The HP1α–CAF1–SetDB1‐containing complex provides H3K9me1 for Suv39‐mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.     

An HP1 isoform-specific feedback mechanism regulates Suv39h1 activity under stress conditions

The presence of H3K9me3 and heterochromatin protein 1 (HP1) are hallmarks of heterochromatin conserved in eukaryotes. The spreading and maintenance of H3K9me3 is effected by the functional interplay between the H3K9me3-specific histone methyltransferase Suv39h1 and HP1. This interplay is complex in mammals because the three HP1 isoforms, HP1α, β, and γ, are thought to play a redundant role in Suv39h1-dependent deposition of H3K9me3 in pericentric heterochromatin (PCH). Here, we demonstrate that despite this redundancy, HP1α and, to a lesser extent, HP1γ have a closer functional link to Suv39h1, compared to HP1β. HP1α and γ preferentially interact in vivo with Suv39h1, regulate its dynamics in heterochromatin, and increase Suv39h1 protein stability through an inhibition of MDM2-dependent Suv39h1-K87 polyubiquitination. The reverse is also observed, where Suv39h1 increases HP1α stability compared HP1β and γ. The interplay between Suv39h1 and HP1 isoforms appears to be relevant under genotoxic stress. Specifically, loss of HP1α and γ isoforms inhibits the upregulation of Suv39h1 and H3K9me3 that is observed under stress conditions. Reciprocally, Suv39h1 deficiency abrogates stress-dependent upregulation of HP1α and γ, and enhances HP1β levels. Our work defines a specific role for HP1 isoforms in regulating Suv39h1 function under stress via a feedback mechanism that likely regulates heterochromatin formation.     

β-Globin cis-elements determine differential nuclear targeting through epigenetic modifications

Increasing evidence points to nuclear compartmentalization as a contributing mechanism for gene regulation, yet mechanisms for compartmentalization remain unclear. In this paper, we use autonomous targeting of bacterial artificial chromosome (BAC) transgenes to reveal cis requirements for peripheral targeting. Three peripheral targeting regions (PTRs) within an HBB BAC bias a competition between pericentric versus peripheral heterochromatin targeting toward the nuclear periphery, which correlates with increased H3K9me3 across the β-globin gene cluster and locus control region. Targeting to both heterochromatin compartments is dependent on Suv39H-mediated H3K9me3 methylation. In different chromosomal contexts, PTRs confer no targeting, targeting to pericentric heterochromatin, or targeting to the periphery. A combination of fluorescent in situ hybridization, BAC transgenesis, and knockdown experiments reveals that peripheral tethering of the endogenous HBB locus depends both on Suv39H-mediated H3K9me3 methylation over hundreds of kilobases surrounding HBB and on G9a-mediated H3K9me2 methylation over flanking sequences in an adjacent lamin-associated domain. Our results demonstrate that multiple cis-elements regulate the overall balance of specific epigenetic marks and peripheral gene targeting.     

HP1γ links histone methylation marks to meiotic synapsis in mice

During meiosis, specific histone modifications at pericentric heterochromatin (PCH), especially histone H3 tri- and dimethylation at lysine 9 (H3K9me3 and H3K9me2, respectively), are required for proper chromosome interactions. However, the molecular mechanism by which H3K9 methylation mediates the synapsis is not yet understood. We have generated a Cbx3-deficient mouse line and performed comparative analysis on Suv39h1/h2-, G9a- and Cbx3-deficient spermatocytes. This study revealed that H3K9me2 at PCH depended on Suv39h1/h2-mediated H3K9me3 and its recognition by the Cbx3 gene product HP1γ. We further found that centromere clustering and synapsis were commonly affected in G9a- and Cbx3-deficient spermatocytes. These genetic observations suggest that HP1γ/G9a-dependent PCH-mediated centromere clustering is an axis for proper chromosome interactions during meiotic prophase. We propose that the role of the HP1γ/G9a axis is to retain centromeric regions of unpaired homologous chromosomes in close alignment and facilitate progression of their pairing in early meiotic prophase. This study also reveals considerable plasticity in the interplay between different histone modifications and suggests that such stepwise and dynamic epigenetic modifications may play a pivotal role in meiosis.     

Uncoupling global and fine-tuning replication timing determinants for mouse pericentric heterochromatin

Mouse chromocenters are clusters of late-replicating pericentric heterochromatin containing HP1 bound to trimethylated lysine 9 of histone H3 (Me3K9H3). Using a cell-free system to initiate replication within G1-phase nuclei, we demonstrate that chromocenters acquire the property of late replication coincident with their reorganization after mitosis and the establishment of a global replication timing program. HP1 dissociated during mitosis but rebound before the establishment of late replication, and removing HP1 from chromocenters by competition with Me3K9H3 peptides did not result in early replication, demonstrating that this interaction is neither necessary nor sufficient for late replication. However, in cells lacking the Suv39h1,2 methyltransferases responsible for K9H3 trimethylation and HP1 binding at chromocenters, replication of chromocenter DNA was advanced by 10-15% of the length of S phase. Reintroduction of Suv39h1 activity restored the later replication time. We conclude that Suv39 activity is required for the fine-tuning of pericentric heterochromatin replication relative to other late-replicating domains, whereas separate factors establish a global replication timing program during early G1 phase.     

Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability.

Histone H3 lysine 9 methylation has been proposed to provide a major "switch" for the functional organization of chromosomal subdomains. Here, we show that 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.     

Altered telomere homeostasis and resistance to skin carcinogenesis in Suv39h1 transgenic mice

The Suv39h1 and Suv39h2 H3K9 histone methyltransferases (HMTs) have a conserved role in the formation of constitutive heterochromatin and gene silencing. Using a transgenic mouse model system we demonstrate that elevated expression of Suv39h1 increases global H3K9me3 levels in vivo. More specifically, Suv39h1 overexpression enhances the imposition of H3K9me3 levels at constitutive heterochromatin at telomeric and major satellite repeats in primary mouse embryonic fibroblasts. Chromatin compaction is paralleled by telomere shortening, indicating that telomere length is controlled by H3K9me3 density at telomeres. We further show that increased Suv39h1 levels result in an impaired clonogenic potential of transgenic epidermal stem cells and Ras/E1A transduced transgenic primary mouse embryonic fibroblasts. Importantly, Suv39h1 overexpression in mice confers resistance to a DMBA/TPA induced skin carcinogenesis protocol that is characterized by the accumulation of activating H-ras mutations. Our results provide genetic evidence that Suv39h1 controls telomere homeostasis and mediates resistance to oncogenic stress in vivo. This identifies Suv39h1 as an interesting target to improve oncogene induced senescence in premalignant lesions.     

Epigenetic regulation of mammalian pericentric heterochromatin in vivo by HP1

We developed a model system whereby HP1 can be targeted to pericentric heterochromatin in ES cells lacking Suv(3)9h1/2 histone methyltransferase (HMTase) activities. HP1 so targeted can reconstitute tri-methylated lysine 9 of histone H3 (Me(3)K9H3) and tri-methylated lysine 20 of histone H4 (Me(3)K20H4) at pericentric heterochromatin, indicating that HP1 can regulate the distribution of these histone modifications in vivo. Both homo- and hetero-typic interactions between the HP1 isotypes were demonstrated in vivo as were HP1 interactions with the ESET/SETDB1 HMTase and the ATRX chromatin remodelling enzyme. We conclude that HP1 not only "deciphers" the histone code but can also "encode it".     

Methylation of Lysine 9 in Histone H3 Directs Alternative Modes of Highly Dynamic Interaction of Heterochromatin Protein hHP1β with the Nucleosome

Binding of heterochromatin protein 1 (HP1) to the histone H3 lysine 9 trimethylation (H3K9me3) mark is a hallmark of establishment and maintenance of heterochromatin. Although genetic and cell biological aspects have been elucidated, the molecular details of HP1 binding to H3K9me3 nucleosomes are unknown. Using a combination of NMR spectroscopy and biophysical measurements on fully defined recombinant experimental systems, we demonstrate that H3K9me3 works as an on/off switch regulating distinct binding modes of hHP1β to the nucleosome. The methyl-mark determines a highly flexible and very dynamic interaction of the chromodomain of hHP1β with the H3-tail. There are no other constraints of interaction or additional multimerization interfaces. In contrast, in the absence of methylation, the hinge region and the N-terminal tail form weak nucleosome contacts mainly with DNA. In agreement with the high flexibility within the hHP1β-H3K9me3 nucleosome complex, the chromoshadow domain does not provide a direct binding interface. Our results report the first detailed structural analysis of a dynamic protein-nucleosome complex directed by a histone modification and provide a conceptual framework for understanding similar interactions in the context of chromatin.     

Role of Swi6/HP1 self-association-mediated recruitment of Clr4/Suv39 in establishment and maintenance of heterochromatin in fission yeast

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.     

PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse

Imprinted genes are important in development and their allelic expression is mediated by imprinting control regions (ICRs). On their DNA-methylated allele, ICRs are marked by trimethylation at H3 Lys 9 (H3K9me3) and H4 Lys 20 (H4K20me3), similar to pericentric heterochromatin. Here, we investigate which histone methyltransferases control this methylation of histone at ICRs. We found that inactivation of SUV4-20H leads to the loss of H4K20me3 and increased levels of its substrate, H4K20me1. H4K20me1 is controlled by PR-SET7 and is detected on both parental alleles. The disruption of SUV4-20H or PR-SET7 does not affect methylation of DNA at ICRs but influences precipitation of H3K9me3, which is suggestive of a trans-histone change. Unlike at pericentric heterochromatin, however, H3K9me3 at ICRs does not depend on SUV39H. Our data show not only new similarities but also differences between ICRs and heterochromatin, both of which show constitutive maintenance of methylation of DNA in somatic cells.     

Partitioning and plasticity of repressive histone methylation states in mammalian chromatin

Methylation of position-specific lysine residues in histone N termini is a central modification for regulating epigenetic transitions in chromatin. Each methylatable lysine residue can exist in a mono-, di-, or trimethylated state, thereby extending the indexing potential of this particular modification. Here, we examine all possible methylation states for histone H3 lysine 9 (H3-K9) and lysine 27 (H3-K27) in mammalian chromatin. Using highly specific antibodies together with quantitative mass spectrometry, we demonstrate that pericentric heterochromatin is selectively enriched for H3-K27 monomethylation and H3-K9 trimethylation. This heterochromatic methylation profile is dependent on the Suv39h histone methyltransferases (HMTases) but independent of the euchromatic G9a HMTase. In Suv39h double null cells, pericentric heterochromatin is converted to alternative methylation imprints and accumulates H3-K27 trimethylation and H3-K9 monomethylation. Our data underscore the selective presence of distinct histone lysine methylation states in partitioning chromosomal subdomains but also reveal a surprising plasticity in propagating methylation patterns in eukaryotic chromatin.     

Mammalian ChlR1 has a role in heterochromatin organization

The ChlR1 DNA helicase, encoded by DDX11 gene, which is responsible for Warsaw breakage syndrome (WABS), has a role in sister-chromatid cohesion. In this study, we show that human ChlR1 deficient cells exhibit abnormal heterochromatin organization. While constitutive heterochromatin is discretely localized at perinuclear and perinucleolar regions in control HeLa cells, ChlR1-depleted cells showed dispersed localization of constitutive heterochromatin accompanied by disrupted centromere clustering. Cells isolated from Ddx11^−/− embryos also exhibited diffuse localization of centromeres and heterochromatin foci. Similar abnormalities were found in HeLa cells depleted of combinations of HP1α and HP1β. Immunofluorescence and chromatin immunoprecipitation showed a decreased level of HP1α at pericentric regions in ChlR1-depleted cells. Trimethyl-histone H3 at lysine 9 (H3K9-me3) was also modestly decreased at pericentric sequences. The abnormality in pericentric heterochromatin was further supported by decreased DNA methylation within major satellite repeats of Ddx11^−/− embryos. Furthermore, micrococcal nuclease (MNase) assay revealed a decreased chromatin density at the telomeres. These data suggest that in addition to a role in sister-chromatid cohesion, ChlR1 is also involved in the proper formation of heterochromatin, which in turn contributes to global nuclear organization and pleiotropic effects.     

Histone H4K20 tri‐methylation at late‐firing origins ensures timely heterochromatin replication

Among other targets, the protein lysine methyltransferase PR‐Set7 induces histone H4 lysine 20 monomethylation (H4K20me1), which is the substrate for further methylation by the Suv4‐20h methyltransferase. Although these enzymes have been implicated in control of replication origins, the specific contribution of H4K20 methylation to DNA replication remains unclear. Here, we show that H4K20 mutation in mammalian cells, unlike in Drosophila, partially impairs S‐phase progression and protects from DNA re‐replication induced by stabilization of PR‐Set7. Using Epstein–Barr virus‐derived episomes, we further demonstrate that conversion of H4K20me1 to higher H4K20me2/3 states by Suv4‐20h is not sufficient to define an efficient origin per se, but rather serves as an enhancer for MCM2‐7 helicase loading and replication activation at defined origins. Consistent with this, we find that Suv4‐20h‐mediated H4K20 tri‐methylation (H4K20me3) is required to sustain the licensing and activity of a subset of ORCA/LRWD1‐associated origins, which ensure proper replication timing of late‐replicating heterochromatin domains. Altogether, these results reveal Suv4‐20h‐mediated H4K20 tri‐methylation as a critical determinant in the selection of active replication initiation sites in heterochromatin regions of mammalian genomes.