Histone H3


Table of contents

Gene-specific targeting of H3K9 methylation is sufficient for initiating repression

Covalent modifications of chromatin have emerged as key determinants of the genome's transcriptional competence. Histone H3 lysine 9 (H3K9) methylation is an epigenetic signal that is recognized by HP1 and correlates with gene silencing in a variety of organisms. Discovery of the enzymes that catalyze H3K9 methylation has identified a second gene-specific function for this modification in transcriptional repression. Whether H3K9 methylation is causative in the initiation and establishment of gene repression or is a byproduct of the process leading to the repressed state remains unknown. To investigate the role of HMTs and specifically H3K9 methylation in gene repression, engineered zinc-finger transcription factors (ZFPs) have been employed to target HMT activity to a specific endogenous gene. By utilizing ZFPs that recognize the promoter of the endogenous VEGF-A gene, and thus employing this chromosomal locus as an in vivo reporter, it has been show that ZFPs linked to a minimal catalytic HMT domain affect local methylation of histone H3K9 and the consequent repression of target gene expression. Furthermore, amino acid substitutions within the HMT that ablate its catalytic activity effectively eliminate the ability of the ZFP fusions to repress transcription. Thus, H3K9 methylation is a primary signal that is sufficient for initiating a gene repression pathway in vivo (Snowden, 2002).

Mutations of leukemia-associated AF9/MLLT3 are implicated in neurodevelopmental diseases, such as epilepsy and ataxia, but little is known about how AF9 influences brain development and function. Analyses of mouse mutants revealed that during cortical development, AF9 is involved in the maintenance of TBR2-positive progenitors (intermediate precursor cells, IPCs) in the subventricular zone and prevents premature cell cycle exit of IPCs. Furthermore, in postmitotic neurons of the developing cortical plate, AF9 is implicated in the formation of the six-layered cerebral cortex by suppressing a TBR1-positive cell fate mainly in upper layer neurons. The molecular mechanism of TBR1 suppression is based on the interaction of AF9 with DOT1L, a protein that mediates transcriptional control through methylation of histone H3 lysine 79 (H3K79). AF9 associates with the transcriptional start site of Tbr1, mediates H3K79 dimethylation of the Tbr1 gene, and interferes with the presence of RNA polymerase II at the Tbr1 transcriptional start site. AF9 expression favors cytoplasmic localization of TBR1 and its association with mitochondria. Increased expression of TBR1 in Af9 mutants is associated with increased levels of TBR1-regulated expression of NMDAR subunit Nr1. Thus, this study identified AF9 as a developmental active epigenetic modifier during the generation of cortical projection neurons (Büttner, 2010).

Methylation of Histone H3 by Su(var)3-9 homologs: The role of HP1 homologs on chromatin structure of heterochromatin

The organization of chromatin into higher-order structures influences chromosome function and epigenetic gene regulation. Higher-order chromatin has been proposed to be nucleated by the covalent modification of histone tails and the subsequent establishment of chromosomal subdomains by non-histone modifier factors. Human SUV39H1 and murine Suv39h1 -- mammalian homologs of Drosophila Su(var)3-9 and of Schizosaccharomyces pombe clr4 -- encode histone H3-specific methyltransferases that selectively methylate lysine 9 of the amino terminus of histone H3 in vitro. The catalytic motif has been mapped to the evolutionarily conserved SET domain, which requires adjacent cysteine-rich regions to confer histone methyltransferase activity. Methylation of lysine 9 interferes with phosphorylation of serine 10, but is also influenced by pre-existing modifications in the amino terminus of H3. In vivo, deregulated SUV39H1 or disrupted Suv39h activity modulates H3 serine 10 phosphorylation in native chromatin and induces aberrant mitotic divisions. These data reveal a functional interdependence of site-specific H3 tail modifications and suggest a dynamic mechanism for the regulation of higher-order chromatin (Rea, 2000).

SUV39H1, a human homolog of the Drosophila position effect variegation modifier Su(var)3-9 and of the Schizosaccharomyces pombe silencing factor clr4, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of SUV39H1 mutant proteins in transfected cells, it has been shown that deregulated SUV39H1 interferes at multiple levels with mammalian higher-order chromatin organization. (1) Forced expression of full-length SUV39H1 (412 amino acids) redistributes endogenous M31 (HP1beta) and induces abundant associations with inter- and meta-phase chromatin. These properties depend on the C-terminal SET domain, although the major portion of the SUV39H1 protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the M31 interaction surface, which maps to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. (2) Cells overexpressing full-length SUV39H1 display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G2-specific distribution of phosphorylated histone H3 at serine 10 (phosH3) is dispersed in these cells. This phosH3 shift is not observed with C-terminally truncated mutant SUV39H1 proteins or with deregulated M31. Together, these data reveal a dominant role(s) for the SET domain of SUV39H1 in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal SU(VAR) protein and histone H3 (Melcher, 2000).

Heterochromatin protein 1 (HP1) is localized at heterochromatin sites where it mediates gene silencing. The chromo domain of HP1 is necessary for both targeting and transcriptional repression. In the fission yeast Schizosaccharomyces pombe, the correct localization of Swi6 (the HP1 equivalent) depends on Clr4, a homolog of the mammalian SUV39H1 histone methylase. Both Clr4 and SUV39H1 specifically methylate lysine 9 of histone H3. In this study it has been shown that mammalian HP1 can bind with high affinity to histone H3 methylated at lysine 9 but not at lysine 4. The chromo domain of HP1 is identified as its methyl-lysine-binding domain. A point mutation in the chromo domain, which destroys the gene silencing activity of HP1 in Drosophila, abolishes methyl-lysine-binding activity. Genetic and biochemical analysis in S. pombe shows that the methylase activity of Clr4 is necessary for the correct localization of Swi6 at centromeric heterochromatin and for gene silencing. These results provide a stepwise model for the formation of a transcriptionally silent heterochromatin: SUV39H1 places a 'methyl marker' on histone H3, which is then recognized by HP1 through its chromo domain. This model may also explain the stable inheritance of the heterochromatic state (Bannister, 2001).

Mammalian methyltransferases that selectively methylate histone H3 on lysine 9 (Suv39h HMTases) generate a binding site for HP1 proteins -- a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated histone H3 peptide by HP1 requires a functional chromo domain; thus, the HP1 chromo domain is a specific interaction motif for the methyl epitope on lysine9 of histone H3. In vivo, heterochromatin association of HP1 proteins is lost in Suv39h double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active SWUV39H1 HMTase. These data define a molecular mechanism through which the SUV39H-HP1 methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin (Lachner, 2001).

The human ISWI-containing factor RSF (remodeling and spacing factor) mediates nucleosome deposition and, in the presence of ATP, generates regularly spaced nucleosome arrays. Using this system, recombinant chromatin was reconstituted with bacterially produced histones. Acetylation of the histone tails was found to play an important role in establishing regularly spaced nucleosome arrays. Recombinant chromatin lacking histone acetylation is impaired in directing transcription. Histone-tail modifications regulate transcription from the recombinant chromatin. Acetylation of the histone tails by p300 increases transcription. Methylation of the histone H3 tail by Suv39H1 represses transcription in an HP1-dependent manner. The effects of histone-tail modifications were observed in nuclear extracts. A highly reconstituted RNA polymerase II transcription system is refractory to the effect imposed by acetylation and methylation (Loyola, 2001).

The establishment of conditions that permit the reconstitution of recombinant chromatin allows for the analysis of the effect of the different histone tail modifications in transcription. Toward this goal, the ability of the recombinant chromatin to be used as template for transcription was analyzed and the effect of two histone-tail modifications was specifically analyzed: p300-mediated acetylation and Suv39H1-mediated methylation (Loyola, 2001).

Although these two modifications can have opposite effects on transcription, these modifications were not recognized in a highly reconstituted transcription system; their effect was observed only in crude extracts. There are different explanations for findings. The most logical explanation is that acetylation and/or methylation per se does not affect template utilization but affects the ability of the chromatin templates to be recognized by the transcription machinery. It is likely that these modifications provide marks on the histone tails that are recognized by factors present in extracts but missing in the reconstituted system that affects transcription. This hypothesis is supported by the findings with methylation and transcription. It was found that HP1-mediated repression of transcription requires Suv39H1-mediated methylation of histone H3. This finding is in perfect agreement with results obtained in vivo showing that the binding of HP1 to chromatin requires methylation of histone H3-Lys 9. Surprisingly, however, chromatin, H3-Lys 9 methylation, and HP1 are not sufficient to establish repression, since this could not be reproduced in a reconstituted transcription system. It is likely that other factors are required to establish repression. Studies in yeast have shown that histone deacetylation is required to establish the appropriate substrate for methylation by Suv39H1. Although the use of chromatin without pre-existing modification bypasses the requirement for the histone deacetylase enzymatic activity, it is possible that the histone deacetylases that target histone H3-lysines 9 and 14 not only function to generate the appropriate substrate but also might be active components of the Suv39H1-repressive complex (Loyola, 2001).

With regards to acetylation, it was observed that chromatin reconstituted with hypoacetylated human histone polypeptides is not optimal for transcription in crude extracts; however, the reconstituted system is indifferent to acetylation of the histone polypeptides. This finding is in agreement with the histone-code hypothesis and strongly suggests that factors in the extract, but lacking in the reconstituted system, might recognize the acetylated mark(s) to stimulate transcription. Using recombinant chromatin, it was observed that acetylation of histone tails, specifically by p300, stimulates transcription in extracts. In agreement with the results obtained using chromatin reconstituted with hypo/hyperacetylated human histones, no effect was observed in a reconstituted transcription system. Although a possible explanation to this observation is the absence of a factor in the reconstituted system, the inability of the reconstituted transcription system to respond to acetylation of the recombinant chromatin might also be the result of the inability of p300 to acetylate specific residues on the histone tails. The recombinant chromatin is devoid of histone-tail modifications, and it is likely that p300-mediated acetylation of a specific residue might require other histone modifications. This possibility is supported by studies showing that phosphorylation of histone H3-Ser 10 modulates acetylation of histone H3-Lys 14. The presence of a specific kinase in the extract might phosphorylate histone H3-Ser 10, resulting in efficient acetylation. Elucidation of the factors necessary for p300-mediated acetylation to result in optimal transcription and of the factor(s) required for Suv39-H1-mediated methylation to result in repression of transcription, and their exact mechanism of action, require further studies. The development of the system described in the present study, capable of generating recombinant chromatin will permit the setting of biochemical complementation assays to isolate the different factors involved in these processes as well as the elucidation of their mechanism of action (Loyola, 2001).

Histone H3 lysine 9 methylation has been proposed to provide a major 'switch' for the functional organization of chromosomal subdomains. The murine Suv39h histone methyltransferases (HMTases) govern H3-K9 methylation at pericentric heterochromatin and induce a specialized histone methylation pattern that differs from the broad H3-K9 methylation present at other chromosomal regions. Suv39h-deficient mice display severely impaired viability and chromosomal instabilities that are associated with an increased tumor risk and perturbed chromosome interactions during male meiosis. These in vivo data assign a crucial role for pericentric H3-K9 methylation in protecting genome stability, and define the Suv39h HMTases as important epigenetic regulators for mammalian development (Peters, 2001).

A novel histone methyltransferase, termed Set9, was isolated from human cells. Set9 contains a SET domain, but lacks the pre- and post-SET domains. Set9 methylates specifically lysine 4 (K4) of histone H3 (H3-K4) and potentiates transcription activation. The histone H3 tail interacts specifically with the histone deacetylase NuRD complex. Methylation of histone H3-K4 by Set9 precludes the association of NuRD with the H3 tail. Moreover, methylation of H3-K4 impairs Suv39h1-mediated methylation at K9 of H3 (H3-K9). The interplay between the Set9 and Suv39h1 histone methyltransferases is specific, since the methylation of H3-K9 by the histone methyltransferase G9a is not affected by Set9 methylation of H3-K4. These studies suggest that Set9-mediated methylation of H3-K4 functions in transcription activation by competing with histone deacetylases and by precluding H3-K9 methylation by Suv39h1. These results suggest that the methylation of histone tails can have distinct effects on transcription, depending on its chromosomal location, the combination of posttranslational modifications, and the enzyme (or protein complex) involved in the particular modification (Nishioka, 2002).

The chromodomain of the HP1 family of proteins recognizes histone tails with specifically methylated lysines. Structural, energetic, and mutational analyses are presented of the complex between the Drosophila HP1 chromodomain and the histone H3 tail with a methyllysine at residue 9, a modification associated with epigenetic silencing. The histone tail inserts as a beta strand, completing the beta-sandwich architecture of the chromodomain. The methylammonium group is caged by three aromatic side chains, whereas adjacent residues form discerning contacts with one face of the chromodomain. Comparison of dimethyl- and trimethyllysine-containing complexes suggests a role for cation-pi and van der Waals interactions, with trimethylation slightly improving the binding affinity (Jacobs, 2002).

Specific modifications to histones are essential epigenetic markers---heritable changes in gene expression that do not affect the DNA sequence. Methylation of lysine 9 in histone H3 is recognized by heterochromatin protein 1 (HP1), which directs the binding of other proteins to control chromatin structure and gene expression. HP1 uses an induced-fit mechanism for recognition of this modification, as revealed by the structure of its chromodomain bound to a histone H3 peptide dimethylated at Nzeta of lysine 9. The binding pocket for the N-methyl groups is provided by three aromatic side chains, Tyr21, Trp42 and Phe45, which reside in two regions that become ordered on binding of the peptide. The side chain of Lys9 is almost fully extended and surrounded by residues that are conserved in many other chromodomains. The QTAR peptide sequence preceding Lys9 makes most of the additional interactions with the chromodomain, with HP1 residues Val23, Leu40, Trp42, Leu58 and Cys60 appearing to be a major determinant of specificity by binding the key buried Ala7. These findings predict which other chromodomains will bind methylated proteins and suggest a motif that they recognize (Nielsen, 2002).

This study provides evidence that H3-K9 methylation and DNA methylation systems can synergize to regulate silenced chromatin domains at major and minor satellite repeats in mammals. Histone H3 lysine 9 (H3-K9) methylation and DNA methylation are characteristic hallmarks of mammalian heterochromatin. H3-K9 methylation is a prerequisite for DNA methylation in Neurospora crassa and Arabidopsis thaliana. Currently, it is unknown whether a similar dependence exists in mammalian organisms. A physical and functional link is demonstrated between the Suv39h-HP1 histone methylation system and DNA methyltransferase 3b (Dnmt3b) in mammals. Whereas in wild-type cells Dnmt3b interacts with HP1alpha and is concentrated at heterochromatic foci, it fails to localize to these regions in Suv39h double null (dn) mouse embryonic stem (ES) cells. Consistently, the Suv39h dn ES cells display an altered DNA methylation profile at pericentric satellite repeats, but not at other repeat sequences. In contrast, H3-K9 trimethylation at pericentric heterochromatin is not impaired in Dnmt1 single- or Dnmt3a/Dnmt3b double-deficient ES cells. Pericentric heterochromatin is not transcriptionally inert and can give rise to transcripts spanning the major satellite repeats. In conclusion, these data demonstrate an evolutionarily conserved pathway between histone H3-K9 methylation and DNA methylation in mammals. While the Suv39h HMTases are required to direct H3-K9 trimethylation and Dnmt3b-dependent DNA methylation at pericentric repeats, DNA methylation at centromeric repeats occurs independent of Suv39h function. Thus, these data also indicate a more complex interrelatedness between histone and DNA methylation systems in mammals. Both methylation systems are likely to be important in reinforcing the stability of heterochromatic subdomains and thereby in protecting genome integrity (Lehnertz, 2003).

The use of immunofluorescence analyses and DNA methylation profiles in wt and mutant murine ES cells has demonstrated that Suv39h-mediated H3-K9 trimethylation can direct Dnmt3b to major satellite repeats present in pericentric heterochromatin. In addition, co-IP data suggest that Dnmt3b and Dnmt3a are part of a repressive complex that is targeted to methylated H3-K9 positions via HP1α and HP1β (Lehnertz, 2003).

The Suv39h-dependent DNA methylation defect at major satellites was only detectable upon digestion with MaeII and reflects a similar deficiency in heterochromatic DNA methylation as compared to Dnmt3a/Dnmt3b mutant ES cells. In contrast, genomic DNA prepared from Dnmt1-deficient ES cells displayed methylation defects that were observed after either MaeII or HpaII digestion. Sequence analyses identified no apparent HpaII sites within the 234 bp major satellite repeat unit, suggesting that they may be interspersed between satellite repeats or present at other repetitive sequences, which together comprise the large blocks of pericentric heterochromatin. It is currently unresolved whether DNA methylation at these HpaII sites is initiated by Dnmt3a/Dnmt3b in an Suv39h-dependent manner and then maintained by Dnmt1, or whether there may be differential target sensitivities of DNMTs to certain DNA sequences or even to chromosomal subdomains (Lehnertz, 2003).

In human embryonic carcinoma cell lines (Tera-1 and NCCIT), Dnmt3b also interacts with HP1α. Mutational inactivation of DNMT3b causes the rare ICF syndrome, which is in part characterized by extensive cytosine demethylation and chromosomal instabilities at pericentric heterochromatin containing satellite 2 and 3 repeats. Since human chromosomes display dense H3-K9 trimethylation at these satellites, it is anticipated that SUV39H-dependent histone methylation may also direct pericentric DNA methylation in humans (Lehnertz, 2003).

In contrast to the major satellites, Dnmt3b-dependent DNA methylation at minor satellites is not impaired in Suv39h dn ES cells. Recent immunofluorescence and chromatin immunoprecipitation analyses with highly specific antibodies that discriminate H3-K9 di- and H3-K9 trimethylation show that the histone methylation pattern differs between centromeric and pericentric heterochromatin. For example, centromeric minor satellites are enriched for H3-K9 dimethylation in both wt and Suv39h dn ES cells, whereas pericentric major satellites display selective H3-K9 trimethylation in an Suv39h-dependent manner. It is possible that Dnmt3b targeting to minor satellites could involve H3-K9 dimethylation, mediated by an HMTase that is distinct from the Suv39h enzymes and maintains a local concentration of HP1α or HP1β. This interpretation would be consistent with the robust HMTase activity associated with Dnmt3b in Suv39h-deficient nuclear extracts (Lehnertz, 2003).

In contrast to Dnmt3b, the pericentric localization of Dnmt1 and the more complete loss of DNA methylation at major satellites observed in Dnmt1 null versus Suv39h dn ES cells indicates that recruitment of Dnmt1 to pericentric regions also occurs independent of the function of the Suv39h HMTases. Indeed, Dnmt1 has been shown to be targeted via PCNA to major satellites during late replication. Similarly, in A. thaliana, maintenance of CpG methylation by the Dnmt1 homolog MET1 is not impaired in mutants of the KYP H3-K9 HMTase. These findings suggests that replication-coupled propagation of CpG methylation may be independent of H3-K9 methylation (Lehnertz, 2003).

Although H3-K9 methylation can be maintained at silent centromeric repeats in CpG (Dnmt1 homolog met1) or CpNpG (cmt3) DNA methylation-deficient mutants in A. thaliana, these studies also show that loss of DNA methylation can feed back on the persistence of H3-K9 methylation patterns if there is significant derepression of silenced loci, e.g., as observed with aberrant transcriptional activity of retro-transposons that had integrated into pericentric domains. Similarly, treatment of human cancer cell lines with the DNA-demethylating compound 5-aza-2'-deoxycytidine (5-aza-dC) results in transcriptional reactivation and reversal of repressive histone methyl marks at silenced tumor suppressor and cell cycle genes. In particular, 5-aza-dC induces a reduction in H3-K9 dimethylation while simultaneously increasing the levels for H3-K4 dimethylation and H3-K9 acetylation. RT-PCR analysis detects a weak upregulation of mouse major satellite transcripts in total RNA prepared from Suv39h dn ES cells, but not from the different DNMT-deficient cells However, pericentric heterochromatin remained underrepresented for H3-K4 dimethylation in wt and in all mutant ES cell lines examined. Together, these observations support a model in which reduced DNA methylation can alter histone methylation marks only if transcriptional reactivation is significantly induced. Since DNA methylation at major satellites is not fully lost in Dnmt1 null or Dnmt3a/Dnmt3b double-deficient ES cells, it remains possible that the complete absence of DNA methylation (e.g., in a triple-deficient Dnmt1/Dnmt3a/Dnmt3b ES cell line) would more drastically affect transcriptional activity and H3-K9 trimethylation patterns at the pericentric satellite repeats (Lehnertz, 2003).

Thus H3-K9 methylation and DNA methylation systems can synergize to regulate silenced chromatin domains at major and minor satellite repeats in mammals. Silencing is likely to be reinforced by binding of the methyl-CpG binding protein MeCP2 and associated histone deacetylases (HDACs) and HMTases. The selective impairment of Suv39h-dependent DNA methylation at the major satellites is intriguingly reminiscent of the recently discovered potential of DNA repeats to target H3-K9 methylation to a chromatin region via the generation of small double-stranded RNAs. Since transcripts spanning the mouse major satellites have been observed, it is conceivable that 'small heterochromatic' RNAs generated from these transcripts may guide recruitment of the Suv39h HMTases to direct H3-K9 trimethylation and, in turn, DNA methylation to pericentric heterochromatin. Ongoing studies are aimed to delineate the molecular mechanism(s) connecting these major epigenetic pathways (Lehnertz, 2003).

In mammalian cells, as in Schizosaccharomyces pombe and Drosophila, HP1 proteins bind histone H3 tails methylated on lysine 9 (K9). However, whereas K9-methylated H3 histones are distributed throughout the nucleus, HP1 proteins are enriched in pericentromeric heterochromatin. This observation suggests that the methyl-binding property of HP1 may not be sufficient for its heterochromatin targeting. The association of HP1alpha with pericentromeric heterochromatin is shown to depend not only on its methyl-binding chromo domain but also on an RNA-binding activity present in the hinge region of the protein that connects the conserved chromo and chromoshadow domains. These data suggest the existence of complex heterochromatin binding sites composed of methylated histone H3 tails and RNA, with each being recognized by a separate domain of HP1alpha (Muchardt, 2002).

Cellular senescence is an extremely stable form of cell cycle arrest that limits the proliferation of damaged cells and may act as a natural barrier to cancer progression. A distinct heterochromatic structure is descibed that accumulates in senescent human fibroblasts, that is designated senescence-associated heterochromatic foci (SAHF). SAHF formation coincides with the recruitment of heterochromatin proteins and the retinoblastoma (Rb) tumor suppressor to E2F-responsive promoters and is associated with the stable repression of E2F target genes. Notably, both SAHF formation and the silencing of E2F target genes depend on the integrity of the Rb pathway and do not occur in reversibly arrested cells. These results provide a molecular explanation for the stability of the senescent state, as well as new insights into the action of Rb as a tumor suppressor (Narita, 2003).

SAHFs are observed in interphase nuclei and contain the heterochromatin-associated proteins H3 methylated on lysine 9 (K9M-H3) and HP1, exclude histones found in euchromatin (e.g., K9Ac-H3 and K4M-H3), and are not sites of active transcription. SAHFs are distinct from pericentric heterochromatin, and their appearance is accompanied by an increase in HP1 incorporation into senescent chromatin and an enhanced resistance of senescent DNA to nuclease digestion (Narita, 2003).

SAHF formation requires an intact Rb pathway, since expression of E1A, or inactivation of either p16INK4a or Rb, can prevent their appearance. During the initial phases of senescence, Rb might control the nucleation of heterochromatin at specific sites throughout the genome, which then spreads by the action of histone methyltransferases and recruitment of HP1 proteins. HP1 proteins have the capacity to dimerize and may interact to form higher order chromatin structures once a critical mass has been reached. A similar pattern of nucleation and spreading occurs during silencing of the mating type locus in S. pombe, position effect variegation in Drosophila, and X inactivation in mammalian cells, although HP1 proteins do not accumulate on the inactive X. Importantly, SAHF formation correlates precisely with cell cycle exit and the silencing of E2F target genes (Narita, 2003).

Much of what is known concerning the regulation of E2F activity comes from studies examining cell cycle transitions into and out of a quiescent state. These transitions are controlled in a reversible manner, in part, by the competing action of HATs and HDACs on the histones of E2F target promoters. This study compares the physical state and regulation of E2F target genes in quiescent and senescent cells. In both cell states, the amount of K9-aceylated histone H3 that associates with E2F target promoters declines, consistent with the downregulation of transcription that accompanies cell cycle exit. However, in senescent IMR90 cells, histone H3 acetylation is ultimately replaced by methylation at lysine 9, an apparently irreversible modification that prevents acetylation by HATs and is barely observed on E2F-responsive promoters in quiescent cells. Methylated lysine 9 forms a docking site for HP1 proteins and, accordingly, HP1gamma preferentially associates with E2F target promoters in senescent cells. These modifications are predicted to form a 'lock' on the transcription of E2F responsive promoters, making them less accessible to the transcription machinery. Accordingly, several E2F-responsive genes in senescent cells are stably repressed and insensitive to enforced E2F expression relative to quiescent cells. Although it remains to be determined whether every E2F target gene behaves as those studied here, their transition to a heterochromatin-like organization may contribute to the insensitivity of senescent cells to mitogenic signals and the apparent irreversibility of the senescence process (Narita, 2003).

SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation

In contrast to stably repressive, constitutive heterochromatin and stably active, euchromatin, facultative heterochromatin has the capacity to alternate between repressive and activated states of transcription1. As such, it is an instructive source to understand the molecular basis for changes in chromatin structure that correlate with transcriptional status. Sirtuin 1 (SIRT1) and suppressor of variegation 3-9 homologue 1 (SUV39H1) are among the enzymes responsible for chromatin modulations associated with facultative heterochromatin formation. SUV39H1 is the principal enzyme responsible for the accumulation of histone H3 containing a tri-methyl group at its lysine 9 position (H3K9me3) in regions of heterochromatin. SIRT1 is an NAD+-dependent deacetylase that targets histone H4 at lysine 16, and through an unknown mechanism facilitates increased levels of H3K9me3. This study shows that the mammalian histone methyltransferase SUV39H1 is itself targeted by the histone deacetylase SIRT1 and that SUV39H1 activity is regulated by acetylation at lysine residue 266 in its catalytic SET domain. SIRT1 interacts directly with, recruits and deacetylates SUV39H1, and these activities independently contribute to elevated levels of SUV39H1 activity resulting in increased levels of the H3K9me3 modification. Loss of SIRT1 greatly affects SUV39H1-dependent H3K9me3 and impairs localization of heterochromatin protein 1. These findings demonstrate a functional link between the heterochromatin-related histone methyltransferase SUV39H1 and the histone deacetylase SIRT1 (Vaguero, 2007).

Histone methyltransferase activity associated with a human multiprotein complex containing the Enhancer of Zeste protein

Enhancer of Zeste [E(z)] is a Polycomb-group transcriptional repressor and one of the founding members of the family of SET domain-containing proteins. Several SET-domain proteins possess intrinsic histone methyltransferase (HMT) activity. However, recombinant E(z) protein was found to be inactive in a HMT assay. A multiprotein E(z) complex has been isolated from humans that contains extra sex combs, suppressor of zeste-12 [Su(z)12], and the histone binding proteins RbAp46/RbAp48. This complex, which has been termed Polycomb repressive complex (PRC) 2, possesses HMT activity with specificity for Lys 9 (K9) and Lys 27 (K27) of histone H3. The HMT activity of PRC2 is dependent on an intact SET domain in the E(z) protein. It is hypothesized that transcriptional repression by the E(z) protein involves methylation-dependent recruitment of PRC1. The presence of Su(z)12, a strong suppressor of position effect variegation, in PRC2 suggests that PRC2 may play a widespread role in heterochromatin-mediated silencing (Kuzmichev, 2002).

The polypeptide composition of the PRC2, specifically the presence of Su(z)12, suggests that PRC2 plays a more general role in transcriptional silencing outside of the repression of HOX genes. Su(z)12 is a protein with dual PcG and Su(var) functions, and this, therefore, suggests that PRC2 has functions other than homeotic gene repression and, in fact, may play a more general role in heterochromatin-mediated silencing. The observation that human E(z) can function as an inducer of silencing in yeast and as an enhancer of PEV in Drosophila supports this notion. It is speculated that the requirement for E(z) and ESC during early embryonic development reflects its function in general transcriptional silencing. The multifunctional nature of both the E(z) and Su(z)12 proteins suggests that they may also display biochemical heterogeneity. For example, the heterogeneous elution profile of E(z) on various columns suggests that E(z) exists in several distinct complexes (Kuzmichev, 2002).

Purified PRC2 displayed specificity for K9 and K27 of the histone H3 tail. The complex, under the conditions of the assays used, displayed a strong preference for K27. However, when the H3-tail was used as a GST-fusion protein, PRC2 displayed apparently equal specificity for K9 and K27. Analyses of the amino acid sequence in which these lysines are embedded shows a great deal of conservation. K9 is present within the sequence QTARK9STG, whereas K27 is present within the sequence KAARK27SAP. Therefore, at least two different possibilities can be postulated to account for the specificity observed. In one case, the specificity of PRC2 is relaxed in vitro, under the assay conditions used, and the methylation of K9 is nonspecific because of the sequence similarity of the residues within which K9 resides. An apparently similar situation was observed in studies analyzing the specificity of the histone methyltransferase G9a, which biochemically behaves as a H3-histone methyltransferase that preferentially targets K9 and, to much lower levels, K27. In vivo, however, G9a clearly targets H3-K9: whether or not the extent of H3-K27 methylation is decreased in G9a-null cells is unknown. A second possibility is that E(z) targets both K9 and K27, but that this is a regulated process such that methylation of K9 and/or K27 is modulated by factors that associate with E(z) and/or by other modifications existing in the nucleosome. This second possibility is favored based on the following observations. First, the E(z) protein can be considered to be a PcG as well as a TrxG. Not surprisingly, the analyses demonstrate that E(z) is present in distinct complexes. One of the complexes containing E(z) is PRC2; however; this complex also includes Su(z)12. Su(z)12 is a polypeptide that has been found in genetic analyses to regulate the expression of the HOX genes, but loss of function of Su(z)12 suppresses PEV. Therefore, the presence of Su(z)12 in PRC2 may regulate the methylation sites within the histone H3 tail (Kuzmichev, 2002).

Methylation of histone H3-K9 was shown to be an essential step in the establishment of inactive X chromosome. H3-Lys 9 methylation of the inactive X chromosome is not mediated by Suv39 or by G9a. Studies have demonstrated that the imprinted inactivation of the X chromosome in females is lost in mutant mice lacking eed (the mammalian homolog of ESC). Moreover, studies have also demonstrated that during imprinted X inactivation, the mammalian ESC-E(z) complex is localized to the inactive X chromosome in a mitotically stable manner. It is speculated, in light of the accumulated data, that H3-K9 methylation of the inactive X chromosome might be mediated by E(z) within PRC2 or a PRC2-like complex. Importantly, however, the function of methylation of histone H3 at K27 has not been analyzed in the establishment and/or maintenance of the inactive X chromosome. In light of the results discussed above, it is postulated that methylation of H3-K27 may also be important in the process of X inactivation (Kuzmichev, 2002).

It is proposed that the role of E(z) HMT activity in the repression of homeotic gene expression is to establish a binding site for other PcG proteins. It is suggested that PRC2 is recruited to the HOX gene cluster by a transiently acting repressor, for example, through an EED-YY1/Pho interaction or an RbAp46/48-HDAC/dMi2/Hb interaction. Once recruited, PRC2 methylates K27 on histone H3, and this mark recruits PC1. The PC1 protein can convert this mark into a permanently repressed state through methylation of K9 through the recruitment of the Su(var)3-9 H3-K9-specific histone methyltransferase and/or the recruitment of PRC1. Alternatively and/or additionally, PC1 may stimulate the H3-K9 HMT activity of PRC2. This hypothesis is supported by studies demonstrating that trimethylation of K27 is necessary for binding of PC1 to an H3 tail peptide. These findings are in full agreement with studies demonstrating loss of chromosome binding for several PRC1 components upon inactivation of E(z). Interestingly, immunolocalization experiments using antibodies specific for methylated H3-K9 suggest that almost all of the H3-K9 methylation is concentrated in the chromocenter of Drosophila polytene chromosomes, with almost no staining detectable on the chromosomal arms. In contrast, E(z) and other PcG proteins, with subnuclear localization that is regulated by E(z), bind only to discrete bands along the arms of polytene chromosomes. These observations suggest that methylation at K27, rather than methylation at K9, is more likely to establish a binding site for the PC1 protein. This may explain why methylation of K9 alone was not sufficient to allow PC1 to recognize specifically the H3 tail in vitro. The observed PC1 binding was independent of DNA. However, repression of HOX genes in vivo is dependent on PRE. From these results, it must be concluded that although methylation of the H3 tail is important in creating a recognition site for PC1 binding, stable and specific binding must require additional factors and or modifications. A likely candidate is a nucleosome on the PRE with the histone H3-tail methylated at position 27 (Kuzmichev, 2002).

The presence of the RbAp46 and RbAp48 proteins in the ESC-E(z) complex may be important for several reasons. First, these histone-binding proteins are often found in complexes with enzymes involved in the covalent modification of histones. For example, RbAp46 is essential for substrate recognition by, and enzymatic activity of, the histone acetyltransferase enzyme Hat1. Therefore, it is speculated that the inability to detect HMT activity in preparations of recombinant E(z) protein is owing, in part, to the lack of the RbAp46/RbAp48. Another implication of the presence of RbAp proteins in the E(z) complex is that they might facilitate interaction with HDACs. During development, GAP proteins facilitate repression of the HOX genes. GAP proteins, such as Hunchback, are short-lived. Hunchback represses HOX genes by recruiting the Drosophila homolog of human Mi-2 protein, a constituent of the NuRD complex which also contains HDACs 1 and 2 and RbAp46/RpAp48. An interesting possibility is that HDACs or RbAp proteins initially recruited by Hunchback can later recruit PRC2 containing HMT activity via interaction with E(z). This may constitute a switch from short-term to long-term repression (Kuzmichev, 2002).

In support of this hypothesis, PRC2 contains E(z) and RbAp proteins. In addition, there is strong experimental evidence for an interaction between HDACs and E(z). One function of the E(z)-HDAC interaction is to deacetylate histones so that the E(z)-containing complex can methylate them. A similar mechanism was found to operate in yeast, in which methylation of H3-K9 by Clr4 requires deacetylation of H3-K9 and Lys 14 (K14) by Clr6 and Clr3, respectively. A similar mechanism is likely to operate in higher eukaryotes because acetylation and methylation are mutually exclusive marks, and methylation of H3-K9 by Suv39h1 requires deacetylation of this residue. The findings demonstrating two distinct ESC-E(z) complexes, one of which coelutes with HDAC1, raises the possibility that the PRC2 can transiently associate with an HDAC complex. This observation raises the possibility that PRC2 HDAC1 may be a highly-specialized complex dedicated to the methylation of H3-K27, which apparently is not acetylated in vivo in higher eukaryotes. Therefore, it is possible that these two different ESC-E(z) multiprotein complexes establish different marks on the histone H3 tail (Kuzmichev, 2002).

Polycomb group protein Ezh2 is an essential epigenetic regulator of embryonic development in mice, but its role in the adult organism is unknown. High expression of Ezh2 in developing murine lymphocytes suggests Ezh2 involvement in lymphopoiesis. Using Cre-mediated conditional mutagenesis, a critical role has been demonstrated for Ezh2 in early B cell development and rearrangement of the immunoglobulin heavy chain gene (Igh). Ezh2 is a key regulator of histone H3 methylation in early B cell progenitors. These data suggest Ezh2-dependent histone H3 methylation as a novel regulatory mechanism controlling Igh rearrangement during early murine B cell development (Su, 2003).

Histone code pathway involving H3 S28 phosphorylation and K27 acetylation activates transcription and antagonizes polycomb silencing

Histone H3 phosphorylation is a critical step that couples signal transduction pathways to gene regulation. To specifically assess the transcriptional regulatory functions of H3 phosphorylation, an in vivo targeting approach was developed and it was found that the H3 kinase MSK1 is a direct and potent transcriptional activator. Targeting of this H3 kinase to the endogenous c-fos promoter is sufficient to activate its expression without the need of upstream signaling. Moreover, targeting MSK1 to the alpha-globin promoter induces H3 S28 phosphorylation and reactivates expression of this polycomb-silenced gene. Importantly, a mechanism was discovered whereby H3 S28 phosphorylation not only displaces binding of the polycomb-repressive complexes, but it also induces a methyl-acetylation switch of the adjacent K27 residue. These findings show that signal transduction activation can directly regulate polycomb silencing through a specific histone code-mediated mechanism (Lau, 2011).

H3 phosphorylation is mostly studied in the context of rapid activation of signal-inducible genes, such as the induction of immediate early (IE) genes upon mitogen or stress stimulation. However, recent studies showed that additional signaling pathways, such as the Toll-like receptor and retinoic acid signaling, also activate MSK1 to regulate non-IE genes. Although many studies have provided excellent correlative data linking this histone modification to transcriptional regulation, they cannot distinguish between direct and indirect effects of H3 phosphorylation. This study shows that the H3 kinase MSK1 is a potent transcription activator when directly targeted to diverse promoters such as the luciferase reporter, the endogenous IE gene c-fos, and the polycomb-silenced α-globin gene. Most studies to date have focused on H3 S10 phosphorylation; however, the current results suggest that phosphorylation of this site alone, mediated by the RSK2 kinase, is not sufficient to directly transactivate the luciferase reporter gene. Instead, it is the induction of H3 S28 phosphorylation that mirrors transcriptional activation. This finding is particularly evident in a ChIP analyses whereby much greater induction of H3S28ph, compared with H3S10ph, was consistently observed at the activated promoters. More importantly, sequential ChIP analyses showed that H3S28ph and H3K27ac/S28ph, but not H3S10ph, are directly associated with the transcription-initiating form of RNAP II (Lau, 2011).

Throughout these studies, a correlation was consistently observed between H3S28ph and transcriptional activation/initiation at the MSK1-targeted genes. However, previous studies also found that H3 S10 phosphorylation at the FOSL1 enhancer and Drosophila heat-shock genes promotes the release of paused RNAP II from a promoter proximal site through the recruitment of the transcription elongation factor P-TEFb. It is possible that H3 S10 phosphorylation facilitates transcriptional elongation of genes that are regulated by polymerase pausing, whereas H3 S28 phosphorylation directly activates transcription at the initiation step. Further studies will be required to distinguish between the roles of H3 phosphorylation at these distinct sites in the transcriptional initiation and elongation steps (Lau, 2011).

To date, little is known about the link between H3 S28 phosphorylation and transcription. ChIP analyses showed that H3S28ph is enriched at IE gene promoters upon their activation. Interestingly, in chicken erythrocytes, H3 S28 phosphorylation preferentially occurs on the transcription-linked H3 variant H3.3, supporting the hypothesis that S28 phosphorylation is functionally linked to transcriptional activation. Originally identified by a H3S10ph peptide pull-down assay, 14-3-3 actually has a much higher binding affinity for the H3S10ph/K14ac di-modified as well as the H3S28ph epitopes. Given the general paradigm that histone modifications recruit modification-specific binding proteins to mediate downstream functions, it is likely that 14-3-3, or additional proteins, bind to the phospho S28 residue to facilitate transcriptional activation. In that regard, how acetylation of K27 might synergistically or antagonistically modulate recruitment of such S28ph binding protein represents yet another potential level of functional cross-talk between histone modifications. The mechanism that couples H3 K27 acetylation and S28 phosphorylation is currently unknown. It is possible that MSK1 and/or its kinase activity recruit an H3 K27 HAT to the promoter of target genes. Alternatively, H3 phosphorylated at S28 may be a better substrate for the H3 K27 HAT. In support of the first scenario, MSK1 was previously shown to coimmunoprecipitate with multiple HATs, including p300 and cAMP response element binding protein (CREB)-binding protein (CBP), which are known to acetylate H3 at K27. As for the second scenario, it has been previously shown that the yeast HAT, Gcn5, preferentially acetylates H3S10ph peptides over the unmodified form. Therefore, H3 K27 HATs may also have a preference for S28-phosphorylated H3 as substrate. These two possibilities are not necessarily mutually exclusive, but further experiments will be required to test these hypotheses (Lau, 2011).

By using the tissue-specific α-globin gene as a model polycomb-regulated gene, this study identified a histone code pathway whereby H3 S28 phosphorylation induces a methyl-acetylation switch on the adjacent K27 residue (see H3 S28 phosphorylation initiates a unique histone code pathway by inducing a methyl-acetylation switch of the adjacent K27 residue). Moreover, this mechanism provides a direct link between signal transduction and polycomb regulation. Activation of polycomb target genes during differentiation is associated with displacement of polycomb group proteins and removal of H3K27me3; however, how this process is regulated is still poorly understood. Recent studies also showed that a switch from methylation to acetylation at H3 K27 often accompanies the activation of these genes. These findings suggest that one way to regulate this switch could be through phosphorylation of the adjacent S28 residue. Given that genome-wide screens showed that many polycomb-regulated genes are downstream of diverse signaling pathways, the finding that H3 S28ph and K27ac are functionally coupled further raises the possibility that signal transduction pathways and activation of polycomb-regulated genes are directly linked through this unique histone code. If so, H3 S28 phosphorylation may have a yet-to-be appreciated function in modulating the epigenome during development and differentiation (Lau, 2011).

MET-2-dependent H3K9 methylation suppresses transgenerational small RNA inheritance

In C. elegans, alterations to chromatin produce transgenerational effects, such as inherited increase in lifespan and gradual loss of fertility. Inheritance of histone modifications can be induced by double-stranded RNA-derived heritable small RNAs. This study shows that the mortal germline phenotype, which is typical of met-2 mutants, defective in H3K9 methylation, depends on HRDE-1, an argonaute that carries small RNAs across generations, and is accompanied by accumulated transgenerational misexpression of heritable small RNAs. It was discovered that MET-2 inhibits small RNA inheritance, and, as a consequence, induction of RNAi in met-2 mutants leads to permanent RNAi responses that do not terminate even after more than 30 generations. Potentiation of heritable RNAi in met-2 animals results from global hyperactivation of the small RNA inheritance machinery. Thus, changes in histone modifications can give rise to drastic transgenerational epigenetic effects, by controlling the overall potency of small RNA inheritance (Lev, 2017).

Evolutionary homologs: Table of contents

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

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