Basis of the H2AK119 specificity of the Polycomb repressive deubiquitinase.

Repression of gene expression by protein complexes of the Polycomb group is a fundamental mechanism that governs embryonic development and cell-type specification1-3. The Polycomb repressive deubiquitinase (PR-DUB) complex removes the ubiquitin moiety from monoubiquitinated histone H2A K119 (H2AK119ub1) on the nucleosome4, counteracting the ubiquitin E3 ligase activity of Polycomb repressive complex 1 (PRC1)5 to facilitate the correct silencing of genes by Polycomb proteins and safeguard active genes from inadvertent silencing by PRC1 (refs. 6-9). The intricate biological function of PR-DUB requires accurate targeting of H2AK119ub1, but PR-DUB can deubiquitinate monoubiquitinated free histones and peptide substrates indiscriminately; the basis for its exquisite nucleosome-dependent substrate specificity therefore remains unclear. Here we report the cryo-electron microscopy structure of human PR-DUB, composed of BAP1 and ASXL1, in complex with the chromatosome. We find that ASXL1 directs the binding of the positively charged C-terminal extension of BAP1 to nucleosomal DNA and histones H3-H4 near the dyad, an addition to its role in forming the ubiquitin-binding cleft. Furthermore, a conserved loop segment of the catalytic domain of BAP1 is situated near the H2A-H2B acidic patch. This distinct nucleosome-binding mode displaces the C-terminal tail of H2A from the nucleosome surface, and endows PR-DUB with the specificity for H2AK119ub1.

Epigenetic regulation of T cells by Polycomb group proteins.

T cells are critical for pathogen elimination, tumor surveillance, and immunoregulation. The development, activation, and differentiation of CD8 and CD4 T lymphocytes are a set of complex and dynamically regulated events that require epigenetic control. The Polycomb group (PcG) proteins are a family of diverse and evolutionarily conserved epigenetic modulators fundamentally involved in several mechanisms of gene regulation. PcG proteins can assemble into distinct repressor complexes, the two most understood being the Polycomb Repressor Complex (PRC)1 and PRC2, which control chromatin structure mainly through posttranslational modifications of histones. In this review, we will summarize the most recent findings regarding the diverse roles performed by PcG proteins in T cell biology. We will focus on PRC1 and PRC2 contribution to the regulation of T cell development in the thymus, CD4 T cell differentiation in helper or regulatory phenotypes and CD8 T cell fate commitment in the context of infections and cancer, highlighting the known mechanisms and knowledge gaps that still need to be addressed.

Polycomb group-mediated histone H2A monoubiquitination in epigenome regulation and nuclear processes.

Histone posttranslational modifications are key regulators of chromatin-associated processes including gene expression, DNA replication and DNA repair. Monoubiquitinated histone H2A, H2Aub (K118 in Drosophila or K119 in vertebrates) is catalyzed by the Polycomb group (PcG) repressive complex 1 (PRC1) and reversed by the PcG-repressive deubiquitinase (PR-DUB)/BAP1 complex. Here we critically assess the current knowledge regarding H2Aub deposition and removal, its crosstalk with PcG repressive complex 2 (PRC2)-mediated histone H3K27 methylation, and the recent attempts toward discovering its readers and solving its enigmatic functions. We also discuss mounting evidence of the involvement of H2A ubiquitination in human pathologies including cancer, while highlighting some knowledge gaps that remain to be addressed.

Analysis of protein-DNA interactions in chromatin by UV induced cross-linking and mass spectrometry.

Protein-DNA interactions are key to the functionality and stability of the genome. Identification and mapping of protein-DNA interaction interfaces and sites is crucial for understanding DNA-dependent processes. Here, we present a workflow that allows mass spectrometric (MS) identification of proteins in direct contact with DNA in reconstituted and native chromatin after cross-linking by ultraviolet (UV) light. Our approach enables the determination of contact interfaces at amino-acid level. With the example of chromatin-associated protein SCML2 we show that our technique allows differentiation of nucleosome-binding interfaces in distinct states. By UV cross-linking of isolated nuclei we determined the cross-linking sites of several factors including chromatin-modifying enzymes, demonstrating that our workflow is not restricted to reconstituted materials. As our approach can distinguish between protein-RNA and DNA interactions in one single experiment, we project that it will be possible to obtain insights into chromatin and its regulation in the future.

Structural basis for histone variant H3tK27me3 recognition by PHF1 and PHF19.

The Polycomb repressive complex 2 (PRC2) is a multicomponent histone H3K27 methyltransferase complex, best known for silencing the Hox genes during embryonic development. The Polycomb-like proteins PHF1, MTF2, and PHF19 are critical components of PRC2 by stimulating its catalytic activity in embryonic stem cells. The Tudor domains of PHF1/19 have been previously shown to be readers of H3K36me3 in vitro. However, some other studies suggest that PHF1 and PHF19 co-localize with the H3K27me3 mark but not H3K36me3 in cells. Here, we provide further evidence that PHF1 co-localizes with H3t in testis and its Tudor domain preferentially binds to H3tK27me3 over canonical H3K27me3 in vitro. Our complex structures of the Tudor domains of PHF1 and PHF19 with H3tK27me3 shed light on the molecular basis for preferential recognition of H3tK27me3 by PHF1 and PHF19 over canonical H3K27me3, implicating that H3tK27me3 might be a physiological ligand of PHF1/19.

Long non-coding RNA-polycomb intimate rendezvous.

The interaction between polycomb-repressive complexes 1/2 (PRC1/2) and long non-coding RNA (lncRNA), such as the X inactive specific transcript Xist and the HOX transcript antisense RNA (HOTAIR), has been the subject of intense debate. While cross-linking, immuno-precipitation and super-resolution microscopy argue against direct interaction of Polycomb with some lncRNAs, there is increasing evidence supporting the ability of both PRC1 and PRC2 to functionally associate with RNA. Recent data indicate that these interactions are in most cases spurious, but nonetheless crucial for a number of cellular activities. In this review, we suggest that while PRC1/2 recruitment by HOTAIR might be direct, in the case of Xist, it might occur indirectly and, at least in part, through the process of liquid-liquid phase separation. We present recent models of lncRNA-mediated PRC1/2 recruitment to their targets and describe potential RNA-mediated roles in the three-dimensional organization of the nucleus.

The Dm-Myb Oncoprotein Contributes to Insulator Function and Stabilizes Repressive H3K27me3 PcG Domains.

Drosophila Myb (Dm-Myb) encodes a protein that plays a key role in regulation of mitotic phase genes. Here, we further refine its role in the context of a developing tissue as a potentiator of gene expression required for proper RNA polymerase II (RNA Pol II) function and efficient H3K4 methylation at promoters. In contrast to its role in gene activation, Myb is also required for repression of many genes, although no specific mechanism for this role has been proposed. We now reveal a critical role for Myb in contributing to insulator function, in part by promoting binding of insulator proteins BEAF-32 and CP190 and stabilizing H3K27me3 Polycomb-group (PcG) domains. In the absence of Myb, H3K27me3 is markedly reduced throughout the genome, leading to H3K4me3 spreading and gene derepression. Finally, Myb is enriched at boundaries that demarcate chromatin environments, including chromatin loop anchors. These results reveal functions of Myb that extend beyond transcriptional regulation.

Genome-Wide Identification, Evolution, and Expression Analysis of RING Finger Gene Family in Solanum lycopersicum.

RING domain proteins generally have E3 ubiquitin ligase activity and are involved in degrading their substrate proteins. The roles of these proteins in growth, development, and responses to different abiotic stresses have been described well in various plant species, but little is available on tomatoes. Here, we identified 474 RING domains in 469 potential proteins encoded in the tomato genome. These RING genes were found to be located in 12 chromosomes and could be divided into 51 and 11 groups according to the conserved motifs outside the RING domain and phylogenetic analysis, respectively. Segmental duplication could be the major driver in the expansion of the tomato RING gene family. Further comparative syntenic analysis suggested that there have been functional divergences of RING genes during plant evolution and most of the RING genes in various species are under negative selection. Expression profiles derived from a transcriptomic analysis showed that most tomato RING genes exhibited tissue-specific expression patterning. Further RT-qPCR validation showed that almost all genes were upregulated by salt treatment, which was consistent with the microarray results. This study provides the first comprehensive understanding of the RING gene family in the tomato genome. Our results pave the way for further investigation of the classification, evolution, and potential functions of the RING domain genes in tomato.

Emerging Roles for Polycomb-Group Proteins in Stem Cells and Cancer.

Polycomb-group (PcG) complexes are multiprotein, evolutionarily conserved epigenetic machineries that regulate stem cell fate decisions and development, and are also implicated in cancer and other maladies. The PcG machinery can be divided into two major complexes: Polycomb repressive complex 1 and 2 (PRC1 and PRC2). Traditionally, PcG complexes have been associated with maintenance of gene repression mainly via histone-modifying activities. However, during the last years, increasing evidence indicates that the PcG complexes can also positively regulate gene transcription and modify non-histone substrates in multiple biological processes, cellular stages, and cancers. In this review, we will illustrate recent findings in PcG-mediated gene regulation, with special focus on the recently described non-classical functions of PcG complexes in stem cells and cancer.

A bidentate Polycomb Repressive-Deubiquitinase complex is required for efficient activity on nucleosomes.

Attachment of ubiquitin to lysine 119 of Histone 2A (H2AK119Ub) is an epigenetic mark characteristic of repressed developmental genes, which is removed by the Polycomb Repressive-Deubiquitinase (PR-DUB) complex. Here we report the crystal structure of the Drosophila PR-DUB, revealing that the deubiquitinase Calypso and its activating partner ASX form a 2:2 complex. The bidentate Calypso-ASX complex is generated by dimerisation of two activated Calypso proteins through their coiled-coil regions. Disrupting the Calypso dimer interface does not affect inherent catalytic activity, but inhibits removal of H2AK119Ub as a consequence of impaired recruitment to nucleosomes. Mutating the equivalent surface on the human counterpart, BAP1, also compromises activity on nucleosomes. Together, this suggests that high local concentrations drive assembly of bidentate PR-DUB complexes on chromatin-providing a mechanistic basis for enhanced PR-DUB activity at specific genomic foci, and the impact of distinct classes of PR-DUB mutations in tumorigenesis.

Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent.

Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

Pho dynamically interacts with Spt5 to facilitate transcriptional switches at the hsp70 locus.

BACKGROUND: Numerous target genes of the Polycomb group (PcG) are transiently activated by a stimulus and subsequently repressed. However, mechanisms by which PcG proteins regulate such target genes remain elusive. RESULTS: We employed the heat shock-responsive hsp70 locus in Drosophila to study the chromatin dynamics of PRC1 and its interplay with known regulators of the locus before, during and after heat shock. We detected mutually exclusive binding patterns for HSF and PRC1 at the hsp70 locus. We found that Pleiohomeotic (Pho), a DNA-binding PcG member, dynamically interacts with Spt5, an elongation factor. The dynamic interaction switch between Pho and Spt5 is triggered by the recruitment of HSF to chromatin. Mutation in the protein-protein interaction domain (REPO domain) of Pho interferes with the dynamics of its interaction with Spt5. The transcriptional kinetics of the heat shock response is negatively affected by a mutation in the REPO domain of Pho. CONCLUSIONS: We propose that a dynamic interaction switch between PcG proteins and an elongation factor enables stress-inducible genes to efficiently switch between ON/OFF states in the presence/absence of the activating stimulus.

Polycomb-like proteins link the PRC2 complex to CpG islands.

The Polycomb repressive complex 2 (PRC2) mainly mediates transcriptional repression and has essential roles in various biological processes including the maintenance of cell identity and proper differentiation. Polycomb-like (PCL) proteins, such as PHF1, MTF2 and PHF19, are PRC2-associated factors that form sub-complexes with PRC2 core components, and have been proposed to modulate the enzymatic activity of PRC2 or the recruitment of PRC2 to specific genomic loci. Mammalian PRC2-binding sites are enriched in CG content, which correlates with CpG islands that display a low level of DNA methylation. However, the mechanism of PRC2 recruitment to CpG islands is not fully understood. Here we solve the crystal structures of the N-terminal domains of PHF1 and MTF2 with bound CpG-containing DNAs in the presence of H3K36me3-containing histone peptides. We show that the extended homologous regions of both proteins fold into a winged-helix structure, which specifically binds to the unmethylated CpG motif but in a completely different manner from the canonical winged-helix DNA recognition motif. We also show that the PCL extended homologous domains are required for efficient recruitment of PRC2 to CpG island-containing promoters in mouse embryonic stem cells. Our research provides the first, to our knowledge, direct evidence to demonstrate that PCL proteins are crucial for PRC2 recruitment to CpG islands, and further clarifies the roles of these proteins in transcriptional regulation in vivo.

Histone Methyltransferase G9a Is Required for Cardiomyocyte Homeostasis and Hypertrophy.

BACKGROUND: Correct gene expression programming of the cardiomyocyte underlies the normal functioning of the heart. Alterations to this can lead to the loss of cardiac homeostasis, triggering heart dysfunction. Although the role of some histone methyltransferases in establishing the transcriptional program of postnatal cardiomyocytes during heart development has been shown, the function of this class of epigenetic enzymes is largely unexplored in the adult heart. In this study, we investigated the role of G9a/Ehmt2, a histone methyltransferase that defines a repressive epigenetic signature, in defining the transcriptional program for cardiomyocyte homeostasis and cardiac hypertrophy. METHODS: We investigated the function of G9a in normal and stressed cardiomyocytes with the use of a conditional, cardiac-specific G9a knockout mouse, a specific G9a inhibitor, and high-throughput approaches for the study of the epigenome (chromatin immunoprecipitation sequencing) and transcriptome (RNA sequencing); traditional methods were used to assess cardiac function and cardiovascular disease. RESULTS: We found that G9a is required for cardiomyocyte homeostasis in the adult heart by mediating the repression of key genes regulating cardiomyocyte function via dimethylation of H3 lysine 9 and interaction with enhancer of zeste homolog 2, the catalytic subunit of polycomb repressive complex 2, and MEF2C-dependent gene expression by forming a complex with this transcription factor. The G9a-MEF2C complex was found to be required also for the maintenance of heterochromatin needed for the silencing of developmental genes in the adult heart. Moreover, G9a promoted cardiac hypertrophy by repressing antihypertrophic genes. CONCLUSIONS: Taken together, our findings demonstrate that G9a orchestrates critical epigenetic changes in cardiomyocytes in physiological and pathological conditions, thereby providing novel therapeutic avenues for cardiac pathologies associated with dysregulation of these mechanisms.

Novel orally bioavailable EZH1/2 dual inhibitors with greater antitumor efficacy than an EZH2 selective inhibitor.

Polycomb repressive complex 2 (PRC2) methylates histone H3 lysine 27 and represses gene expression to regulate cell proliferation and differentiation. Enhancer of zeste homolog 2 (EZH2) or its close homolog EZH1 functions as a catalytic subunit of PRC2, so there are two PRC2 complexes containing either EZH2 or EZH1. Tumorigenic functions of EZH2 and its synthetic lethality with some subunits of SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeling complexes have been observed. However, little is known about the function of EZH1 in tumorigenesis. Herein, we developed novel, orally bioavailable EZH1/2 dual inhibitors that strongly and selectively inhibited methyltransferase activity of both EZH2 and EZH1. EZH1/2 dual inhibitors suppressed trimethylation of histone H3 lysine 27 in cells more than EZH2 selective inhibitors. They also showed greater antitumor efficacy than EZH2 selective inhibitor in vitro and in vivo against diffuse large B-cell lymphoma cells harboring gain-of-function mutation in EZH2. A hematological cancer panel assay indicated that EZH1/2 dual inhibitor has efficacy against some lymphomas, multiple myeloma, and leukemia with fusion genes such as MLL-AF9, MLL-AF4, and AML1-ETO. A solid cancer panel assay demonstrated that some cancer cell lines are sensitive to EZH1/2 dual inhibitor in vitro and in vivo. No clear correlation was detected between sensitivity to EZH1/2 dual inhibitor and SWI/SNF mutations, with a few exceptions. Severe toxicity was not seen in rats treated with EZH1/2 dual inhibitor for 14 days at drug levels higher than those used in the antitumor study. Our results indicate the possibility of EZH1/2 dual inhibitors for clinical applications.

PHF1 Tudor and N-terminal domains synergistically target partially unwrapped nucleosomes to increase DNA accessibility.

The Tudor domain of human PHF1 recognizes trimethylated lysine 36 on histone H3 (H3K36me3). PHF1 relies on this interaction to regulate PRC2 methyltransferase activity, localize to DNA double strand breaks and mediate nucleosome accessibility. Here, we investigate the impact of the PHF1 N-terminal domain (NTD) on the Tudor domain interaction with the nucleosome. We show that the NTD is partially ordered when it is natively attached to the Tudor domain. Through a combination of FRET and single molecule studies, we find that the increase of DNA accessibility within the H3K36me3-containing nucleosome, instigated by the Tudor binding to H3K36me3, is dramatically enhanced by the NTD. We demonstrate that this nearly order of magnitude increase is due to preferential binding of PHF1 to partially unwrapped nucleosomes, and that PHF1 alters DNA-protein binding within the nucleosome by decreasing dissociation rates. These results highlight the potency of a PTM-binding protein to regulate DNA accessibility and underscores the role of the novel mechanism by which nucleosomes control DNA-protein binding through increasing protein dissociation rates.

Polycomb complexes PRC1 and their function in hematopoiesis.

Hematopoiesis, the process by which blood cells are continuously produced, is one of the best studied differentiation pathways. Hematological diseases are associated with reiterated mutations in genes encoding important gene expression regulators, including chromatin regulators. Among them, the Polycomb group (PcG) of proteins is an essential system of gene silencing involved in the maintenance of cell identities during differentiation. PcG proteins assemble into two major types of Polycomb repressive complexes (PRCs) endowed with distinct histone-tail-modifying activities. PRC1 complexes are histone H2A E3 ubiquitin ligases and PRC2 trimethylates histone H3. Established conceptions about their activities, mostly derived from work in embryonic stem cells, are being modified by new findings in differentiated cells. Here, we focus on PRC1 complexes, reviewing recent evidence on their intricate architecture, the diverse mechanisms of their recruitment to targets, and the different ways in which they engage in transcriptional control. We also discuss hematopoietic PRC1 gain- and loss-of-function mouse strains, including those that model leukemic and lymphoma diseases, in the belief that these genetic analyses provide the ultimate test for molecular mechanisms driving normal hematopoiesis and hematological malignancies.

Genome-wide chemical mapping of O-GlcNAcylated proteins in Drosophila melanogaster.

N-Acetylglucosamine ß-O-linked to nucleocytoplasmic proteins (O-GlcNAc) is implicated in the regulation of gene expression in organisms, from humans to Drosophila melanogaster. Within Drosophila, O-GlcNAc transferase (OGT) is one of the Polycomb group proteins (PcGs) that act through Polycomb group response elements (PREs) to silence homeotic (HOX) and other PcG target genes. Using Drosophila, we identify new O-GlcNAcylated PcG proteins and develop an antibody-free metabolic feeding approach to chemoselectively map genomic loci enriched in O-GlcNAc using next-generation sequencing. We find that O-GlcNAc is distributed to specific genomic loci both in cells and in vivo. Many of these loci overlap with PREs, but O-GlcNAc is also present at other loci lacking PREs. Loss of OGT leads to altered gene expression not only at loci containing PREs but also at loci lacking PREs, including several heterochromatic genes. These data suggest that O-GlcNAc acts through multiple mechanisms to regulate gene expression in Drosophila.

Mapping the Function of Polycomb Proteins.

Polycomb group (PcG) proteins are master regulators of proliferation and development that play essential roles in human pathologies including cancers. PcGs act as gatekeepers of cellular identity, maintaining repression of a multitude of target genes. However, these properties have only been recently uncovered thanks to technological advances, first of all chromatin immunoprecipitations (ChIP), that allowed a systematic characterization of the activity of these factors in an unbiased manner at a genome-wide level. Using PcG protein as example, this chapter introduces the readers to the use of chromatin analysis (ChIP assays and replication timing) and how to move these approaches to a level of genome-wide interpretation.

Analysis of Endogenous Protein Interactions of Polycomb Group of Proteins in Mouse Embryonic Stem Cells.

PRC1 complexes contain four core subunits: Pcgf, Phc, Ring1, and Cbx proteins. Interestingly, mammalian genomes have several paralogues for each subunit, which are differentially expressed depending on the cell type, differentiation program, and cellular stimuli. Therefore, identification and characterization of the specific architecture of different PRC1 complexes during cellular differentiation are essential to better understand the function and recruitment mechanism of PRC1 complexes. In this chapter we describe several methods to study Polycomb architecture, and identification of novel interactors in both pluripotent and differentiating mouse embryonic stem cells.