Zhuqiang Zhang

Dense Chromatin Activates Polycomb Repressive Complex 2 to Regulate H3 Lysine 27 Methylation

Maintaining Repression The Polycomb Repressive Complex 2 (PRC2) plays a critical role in gene silencing in metazoans, methylating histone H3 on lysine 27 (H3K27) to generate a repressive chromatin mark. The catalytic subunit E(z)/Ezh2 requires the presence of two other subunits—ESC/EED and Su(z)12—for enzyme activity. Yuan et al. (p. 971; see the Perspective by Pirrotta) show that both a fragment of the histone H3 N-terminal tail, and histone H1 stimulated PRC2 enzyme activity on poor, low-density chromatin substrates, indicating that that PRC2 is regulated by the density and compaction states of chromatin. The histone H3 fragment binds to the Su(z)12 subunit of PRC2 to stimulate E(z)/Ezh2. Local chromatin compaction preceded establishment of histone H3K27 methylation indicating how PRC2 might maintain the repressed state. The density and compaction state of chromatin directly regulates the activity of a transcription repressor protein complex. Polycomb repressive complex 2 (PRC2)–mediated histone H3 lysine 27 (H3K27) methylation is vital for Polycomb gene silencing, a classic epigenetic phenomenon that maintains transcriptional silencing throughout cell divisions. We report that PRC2 activity is regulated by the density of its substrate nucleosome arrays. Neighboring nucleosomes activate the PRC2 complex with a fragment of their H3 histones (Ala31 to Arg42). We also identified mutations on PRC2 subunit Su(z)12, which impair its binding and response to the activating peptide and its ability in establishing H3K27 trimethylation levels in vivo. In mouse embryonic stem cells, local chromatin compaction occurs before the formation of trimethylated H3K27 upon transcription cessation of the retinoic acid–regulated gene CYP26a1. We propose that PRC2 can sense the chromatin environment to exert its role in the maintenance of transcriptional states.

Recognition of H3K9 methylation by GLP is required for efficient establishment of H3K9 methylation, rapid target gene repression, and mouse viability.

GLP and G9a are major H3K9 dimethylases and are essential for mouse early embryonic development. GLP and G9a both harbor ankyrin repeat domains that are capable of binding H3K9 methylation. However, the functional significance of their recognition of H3K9 methylation is unknown. Here, we report that the histone methyltransferase activities of GLP and G9a are stimulated by neighboring nucleosomes that are premethylated at H3K9. These stimulation events function in cis and are dependent on the H3K9 methylation binding activities of ankyrin repeat domains of GLP and G9a. Disruption of the H3K9 methylation-binding activity of GLP in mice causes growth retardation of embryos, ossification defects of calvaria, and postnatal lethality due to starvation of the pups. In mouse embryonic stem cells (ESCs) harboring a mutant GLP that lacks H3K9me1-binding activity, critical pluripotent genes, including Oct4 and Nanog, display inefficient establishment of H3K9me2 and delayed gene silencing during differentiation. Collectively, our study reveals a new activation mechanism for GLP and G9a that plays an important role in ESC differentiation and mouse viability.

Anp32e, a higher eukaryotic histone chaperone directs preferential recognition for H2A.Z

H2A.Z is a highly conserved histone variant in all species. The chromatin deposition of H2A.Z is specifically catalyzed by the yeast chromatin remodeling complex SWR1 and its mammalian counterpart SRCAP. However, the mechanism by which H2A.Z is preferentially recognized by non-histone proteins remains elusive. Here we identified Anp32e, a novel higher eukaryote-specific histone chaperone for H2A.Z. Anp32e preferentially associates with H2A.Z-H2B dimers rather than H2A-H2B dimers in vitro and in vivo and dissociates non-nucleosomal aggregates formed by DNA and H2A-H2B. We determined the crystal structure of the Anp32e chaperone domain (186-232) in complex with the H2A.Z-H2B dimer. In this structure, the region containing Anp32e residues 214-224, which is absent in other Anp32 family proteins, specifically interacts with the extended H2A.Z αC helix, which exhibits an unexpected conformational change. Genome-wide profiling of Anp32e revealed a remarkable co-occupancy between Anp32e and H2A.Z. Cells overexpressing Anp32e displayed a strong global H2A.Z loss at the +1 nucleosomes, whereas cells depleted of Anp32e displayed a moderate global H2A.Z increase at the +1 nucleosomes. This suggests that Anp32e may help to resolve the non-nucleosomal H2A.Z aggregates and also facilitate the removal of H2A.Z at the +1 nucleosomes, and the latter may help RNA polymerase II to pass the first nucleosomal barrier.

H3.3-H4 Tetramer Splitting Events Feature Cell-Type Specific Enhancers

Previously, we reported that little canonical (H3.1–H4)2 tetramers split to form “hybrid” tetramers consisted of old and new H3.1–H4 dimers, but approximately 10% of (H3.3–H4)2 tetramers split during each cell cycle. In this report, we mapped the H3.3 nucleosome occupancy, the H3.3 nucleosome turnover rate and H3.3 nucleosome splitting events at the genome-wide level. Interestingly, H3.3 nucleosome turnover rate at the transcription starting sites (TSS) of genes with different expression levels display a bimodal distribution rather than a linear correlation towards the transcriptional activity, suggesting genes are either active with high H3.3 nucleosome turnover or inactive with low H3.3 nucleosome turnover. H3.3 nucleosome splitting events are enriched at active genes, which are in fact better markers for active transcription than H3.3 nucleosome occupancy itself. Although both H3.3 nucleosome turnover and splitting events are enriched at active genes, these events only display a moderate positive correlation, suggesting H3.3 nucleosome splitting events are not the mere consequence of H3.3 nucleosome turnover. Surprisingly, H3.3 nucleosomes with high splitting index are remarkably enriched at enhancers in a cell-type specific manner. We propose that the H3.3 nucleosomes at enhancers may be split by an active mechanism to regulate cell-type specific transcription.

Histone H2A ubiquitination inhibits the enzymatic activity of H3 Lysine 36 methyltransferases

Background: H3K36 methylation antagonizes Polycomb function, but it is not clear whether the reverse is true. Results: H3K36-specific histone methyltransferases display poor enzymatic activities on nucleosome substrates containing H2A ubiquitination, an important Polycomb modification. Conclusion: H3K36-specific histone methyltransferases can respond to chromatin environment. Significance: It provides additional understanding about interplays among chromatin modifications and their roles in transcription regulation. Histone H3 lysine 27 (H3K27) methylation and H2A monoubiquitination (ubH2A) are two closely related histone modifications that regulate Polycomb silencing. Previous studies reported that H3K27 trimethylation (H3K27me3) rarely coexists with H3K36 di- or tri-methylation (H3K36me2/3) on the same histone H3 tails, which is partially controlled by the direct inhibition of the enzymatic activity of H3K27-specific methyltransferase PRC2. By contrast, H3K27 methylation does not affect the catalytic activity of H3K36-specific methyltransferases, suggesting other Polycomb mechanism(s) may negatively regulate the H3K36-specific methyltransferase(s). In this study, we established a simple protocol to purify milligram quantities of ubH2A from mammalian cells, which were used to reconstitute nucleosome substrates with fully ubiquitinated H2A. A number of histone methyltransferases were then tested on these nucleosome substrates. Notably, all of the H3K36-specific methyltransferases, including ASH1L, HYPB, NSD1, and NSD2 were inhibited by ubH2A, whereas the other histone methyltransferases, including PRC2, G9a, and Pr-Set7 were not affected by ubH2A. Together with previous reports, these findings collectively explain the mutual repulsion of H3K36me2/3 and Polycomb modifications.

Structural basis of H2A.Z recognition by SRCAP chromatin-remodeling subunit YL1

Histone variant H2A.Z, a universal mark of dynamic nucleosomes flanking gene promoters and enhancers, is incorporated into chromatin by SRCAP (SWR1), an ATP-dependent, multicomponent chromatin-remodeling complex. The YL1 (Swc2) subunit of SRCAP (SWR1) plays an essential role in H2A.Z recognition, but how it achieves this has been unclear. Here, we report the crystal structure of the H2A.Z-binding domain of Drosophila melanogaster YL1 (dYL1-Z) in complex with an H2A.Z–H2B dimer at 1.9-Å resolution. The dYL1-Z domain adopts a new whip-like structure that wraps over H2A.Z–H2B, and preferential recognition is largely conferred by three residues in loop 2, the hyperacidic patch and the extended αC helix of H2A.Z. Importantly, this domain is essential for deposition of budding yeast H2A.Z in vivo and SRCAP (SWR1)-catalyzed histone H2A.Z replacement in vitro. Our studies distinguish YL1-Z from known H2A.Z chaperones and suggest a hierarchical mechanism based on increasing binding affinity facilitating H2A.Z transfer from SRCAP (SWR1) to the nucleosome.

Preferential Protection of Genetic Fidelity within Open Chromatin by the Mismatch Repair Machinery

Epigenetic systems are well known for the roles they play in regulating the differential expression of the same genome in different cell types. However, epigenetic systems can also directly impact genomic integrity by protecting genetic sequences. Using an experimental evolutionary approach, we studied rates of mutation in the fission yeast Schizosaccharomyces pombe strains that lacked genes encoding several epigenetic regulators or mismatch repair components. We report that loss of a functional mismatch repair pathway in S. pombe resulted in the preferential enrichment of mutations in euchromatin, indicating that the mismatch repair machinery preferentially protected genetic fidelity in euchromatin. This preference is probably determined by differences in the accessibility of chromatin at distinct chromatin regions, which is supported by our observations that chromatin accessibility positively correlated with mutation rates in S. pombe or human cancer samples with deficiencies in mismatch repair. Importantly, such positive correlation was not observed in S. pombe strains or human cancer samples with functional mismatch repair machinery.

Mrg15 stimulates Ash1 H3K36 methyltransferase activity and facilitates Ash1 Trithorax group protein function in Drosophila

Ash1 is a Trithorax group protein that possesses H3K36-specific histone methyltransferase activity, which antagonizes Polycomb silencing. Here we report the identification of two Ash1 complex subunits, Mrg15 and Nurf55. In vitro, Mrg15 stimulates the enzymatic activity of Ash1. In vivo, Mrg15 is recruited by Ash1 to their common targets, and Mrg15 reinforces Ash1 chromatin association and facilitates the proper deposition of H3K36me2. To dissect the functional role of Mrg15 in the context of the Ash1 complex, we identify an Ash1 point mutation (Ash1-R1288A) that displays a greatly attenuated interaction with Mrg15. Knock-in flies bearing this mutation display multiple homeotic transformation phenotypes, and these phenotypes are partially rescued by overexpressing the Mrg15-Nurf55 fusion protein, which stabilizes the association of Mrg15 with Ash1. In summary, Mrg15 is a subunit of the Ash1 complex, a stimulator of Ash1 enzymatic activity and a critical regulator of the TrxG protein function of Ash1 in Drosophila.Ash1 is an H3K36 methyltransferase, Trithorax group protein and is critical in antagonizing Polycomb silencing. Here, the authors purify the Drosophila Ash1 complex and identify Mrg15 and Nurf55 as subunits, finding that Mrg15 is recruited by Ash1 and reinforces Ash1 activity.

Polycomb “polypacks” the chromatin

For decades, researchers have made a tremendous effort to characterize the chromosome organization in the nucleus. With the development of chromosome conformation capture (1) and related techniques, researchers have begun to gain insight at the molecular level into how inter- and intrachromosome contacts are formed. Quantitative measurement of the contact frequencies in metazoans suggested the existence of topologically associating domains (TADs) as the building blocks of chromosome organization (1⇓–3). These domains are often demarcated by distinct patterns of histone modifications. For example, compartments that are enriched with H3K27me3 and have depleted H3K36me3 are signatures of facultative heterochromatin in the human genome. Although the features of chromosome organization have been characterized in several organisms, the principles and mechanisms governing the formation of chromosome organization have remained a mystery. In PNAS, Klocko et al. (4) report that the Polycomb Repressive Complex 2 (PRC2) and H3K27me3 have a role in shaping genome organization in Neurospora crassa . In mammalian and Drosophila cells, chromosomes are segregated into large TADs, and boundaries between adjacent TADs are characterized by enriched insulator protein CTCF occupancy (2, 3). Plants, yeast, and N. crassa lack large local interactive domains in their genomes, which is likely a result of the loss of genes encoding the CTCF protein (5, 6). The most obvious chromosomal contacts in plants and N. crassa genomes are formed between heterochromatin regions. However, H3K9me3 and the constitutive heterochromatin machinery seemed to be dispensable for this contact (5, 6). Fission yeast does not have H3K27 methylation, and its sole H3K9 methyltransferase is needed for proper genome organization (7). This result raises the question of whether H3K27 methylation and facultative heterochromatin contribute to genome structure formation. Klocko et al. (4) first profile chromosome organization with a high-throughput chromosome conformation capture (Hi-C) technique in … [↵][1]1To whom correspondence should be addressed. Email: zhubing{at}ibp.ac.cn. [1]: #xref-corresp-1-1

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing

Significance Regulators essential for facilitating gene silencing are interesting targets of epigenetic studies. Our work describes a regulator, CSE1L, that is essential for the silencing of many endogenous methylated genes. Depletion of CSE1L reactivates these genes without causing DNA demethylation. Interestingly, such reactivation is not due to a direct chromatin role of CSE1L. Instead, it depends on the role of CSE1L in importin-mediated protein nuclear transportation, which is confirmed by similar effects observed in cells depleted of other players in the same protein transportation pathway. Intriguingly, importin-mediated protein nuclear transportation preferentially facilitates gene silencing with specificity for a subset of genes, suggesting that the cargo specificity of protein nuclear import systems may impact the selectivity of gene regulation. Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.