Polycomb Repressive Complex 1 Subunits Research Articles

Balanced Epigenetic Regulation of MHC Class I Expression in Tumor Cells by the Histone Ubiquitin Modifiers BAP1 and PCGF1.

MHC class I (MHC-I) molecules are critical for CD8+ T cell responses to viral infections and malignant cells, and tumors can downregulate MHC-I expression to promote immune evasion. In this study, using a genome-wide CRISPR screen on a human melanoma cell line, we identified the polycomb repressive complex 1 (PRC1) subunit PCGF1 and the deubiquitinating enzyme BAP1 as opposite regulators of MHC-I transcription. PCGF1 facilitates deposition of ubiquitin at H2AK119 at the MHC-I promoters to silence MHC-I, whereas BAP1 removes this modification to restore MHC-I expression. PCGF1 is widely expressed in tumors and its depletion increased MHC-I expression in multiple tumor lines, including MHC-Ilow tumors. In cells characterized by poor MHC-I expression, PRC1 and PRC2 act in parallel to impinge low transcription. However, PCGF1 depletion was sufficient to increase MHC-I expression and restore T cell-mediated killing of the tumor cells. Taken together, our data provide an additional layer of regulation of MHC-I expression in tumors: epigenetic silencing by PRC1 subunit PCGF1.

Functional dissection of PRC1 subunits RYBP and YAF2 during neural differentiation of embryonic stem cells

Polycomb repressive complex 1 (PRC1) comprises two different complexes: CBX-containing canonical PRC1 (cPRC1) and RYBP/YAF2-containing variant PRC1 (vPRC1). RYBP-vPRC1 or YAF2-vPRC1 catalyzes H2AK119ub through a positive-feedback model; however, whether RYBP and YAF2 have different regulatory functions is still unclear. Here, we show that the expression of RYBP and YAF2 decreases and increases, respectively, during neural differentiation of embryonic stem cells (ESCs). Rybp knockout impairs neural differentiation by activating Wnt signaling and derepressing nonneuroectoderm-associated genes. However, Yaf2 knockout promotes neural differentiation and leads to redistribution of RYBP binding, increases enrichment of RYBP and H2AK119ub on the RYBP-YAF2 cotargeted genes, and prevents ectopic derepression of nonneuroectoderm-associated genes in neural-differentiated cells. Taken together, this study reveals that RYBP and YAF2 function differentially in regulating mESC neural differentiation.

Dispersal of PRC1 condensates disrupts polycomb chromatin domains and loops.

Polycomb repressive complex 1 (PRC1) strongly influences 3D genome organization, mediating local chromatin compaction and clustering of target loci. Several PRC1 subunits have the capacity to form biomolecular condensates through liquid-liquid phase separation in vitro and when tagged and over-expressed in cells. Here, we use 1,6-hexanediol, which can disrupt liquid-like condensates, to examine the role of endogenous PRC1 biomolecular condensates on local and chromosome-wide clustering of PRC1-bound loci. Using imaging and chromatin immunoprecipitation, we show that PRC1-mediated chromatin compaction and clustering of targeted genomic loci-at different length scales-can be reversibly disrupted by the addition and subsequent removal of 1,6-hexanediol to mouse embryonic stem cells. Decompaction and dispersal of polycomb domains and clusters cannot be solely attributable to reduced PRC1 occupancy detected by chromatin immunoprecipitation following 1,6-hexanediol treatment as the addition of 2,5-hexanediol has similar effects on binding despite this alcohol not perturbing PRC1-mediated 3D clustering, at least at the sub-megabase and megabase scales. These results suggest that weak hydrophobic interactions between PRC1 molecules may have a role in polycomb-mediated genome organization.

Rare deleterious de novo missense variants in Rnf2/Ring2 are associated with a neurodevelopmental disorder with unique clinical features.

The Polycomb group (PcG) gene RNF2 (RING2) encodes a catalytic subunit of the Polycomb repressive complex 1 (PRC1), an evolutionarily conserved machinery that post-translationally modifies chromatin to maintain epigenetic transcriptional repressive states of target genes including Hox genes. Here, we describe two individuals, each with rare de novo missense variants in RNF2. Their phenotypes include intrauterine growth retardation, severe intellectual disabilities, behavioral problems, seizures, feeding difficulties and dysmorphic features. Population genomics data suggest that RNF2 is highly constrained for loss-of-function (LoF) and missense variants, and both p.R70H and p.S82R variants have not been reported to date. Structural analyses of the two alleles indicate that these changes likely impact the interaction between RNF2 and BMI1, another PRC1 subunit or its substrate Histone H2A, respectively. Finally, we provide functional data in Drosophila that these two missense variants behave as LoF alleles in vivo. The evidence provide support for deleterious alleles in RNF2 being associated with a new and recognizable genetic disorder. This tentative gene-disease association in addition to the 12 previously identified disorders caused by PcG genes attests to the importance of these chromatin regulators in Mendelian disorders.

The heterochronic gene Oryza sativa LIKE HETEROCHROMATIN PROTEIN 1 modulates miR156b/c/i/e levels.

The juvenile-to-adult transition in plants involves changes in vegetative growth and plant architecture; the timing of this transition has important implications for agriculture. The microRNA miR156 regulates this transition and shoot maturation in plants. In Arabidopsis thaliana, deposition of histone H3 trimethylation on lysine 27 (H3K27me3, a repressive mark) at the MIR156A/C loci is regulated by Polycomb Repressive Complex 1 (PRC1) or PRC2, depending on the developmental stage. The levels of miR156 progressively decline during shoot maturation. The amount of H3K27me3 at MIR156A/C loci affects miR156 levels; however, whether this epigenetic regulation is conserved remains unclear. Here, we found that in rice (Oryza sativa), the putative PRC1 subunit LIKE HETEROCHROMATIN PROTEIN 1 (OsLHP1), with the miR156-SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) module, affects developmental phase transitions. Loss of OsLHP1 function results in ectopic expression of MIR156B/C/I/E, phenocopy of miR156 overexpression, and reduced H3k27me3 levels at MIR156B/C/I/E. This indicates that OsLHP1 has functionally diverged from Arabidopsis LHP1. Genetic and transcriptome analyses of wild-type, miR156b/c-overexpression, and Oslhp1-2 mutant plants suggest that OsLHP1 acts upstream of miR156 and SPL during the juvenile-to-adult transition. Therefore, modifying the OsLHP1-miR156-SPL pathway may enable alteration of the vegetative period and plant architecture.

Multiple polycomb epigenetic regulatory proteins are active in normal and regenerating adult olfactory epithelium.

ObjectivesTo investigate epigenetic mechanisms contributing to regulation of cellular renewal and neurogenesis in adult olfactory epithelium (OE).Study DesignProspective basic science study.MethodsOlfactory basal cell cultures were prepared from adult mice per established protocols. in vivo studies were performed using the mouse methimazole lesion–regeneration paradigm. Nasal tissue sections were prepared from adult mice 7 days following lesion, or from unlesioned controls. Polycomb proteins were assessed by Western blot from culture or nasal tissue lysates, and by gene expression studies from cultures. In addition, in vivo expression patterns of Polycomb proteins were examined using immunohistochemistry. Chromosome immunoprecipitation (ChIP) was performed to investigate epigenetic modifications and specific chromatin interactions for Polycomb proteins in olfactory basal cells.ResultsSubunits of Polycomb Repressive Complex 1 (PRC1) and Polycomb Repressive Complex 2 (PRC2) were identified in basal cell cultures and in vivo. In regenerating OE, basal progenitor cells identified by expression of the c–KIT receptor were found to coexpress the PRC2 protein EZH2. Because multiple variants of PRC1 subunits give rise to diverse PRC1 complexes serving different functions, expression of specific PRC1 variants was further examined. We identified PRC1 components including MEL18 (PCGF2) in immature neurons, and confirm BMI1 (PCGF4) expression in mature neurons. Moreover, we identified CBX8 as a neuron–specific PRC1 subunit. ChIP assays from OE cells demonstrated binding of PRC proteins to regulatory regions of specific transcription factors, consistent with PRC–mediated epigenetic silencing mechanisms active in adult OE.ConclusionsMultiple Polycomb proteins have cell type–specific expression patterns in the adult OE. Findings presented here, together with evidence from prior studies, suggest that PRC–mediated epigenetic silencing contributes to regulation of cellular renewal and tissue homeostasis in the OE. Efforts to define the mechanisms that regulate repair in the OE are essential for development of new therapeutic strategies for olfactory disorders.Level of EvidenceN/A

The polycomb group protein BMI-1 inhibitor PTC-209 is a potent anti-myeloma agent alone or in combination with epigenetic inhibitors targeting EZH2 and the BET bromodomains.

Multiple myeloma (MM) is a tumor of plasmablasts/plasma cells (PCs) characterized by the expansion of malignant PCs with complex genetic aberrations in the bone marrow (BM). Recent reports, by us and others, have highlighted the polycomb group (PcG) proteins as potential targets for therapy in MM. The PcG protein BMI-1 of the polycomb repressive complex 1 (PRC1) has been reported to be overexpressed and to possess oncogenic functions in MM. Herein, we report on the anti-myeloma effects of the BMI-1 inhibitor PTC-209 and demonstrate that PTC-209 is a potent anti-myeloma agent in vitro using MM cell lines and primary MM cells. We show that PTC-209 reduces the viability of MM cells via induction of apoptosis and reveal that the anti-MM actions of PTC-209 are mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, leaving other PRC1 subunits such as CBX-7 and the catalytic subunit RING1B unaffected. Importantly, we demonstrate that PTC-209 exhibits synergistic and additive anti-myeloma activity when combined with other epigenetic inhibitors targeting EZH2 and BET bromodomains. Collectively, these data qualify BMI-1 as a candidate for targeted therapy in MM alone or in combinations with epigenetic inhibitors directed to PRC2/EZH2 or BET bromodomains.

Polycomb repressive complex 1 modifies transcription of active genes.

This study examines the role of Polycomb repressive complex 1 (PRC1) at active genes. The PRC1 and PRC2 complexes are crucial for epigenetic silencing during development of an organism. They are recruited to Polycomb response elements (PREs) and establish silenced domains over several kilobases. Recent studies show that PRC1 is also directly recruited to active genes by the cohesin complex. Cohesin participates broadly in control of gene transcription, but it is unknown whether cohesin-recruited PRC1 also plays a role in transcriptional control of active genes. We address this question using genome-wide RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq). The results show that PRC1 influences transcription of active genes, and a significant fraction of its effects are likely direct. The roles of different PRC1 subunits can also vary depending on the gene. Depletion of PRC1 subunits by RNA interference alters phosphorylation of RNA polymerase II (Pol II) and occupancy by the Spt5 pausing-elongation factor at most active genes. These effects on Pol II phosphorylation and Spt5 are likely linked to changes in elongation and RNA processing detected by nascent RNA-seq, although the mechanisms remain unresolved. The experiments also reveal that PRC1 facilitates association of Spt5 with enhancers and PREs. Reduced Spt5 levels at these regulatory sequences upon PRC1 depletion coincide with changes in Pol II occupancy and phosphorylation. Our findings indicate that, in addition to its repressive roles in epigenetic gene silencing, PRC1 broadly influences transcription of active genes and may suppress transcription of nonpromoter regulatory sequences.

UbE2E1/UBCH6 Is a Critical in Vivo E2 for the PRC1-catalyzed Ubiquitination of H2A at Lys-119

UbE2E1/UbcH6 is an E2 ubiquitin-conjugating enzyme that is regulated by USP7. We identified UbE2E1 as a novel component of Polycomb repressive complex 1 (PRC1), the E3 ligase complex responsible for histone H2A ubiquitination and gene silencing. We demonstrate that UbE2E1 is critical for the monoubiquitination of H2A at residue Lys-119 (uH2AK119) through its association with the PRC1 complex. UbE2E1 interacts with PRC1 subunits including Ring1A and Ring1B. Overexpression of UbE2E1 results in increased levels of uH2AK119, whereas overexpression of catalytically inactive UbE2E1_C131A or UbE2E1 knockdown results in decreased levels of uH2AK119. The down-regulation of H2A ubiquitination by loss of function of UbE2E1 is correlated with alleviated p16INK4a promoter repression and induced growth inhibition in HCT116 cells. These results are specific to UbE2E1 as knockdown of UbE2D E2s does not show any effect on uH2AK119. We extended the UbE2E1 regulation of uH2AK119 to USP7 and showed that USP7 is also a key regulator for monoubiquitination at H2A Lys-119 as both knockdown and deletion of USP7 results in decreased levels of uH2AK119. This study reveals that UbE2E1 is an in vivo E2 for the PRC1 ligase complex and thus plays an important role in the regulation of H2A Lys-119 monoubiquitination.

The Arabidopsis hnRNP-Q Protein LIF2 and the PRC1 Subunit LHP1 Function in Concert to Regulate the Transcription of Stress-Responsive Genes.

LHP1-INTERACTING FACTOR2 (LIF2), a heterogeneous nuclear ribonucleoprotein involved in Arabidopsis thaliana cell fate and stress responses, interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a Polycomb Repressive Complex1 subunit. To investigate LIF2-LHP1 functional interplay, we mapped their genome-wide distributions in wild-type, lif2, and lhp1 backgrounds, under standard and stress conditions. Interestingly, LHP1-targeted regions form local clusters, suggesting an underlying functional organization of the plant genome. Regions targeted by both LIF2 and LHP1 were enriched in stress-responsive genes, the H2A.Z histone variant, and antagonistic histone marks. We identified specific motifs within the targeted regions, including a G-box-like motif, a GAGA motif, and a telo-box. LIF2 and LHP1 can operate both antagonistically and synergistically. In response to methyl jasmonate treatment, LIF2 was rapidly recruited to chromatin, where it mediated transcriptional gene activation. Thus, LIF2 and LHP1 participate in transcriptional switches in stress-response pathways.

The histone H2A deubiquitinase Usp16 regulates hematopoiesis and hematopoietic stem cell function

Epigenetic mechanisms play important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it is unclear which of the several counteracting H2A deubiquitinases functions along with PRC1 to control H2A ubiquitination (ubH2A) level and regulates hematopoiesis in vivo. Here we investigated the function of Usp16 in mouse hematopoiesis. Conditional deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and lethality. Usp16 deletion did not change HSC number but was associated with a dramatic reduction of mature and progenitor cell populations, revealing a role in governing HSC lineage commitment. ChIP- and RNA-sequencing studies in HSC and progenitor cells revealed that Usp16 bound to many important hematopoietic regulators and that Usp16 deletion altered the expression of genes in transcription/chromosome organization, immune response, hematopoietic/lymphoid organ development, and myeloid/leukocyte differentiation. The altered gene expression was partly rescued by knockdown of PRC1 subunits, suggesting that Usp16 and PRC1 counterbalance each other to regulate cellular ubH2A level and gene expression in the hematopoietic system. We further discovered that knocking down Cdkn1a (p21cip1), a Usp16 target and regulated gene, rescued the altered cell cycle profile and differentiation defect of Usp16-deleted HSCs. Collectively, these studies identified Usp16 as one of the histone H2A deubiquitinases, which coordinates with the H2A ubiquitin ligase PRC1 to regulate hematopoiesis, and revealed cell cycle regulation by Usp16 as key for HSC differentiation.

Regulation of Hematopoietic Stem Cell Function By the Histone H2A Deubiquitinase Usp16

Epigenetic mechanism plays important regulatory roles in hematopoiesis and hematopoietic stem cell (HSC) function. Subunits of Polycomb repressive complex 1 (PRC1), the major histone H2A ubiquitin ligase, are critical for both normal and pathological hematopoiesis; however, it was unclear which H2A deubiquitinase pairs with PRC1 to control H2A ubiquitination (ubH2A) level in vivo and regulates hematopoiesis. Here we investigated the function of Usp16 in mouse hematopoiesis. Deletion of Usp16 in bone marrow resulted in a significant increase of global ubH2A level and mouse lethality. Usp16 deletion was associated with a dramatic reduction of progenitor cell populations and unchanged HSC number, revealing a critical role for Usp16 in HSC differentiation. The HSC differentiation defect was correlated with a great reduction of G1 phase cell population. RNA-seq and RT-qPCR studies revealed that Usp16 regulates the expression of many genes associated with HSC differentiation and self-renewal including cell cycle regulator p21. Significantly, knockdown of p21 largely rescued the undifferentiated phenotype of Usp16 deleted HSCs. Usp16 binds to regions flanking transcription start site and knockdown of PRC1 subunits, which reduced ubH2A levels, largely rescued the altered gene expression pattern associated with Usp16 deletion. Therefore, these studies identified Usp16 as the H2A deubiquitinase that pairs with PRC1 to regulate hematopoiesis in vivo and revealed that Usp16 regulates HSC function by affecting HSC cell cycle progression. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional repression by PRC1 in the absence of H2A monoubiquitylation.

Histone H2A monoubiquitylation (H2Aub) is considered to be a key effector in transcriptional repression by Polycomb-repressive complex 1 (PRC1). We analyzed Drosophila with a point mutation in the PRC1 subunit Sce that abolishes its H2A ubiquitylase activity or with point mutations in the H2A and H2Av residues ubiquitylated by PRC1. H2Aub is essential for viability and required for efficient histone H3 Lys27 trimethylation by PRC2 early in embryogenesis. However, H2Aub-deficient animals fully maintain repression of PRC1 target genes and do not show phenotypes characteristic of Polycomb group mutants. PRC1 thus represses canonical target genes independently of H2Aub.

Solution NMR Structure of the DNA-binding Domain from Scml2 (Sex Comb on Midleg-like 2)

Scml2 is a member of the Polycomb group of proteins involved in epigenetic gene silencing. Human Scml2 is a part of a multisubunit protein complex, PRC1 (Polycomb repressive complex 1), which is responsible for maintenance of gene repression, prevention of chromatin remodeling, preservation of the "stemness" of the cell, and cell differentiation. Although the majority of PRC1 subunits have been recently characterized, the structure of Scml2 and its role in PRC1-mediated gene silencing remain unknown. In this work a conserved protein domain within human Scml2 has been identified, and its structure was determined by solution NMR spectroscopy. This module was named Scm-like embedded domain, or SLED. Evolutionarily, the SLED domain emerges in the first multicellular organisms, consistent with the role of Scml2 in cell differentiation. Furthermore, it is exclusively found within the Scm-like family of proteins, often accompanied by malignant brain tumor domain (MBT) and sterile α motif (SAM) domains. The domain adopts a novel α/β fold with no structural analogues found in the Protein Data Bank (PDB). The ability of the SLED to bind double-stranded DNA was also examined, and the isolated domain was shown to interact with DNA in a sequence-specific manner. Because PRC1 complexes localize to the promoters of a specific subset of developmental genes in vivo, the SLED domain of Scml2 may provide an important link connecting the PRC1 complexes to their target genes.

Compaction of chromatin by diverse Polycomb group proteins requires localized regions of high charge

Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.