provides an additional mechanism for promoter selectivity during differentiation (Yao, 2011).

The MLL3/MLL4 branch of the COMPASS family is a major H3K4 monomethylase at enhancers

Histone H3 lysine 4 (H3K4) can be mono-, di-, and trimethylated by members of the COMPASS (COMplex of Proteins ASsociated with Set1) family from yeast to human and these modifications can be found at distinct regions of the genome. Monomethylation of histone H3K4 (H3K4me1) is relatively more enriched at metazoan enhancer regions compared to trimethylated histone H3K4 (H3K4me3), which are found at transcription start sites in all eukaryotes. Recent studies in Drosophila demonstrated that the Trithorax-related (Trr) branch of the COMPASS family regulates enhancer activity and is responsible for the implementation of H3K4me1 at these regions. There are six COMPASS family members in mammals, two of which, MLL3 and MLL4, are most closely related to Drosophila Trr. This study used ChIP-seq of this class of COMPASS family members in both human HCT116 cells and mouse embryonic stem cells and found that MLL4 is preferentially found at enhancer regions. MLL3 and MLL4 are frequently mutated in cancer, and indeed, the widely used HCT116 cancer cell line contains inactivating mutations in the MLL3 gene. Using HCT116 cells in which MLL4 has also been knocked out, it was demonstrated that MLL3 and MLL4 are major regulators of H3K4me1 in these cells, with the greatest loss of monomethylation at enhancer regions. Moreover, a redundant role was found between Mll3 and Mll4 in enhancer H3K4 monomethylation in mouse embryonic fibroblast (MEF) cells. These findings suggest that mammalian MLL3/MLL4 function in the regulation of enhancer activity and enhancer-promoter communication during gene expression and that mutations of MLL3 and MLL4 found in cancer could exert their properties through enhancer malfunction (Hu, 2013).

Context dependency of Set1/COMPASS-mediated histone H3 Lys4 trimethylation

The stimulation of trimethylation of histone H3 Lys4 (H3K4) by H2B monoubiquitination (H2Bub) has been widely studied, with multiple mechanisms having been proposed for this form of histone cross-talk. Cps35/Swd2 within COMPASS (complex of proteins associated with Set1) is considered to bridge these different processes. However, a truncated form of Set1 (762-Set1) is reported to function in H3K4 trimethylation (H3K4me3) without interacting with Cps35/Swd2, and such cross-talk is attributed to the n-SET domain of Set1 and its interaction with the Cps40/Spp1 subunit of COMPASS. This study used biochemical, structural, in vivo, and chromatin immunoprecipitation (ChIP) sequencing (ChIP-seq) approaches to demonstrate that Cps40/Spp1 and the n-SET domain of Set1 are required for the stability of Set1 and not the cross-talk. Furthermore, the apparent wild-type levels of H3K4me3 in the 762-Set1 strain are due to the rogue methylase activity of this mutant, resulting in the mislocalization of H3K4me3 from the promoter-proximal regions to the gene bodies and intergenic regions. Detailed screens were performed, and yeast strains were identified lacking H2Bub but containing intact H2Bub enzymes that have normal levels of H3K4me3, suggesting that monoubiquitination may not directly stimulate COMPASS but rather works in the context of the PAF and Rad6/Bre1 complexes. This study demonstrates that the monoubiquitination machinery and Cps35/Swd2 function to focus COMPASS's H3K4me3 activity at promoter-proximal regions in a context-dependent manner (Thorton, 2014).

Crosstalk between NSL histone acetyltransferase and MLL/SET complexes: NSL complex functions in promoting histone H3K4 di-methylation activity by MLL/SET complexes

hMOF (MYST1), a histone acetyltransferase (HAT), forms at least two distinct multiprotein complexes in human cells. The male specific lethal (MSL) HAT complex plays a key role in dosage compensation in Drosophila and is responsible for histone H4K16ac in vivo. A second hMOF-containing HAT complex has been described, the non-specific lethal (NSL) HAT complex. The NSL complex has a broader substrate specificity, can acetylate H4 on K16, K5, and K8. The WD (tryptophan-aspartate) repeat domain 5 (WDR5) and host cell factor 1 (HCF1) are shared among members of the MLL/SET (mixed-lineage leukemia/set-domain containing) family of histone H3K4 methyltransferase complexes. The presence of these shared subunits raises the possibility that there are functional links between these complexes and the histone modifications they catalyze; however, the degree to which NSL and MLL/SET influence one another's activities remains unclear. This study presents evidence from biochemical assays and knockdown/overexpression approaches arguing that the NSL HAT promotes histone H3K4me2 by MLL/SET complexes by an acetylation-dependent mechanism. In genomic experiments, a set of genes was identified, including ANKRD2, that are affected by knockdown of both NSL and MLL/SET subunits, suggested they are co-regulated by NSL and MLL/SET complexes. In ChIP assays, it was observed that depletion of the NSL subunits hMOF or NSL1 resulted in a significant reduction of both H4K16ac and H3K4me2 in the vicinity of the ANKRD2 transcriptional start site proximal region. However, depletion of RbBP5 (a core component of MLL/SET complexes) only reduced H3K4me2 marks, but not H4K16ac in the same region of ANKRD2, consistent with the idea that NSL acts upstream of MLL/SET to regulate H3K4me2 at certain promoters, suggesting coordination between NSL and MLL/SET complexes is involved in transcriptional regulation of certain genes. Taken together, these results suggest a crosstalk between the NSL and MLL/SET complexes in cells (Zhao, 2013).

Large hypomethylated domains serve as strong repressive machinery for key developmental genes in vertebrates

DNA methylation is a fundamental epigenetic modification in vertebrate genomes and a small fraction of genomic regions is hypomethylated. Previous studies have implicated hypomethylated regions in gene regulation, but their functions in vertebrate development remain elusive. To address this issue, epigenomic profiles were generated that include base-resolution DNA methylomes and histone modification maps from both pluripotent cells and mature organs of medaka fish, and the profiles were compared with those of human ES cells. It was found that a subset of hypomethylated domains harbor H3K27me3 (K27HMDs) and their size positively correlates with the accumulation of H3K27me3. Large K27HMDs are conserved between medaka and human pluripotent cells and predominantly contain promoters of developmental transcription factor genes. These key genes were found to be under strong transcriptional repression, when compared with other developmental genes with smaller K27HMDs. Furthermore, human-specific K27HMDs show an enrichment of neuronal activity-related genes, which suggests a distinct regulation of these genes in medaka and human. In mature organs, some of the large HMDs become shortened by elevated DNA methylation and associate with sustained gene expression. This study highlights the significance of domain size in epigenetic gene regulation. It is proposed that large K27HMDs play a crucial role in pluripotent cells by strictly repressing key developmental genes, whereas their shortening consolidates long-term gene expression in adult differentiated cells (Nakamura, 2014).

Evolutionary homologs: Table of contents

Histone H3: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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