Polycomb Repressive Complex 2 Complex Research Articles

How CBX proteins regulate normal and leukemic blood cells.

Hematopoietic stem cell (HSC) fate decisions are dictated by epigenetic landscapes. The Polycomb Repressive Complex 1 (PRC1) represses genes that induce differentiation, thereby maintaining HSC self-renewal. Depending on which chromobox (CBX) protein (CBX2, CBX4, CBX6, CBX7, or CBX8) is part of the PRC1 complex, HSC fate decisions differ. Here, we review how this occurs. We describe how CBX proteins dictate age-related changes in HSCs and stimulate oncogenic HSC fate decisions, either as canonical PRC1 members or by alternative interactions, including non-epigenetic regulation. CBX2, CBX7, and CBX8 enhance leukemia progression. To target, reprogram, and kill leukemic cells, we suggest and describe multiple therapeutic strategies to interfere with the epigenetic functions of oncogenic CBX proteins. Future studies should clarify to what extent the non-epigenetic function of cytoplasmic CBX proteins is important for normal, aged, and leukemic blood cells.

Upregulation of Enhancer of Zeste Homolog 2 (EZH2) with Associated pERK Co-Expression and PRC2 Complex Protein SUZ12 Correlation in Adult T-Cell Leukemia/Lymphoma

EZH2, a subunit of the polycomb repressive complex 2 (PRC2), is an important methyltransferase that catalyzes the trimethylation of histone H3 at lysine 27 (H3K27me3). EZH2 is overexpressed in various malignancies. Here, we investigated EZH2 expression and potential signaling molecules that correlate with EZH2 expression in ATLL and other T-cell neoplasms. Immunohistochemical staining (IHC) was performed for EZH2, pERK, MYC, and pSTAT3 on 43 ATLL cases and 104 cases of other T-cell neoplasms. Further IHC studies were conducted for Ki-67, SUZ12, and H3K27me3 on ATLL cases. All ATLL cases showed EZH2 overexpression. In other T-cell neoplasms, a high prevalence of EZH2 overexpression was identified (86%), except for T-PLL (33%). In ATLL, EZH2 overexpression correlated with pERK co-expression (86%), while only a small subset of cases showed MYC (7%) or pSTAT3 (14%) co-expression. In the other T-cell neoplasms, there was a variable, but higher, co-expression of EZH2 with pERK, MYC, and pSTAT3. In ATLL, enhanced EZH2 expression correlated with higher Ki-67 staining, SUZ12 (another PRC2 subunit), and H3K27me3 co-expression. In conclusion, EZH2 is overexpressed in ATLL and is associated with pERK expression. It correlates with an increased proliferation index, indicating an aggressive clinical course. EZH2 also correlates with SUZ12 and H3K27me3 co-expression, suggesting its PRC2-dependent catalytic activity through trimethylation. Additionally, EZH2 is overexpressed in most T-cell neoplasms, suggesting that EZH2 could function as an oncogenic protein in T-cell tumorigenesis. EZH2 and pERK could serve as potential therapeutic targets for treating aggressive ATLL. EZH2 could also be targeted in other T-cell neoplasms.

Epigenetic vulnerabilities of leukemia harboring inactivating EZH2 mutations

Epigenetic regulators, such as the polycomb repressive complex 2 (PRC2), play a critical role in both normal development and carcinogenesis. Mutations and functional dysregulation of PRC2 complex components, such as EZH2, are implicated in various forms of cancer and associated with poor prognosis. This study investigated the epigenetic vulnerabilities of acute myeloid leukemia (AML) and myelodysplastic/myeloproliferative disorders (MDS/MPN) by performing a chemical probe screen in patient cells. Paradoxically, we observed increased sensitivity to EZH2 and embryonic ectoderm development (EED) inhibitors in AML and MDS/MPN patient cells harboring EZH2 mutations. Expression analysis indicated that EZH2 inhibition elicited upregulation of pathways responsible for cell death and growth arrest, specifically in patient cells with mutant EZH2. The identified EZH2 mutations had drastically reduced catalytic activity, resulting in lower cellular H3K27me3 levels, and were associated with decreased EZH2 and PRC2 component EED protein levels. Overall, this study provides an important understanding of the role of EZH2 dysregulation in blood cancers and may indicate disease etiology for these poor prognosis AML and MDS/MPN cases.

Understanding the Functional Differences between PRC2.1 and PRC2.2 in Hematopoiesis

Epigenetic mechanisms play an important role in the determination of hematopoietic stem and progenitor cell (HSPC) fate through the modification of chromatin architecture. Polycomb repressive complex 2 (PRC2) is an indispensable epigenetic modifier that catalyzes histone 3 lysine 27 tri-methylation (H3K27me3) repressive mark through its catalytic subunit EZH2. PRC2 is divided into two independent complexes, PRC2.1 and PRC2.2, based on the specific interacting accessory proteins. PRC2.1 comprises one of the three polycomb-like proteins (PCL1/PHF1, PCL2/MTF2, or PCL3/PHF19), as well as one of the two recently identified accessory proteins, EPOP or PALI1. However, PRC2.2 contains either JARID2 or AEBP2 as co-factors. These accessory proteins aid in the recruitment of the PRC2 complexes to different sites in the genome to catalyze H3K27me3. Recent studies performed on mouse embryonic stem cells showed that PRC2.1 and PRC2.2 are involved in distinct lineage specification and commitment through their specific co-factors. However, the functional differences between PRC2.1 and PRC2.2 in HSPC differentiation and lineage commitment has not been thoroughly characterized. To compare the complete loss of PRC2 versus PRC2.2-specific loss of function in normal hematopoiesis, we generated Mx1-Cre: Jarid2fl/fl and Mx1-Cre: Ezh2fl/fl mouse strains. Two-month old Mx1-Cre(+) control, Mx1-Cre(+): Jarid2fl/fl, and Mx1-Cre(+): Ezh2fl/fl mice were injected with six doses of polyinosinic:polycytidylic acid (pIpC) to promote conditional deletion of Jarid2 and Ezh2. Two months post-pIpC injection, whole bone marrow (WBM) cells were used to perform competitive transplantation (5x10 5 CD45.2 test WBM plus 5x10 5 CD45.1 competitor WBM cells). Jarid2-KO primary recipients showed the highest CD45.2 peripheral blood (PB) engraftment (~80%), and the highest tri-lineage (myeloid 80%, B cell 70%, T cell 80%) chimerism compared to the control (myeloid 50%, B cell 45%, T cell 25%) and Ezh2-KO (myeloid 65%, B cell 5%, T cell 5%) recipients. Ezh2-KO recipients showed the lowest CD45.2 PB engraftment (~20%), and were deficient for PB lymphoid cell production. Jarid2-KO recipients showed the highest bone marrow (BM) engraftment (~ 90%), while Ezh2-KO recipients showed significant BM lymphoid engraftment (B cell 55% and T cell 40%) inverse to what was observed in the PB (B cell 5% and T cell 5%). This suggested that complete loss of PRC2 function promotes blockage in the migration of mature lymphoid cells from BM to the PB. Jarid2-KO and Ezh2-KO recipients showed significant reductions in phenotypically defined HSC cell number and frequency compared to control recipients suggesting that PRC2.2 function through its co-factor Jarid2 to maintain HSC pool. To further investigate the functional effect of both genes' loss of function at the molecular level, we performed single cell transcriptomic analysis from KSL (lineage-, c-Kit+, Sca1+, CD45.2+) cells sorted from the aforementioned recipients. Based on transcriptomic analysis, Ezh2 loss of function promoted an increase in MPP4 and precursor B cell populations in the BM of recipient mice. However, no significant changes was observed between control and Jarid2-KO clusters. Analysis showed that the Mtf2 co-factor has the highest expression level throughout the whole KSL clusters compared to other PRC2.1 and PRC2.2 accessory proteins. These results suggested that Mtf2-associated PRC2.1 is necessary to restrict MPP4 self-renewal and to repress B cell priming in HSPCs. Although no significant change was observed in the HSC cluster between Jarid2-KO and Ezh2-KO genotypes, Jarid2 and Ezh2 loss of functions showed significant differential gene expression in the HSC population when compared to each other and to the control suggesting that Jarid2-associated PRC2.2 regulates a unique molecular program in HSCs independently from PRC2.1. Combined with our functional data, these results suggested that PRC2.2 restricts HSC-specific gene expression in non-self-renewing progenitor cells through its co-factor Jarid2; while PRC2.1 restricts MPP4 fate and B cell priming through its co-factor Mtf2. Ongoing studies are aimed to delineate the distinct molecular programs regulated by PRC2.1 and PRC2.2 in HSPCs using CRISPR-Cas9 sgRNA lentiviral system targeting each of the PRC2.1 and PRC2.2 accessory proteins independently and validation in lineage-specific functional assays.

Polycom Group Protein Mel18 Inhibits Hematopoietic Stem Cell Self-Renewal through Repressing the Expression of Genes Important for Cellular Senescence and Proliferation

Polycomb group (PcG) proteins are epigenetic gene silencers that have been implicated in stem cell maintenance and cancer development. Genetic and biochemical studies indicate that Polycomb group proteins exist in at least two protein complexes, Polycomb repressive complex 2 (PRC2) and Polycomb repressive complex 1 (PRC1), that act in concert to initiate and maintain stable gene repression. Bmi1 and Mel18 are two major homologs of the PCGF subunit within the canonical PRC1 complex. While Bmi1 is a positive regulator of hematopoietic stem cell (HSC) and leukemia stem cell (LSC) self-renewal, the role of Mel18 in normal and malignant hematopoiesis is not fully understood. To further determine the role of Mel18 in hematopoiesis, we have generated Mel18 conditional knockout mice ( Mel18 f/f-Mx1Cre +). Acute deletion of Mel18 in the hematopoietic compartment did not affect the frequency and survival of hematopoietic stem and progenitor cells (HSPCs). To determine the impact of Mel18 deficiency on HSPC proliferation, we performed serial replating assays and found that loss of Mel18 increases the replating potential of HSPCs in vitro. To determine the role of Mel18 in HSC self-renewal, we first performed serial bone marrow (BM) transplantation assays and found that Mel18 null BM cells show increased engraftment in both primary and secondary transplantation assays. Limiting dilution transplantation showed that there were more functional HSCs in the BM of Mel18 knockout mice. We then performed HSC transplantation assays and found that loss Mel18 enhances HSC self-renewal. Thus, we demonstrated that Mel18 inhibits HSC self-renewal and decreases the number of functional HSCs in vivo. To understand the mechanism by which Mel18 inhibits HSC self-renewal and proliferation, we performed RNA-seq using Mel18 +/+ and Mel18 -/- HSCs. We then performed gene set enrichment analysis (GSEA) to identify potential Mel18 target genes important for HSC behavior. We found that HSC gene signatures are enriched in Mel18 -/- HSCs. We also found that leukemia stem cell gene signatures and CBF-mutant-AML genes are significantly enriched in Mel18 -/- HSCs, suggesting that loss of Mel18 may prime normal HSCs for leukemic transformation. Notably, our RNA-seq assay showed that senescence-related genes, including S100a9 and S100a8, were down-regulated in Mel18 -/- HSPCs. Importantly, loss of Mel18 significantly decreased the number of senescent HSPCs. To understand how Mel18 regulates gene transcription, we performed ATAC-seq assays in Mel18 +/+ and Mel18 -/- HSPCs. Mel18 null HSPCs showed decreased chromatin accessibility near both S100a8 and S100a9 genes. We confirmed that both S100a8 and S100a9 are downregulated in Mel18 -/- HSPCs compared to Mel18 +/+ HSPCs. Given that cell cycle arrest is a common feature of cellular senescence, we also examined the expression of cell cycle related genes in Mel18 -/- HSPCs. The expression of cell cycle regulators, including CDK4 and CCND2, is increased in Mel18 -/- HSPCs. Mel18 can bind to a specific DNA sequence, 5‘-GACTNGACT-3‘, thereby repressing the expression of target genes. Mel18 directly binds to the CCND2 gene locus as revealed by ChIP-seq assays in embryonic stem cells, suggesting that Mel18 may directly repress CCND2 expression in HSPCs. In summary, we demonstrate that Mel18 inhibits HSC self-renewal via repressing the expression of genes that are important for cellular senescence and proliferation.

Tissue-Specific Tumour Suppressor and Oncogenic Activities of the Polycomb-like Protein MTF2.

The Polycomb repressive complex 2 (PRC2) is a conserved chromatin-remodelling complex that catalyses the trimethylation of histone H3 lysine 27 (H3K27me3), a mark associated with gene silencing. PRC2 regulates chromatin structure and gene expression during organismal and tissue development and tissue homeostasis in the adult. PRC2 core subunits are associated with various accessory proteins that modulate its function and recruitment to target genes. The multimeric composition of accessory proteins results in two distinct variant complexes of PRC2, PRC2.1 and PRC2.2. Metal response element-binding transcription factor 2 (MTF2) is one of the Polycomb-like proteins (PCLs) that forms the PRC2.1 complex. MTF2 is highly conserved, and as an accessory subunit of PRC2, it has important roles in embryonic stem cell self-renewal and differentiation, development, and cancer progression. Here, we review the impact of MTF2 in PRC2 complex assembly, catalytic activity, and spatiotemporal function. The emerging paradoxical evidence suggesting that MTF2 has divergent roles as either a tumour suppressor or an oncogene in different tissues merits further investigations. Altogether, our review illuminates the context-dependent roles of MTF2 in Polycomb group (PcG) protein-mediated epigenetic regulation. Its impact on disease paves the way for a deeper understanding of epigenetic regulation and novel therapeutic strategies.

Context-defined cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Interplay between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 145 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex composition and function, and predict new functional interactions, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality.

PGC7 regulates H3K27me3 modification by inhibiting the interaction of YY1 with PRC2.

Primordial germ cell 7 (PGC7)(Dppa3 or Stella) is a small inherently disordered protein that is mainly expressed in oocytes and plays a vital role in the regulation of DNA methylation reprogramming in imprinted loci through interaction with other proteins. Most of PGC7-deficient zygotes are blocked at two-cell stage with an increased tri-methylation at lysine 27 of histone H3 (H3K27me3) level in the nucleus. Our previous work has indicated that PGC7 interacts with yin-yang1 (YY1) that is essential for the recruitment of enhancer of zeste homolog 2 (EZH2)-containing Polycomb repressive complex 2 (PRC2) to H3K27me3 modification sites. Here, we found that the presence of PGC7 weakened the interaction between YY1 and PRC2 without disrupting the assembly of core subunits of the PRC2 complex. In addition, PGC7 promoted AKT to phosphorylate serine 21 of EZH2, resulting in inhibition of EZH2 activity and the dissociation of EZH2 from YY1, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 both promoted EZH2 to enter the pronuclei but without disturbing the subcellular localization of YY1 and caused an increase in the level of H3K27me3 in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos. In summary, PGC7 could affect zygotic genome activation during early embryonic development by regulating the level of H3K27me3 through regulation of PRC2 recruitment, EZH2 activity, and subcellular localization.NEW & NOTEWORTHY PGC7 and YY1 interaction inhibits recruitment of PRC2 by YY1. PGC7 promotes AKT and EZH2 interaction to increase pEZH2-S21 level, which weakens YY1 and EZH2 interaction, thereby decreasing H3K27me3 level. In zygotes, the PGC7-deficient and AKT inhibitor MK2206 promote EZH2 to enter the pronuclei, and increase H3K27me3 level in the pronuclei, as well as inhibition of the expression of zygote-activating genes regulated by H3K27me3 in two-cell embryos, which ultimately affects early embryo development.

Deregulated expression of polycomb repressive complex 2 target genes in a NF1 patient with microdeletion generating the RNF135-SUZ12 chimeric gene

Neurofibromatosis type I (NF1) microdeletion syndrome, accounting for 5–11% of NF1 patients, is caused by the heterozygous deletion of NF1 and a variable number of flanking genes in the 17q11.2 region. This syndrome is characterized by more severe symptoms than those shown by patients with intragenic NF1 mutation and by variable expressivity, which is not fully explained by the haploinsufficiency of the genes included in the deletions. We here reevaluate an 8-year-old NF1 patient, who carries an atypical deletion generating the RNF135-SUZ12 chimeric gene, previously described when he was 3 years old. As the patient has developed multiple cutaneous/subcutaneous neurofibromas over the past 5 years, we hypothesized a role of RNF135-SUZ12 chimeric gene in the onset of the patient’s tumor phenotype. Interestingly, SUZ12 is generally lost or disrupted in NF1 microdeletion syndrome and frequently associated to cancer as RNF135. Expression analysis confirmed the presence of the chimeric gene transcript and revealed hypo-expression of five out of the seven analyzed target genes of the polycomb repressive complex 2 (PRC2), to which SUZ12 belongs, in the patient’s peripheral blood, indicating a higher transcriptional repression activity mediated by PRC2. Furthermore, decreased expression of tumor suppressor gene TP53, which is targeted by RNF135, was detected. These results suggest that RNF135-SUZ12 chimera may acquire a gain of function, compared with SUZ12 wild type in the PRC2 complex, and a loss of function relative to RNF135 wild type. Both events may have a role in the early onset of the patient’s neurofibromas.

BRD4-PRC2 represses transcription of T-helper 2-specific negative regulators during T-cell differentiation.

BRD4 is a well-recognized transcriptional activator, but how it regulates gene transcriptional repression in a cell type-specific manner has remained elusive. In this study, we report that BRD4 works with Polycomb repressive complex 2 (PRC2) to repress transcriptional expression of the T-helper 2 (Th2)-negative regulators Foxp3 and E3-ubiqutin ligase Fbxw7 during lineage-specific differentiation of Th2 cells from mouse primary naïve CD4+ T cells. Brd4 binds to the lysine-acetylated-EED subunit of the PRC2 complex via its second bromodomain (BD2) to facilitate histone H3 lysine 27 trimethylation (H3K27me3) at target gene loci and thereby transcriptional repression. We found that Foxp3 represses transcription of Th2-specific transcription factor Gata3, while Fbxw7 promotes its ubiquitination-directed protein degradation. BRD4-mediated repression of Foxp3 and Fbxw7 in turn promotes BRD4- and Gata3-mediated transcriptional activation of Th2 cytokines including Il4, Il5, and Il13. Chemical inhibition of the BRD4 BD2 induces transcriptional de-repression of Foxp3 and Fbxw7, and thus transcriptional downregulation of Il4, Il5, and Il13, resulting in inhibition of Th2 cell lineage differentiation. Our study presents a previously unappreciated mechanism of BRD4's role in orchestrating a Th2-specific transcriptional program that coordinates gene repression and activation, and safeguards cell lineage differentiation.

Dynamic Interactome of PRC2-EZH1 Complex Using Tandem-Affinity Purification and Quantitative Mass Spectrometry.

The Polycomb repressive complex 2 (PRC2) is a well-characterized chromatin regulator of transcription programs acting through H3K27me3 deposition. In mammals, there are two main versions of PRC2 complexes: PRC2-EZH2, which is prevalent in cycling cells, and PRC2-EZH1 where EZH1 replaces EZH2 in post-mitotic tissues. Stoichiometry of PRC2 complex is dynamically modulated during cellular differentiation and various stress conditions. Therefore, unraveling unique architecture of PRC2 complexes under specific biological context through comprehensive and quantitative characterization could provide insight into the underlying mechanistic molecular mechanism in regulation of transcription process. In this chapter, we describe an efficient method which combines tandem-affinity purification (TAP) with label-free quantitative proteomics strategy for studying PRC2-EZH1 complex architecture alterations and identifying novel protein regulators in post-mitotic C2C12 skeletal muscle cells.

Abstract A015: Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma

Abstract Interplays between chromatin-associated complexes and modifications critically contribute to the partitioning of epigenome into stable and functionally distinct domains. Yet there is a lack of systematic identification of chromatin crosstalk mechanisms, limiting our understanding of the dynamic transition between chromatin states during development and disease. Here we perform co-dependency mapping of genes encoding nuclear proteins using CRISPR-Cas9-mediated fitness screens in pan-cancer cell lines to quantify gene-gene functional relationships. We identify 84 co-dependency modules and further define the molecular context underlying the essentiality of these modules by incorporating mutational, epigenome, gene expression and drug sensitivity profiles of cell lines. These analyses assign new protein complex functions and biochemical interactions, and predict new chromatin crosstalk, including an unexpected co-dependency between two transcriptionally counteracting chromatin complexes - polycomb repressive complex 2 (PRC2) and MLL-MEN1 complex. We show that PRC2-mediated H3K27 tri-methylation regulates the genome-wide distribution of MLL1 and MEN1. In lymphoma cells with EZH2 gain-of-function mutations, the re-localization of MLL-MEN1 complex drives oncogenic gene expression and results in a hypersensitivity to pharmacologic inhibition of MEN1. Together, our findings provide a resource for discovery of trans-regulatory interactions as mechanisms of chromatin regulation and potential targets of synthetic lethality. Citation Format: Xiao Chen, Yinglu Li, Chao Lu. Cancer co-dependency mapping identifies a functional interplay between PRC2 and MLL-MEN1 complex in lymphoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A015.

Mechanisms of Polycomb Group Protein-Mediated Fetal Hemoglobin Silencing

Reactivation of fetal hemoglobin (HbF) is beneficial for patients with β-globin gene disorders, such as sickle cell disease and β-thalassemia. Polycomb repressive complex 1 (PRC1) and 2 (PRC2) are multi-subunit protein complexes that function as transcriptional repressors to maintain lineage-specific gene expression programs. Prior works showed that genetic or pharmacologic manipulation of select PRC2 subunits can induce HbF expression in adult erythroid cells, and an inhibitor targeting the EED subunit of PRC2 has entered a clinical trial. Yet the molecular basis underlying HbF regulation by PRC1/2 complexes is unknown. Using a CRISPR-Cas9-based genetic screen, we identified BMI1, a subunit of the PRC1 complex as novel HbF repressor. Disrupting BMI1 in the adult-type erythroid cell line HUDEP2 or primary human erythroblast cells robustly increased HbF production. We failed to detect BMI1 binding at the β-globin gene locus by CUT&RUN, indicating that BMI1 functions via an indirect mechanism. RNA-seq analysis of BMI1-deficient HUDEP2 cells uncovered upregulation of two known HbF regulators: IGF2BP1 and LIN28B, both of which induce HbF when forcibly expressed in adult erythroid cells. Depletion of either IGF2BP1 or LIN28B partially restored HbF silencing in BMI1-deficient HUDEP2 cells, whereas simultaneous depletion of IGF2BP1 and LIN28B lowered HbF production virtually to basal levels in these cells. In primary erythroid cells, IGF2BP3, which is functionally related to IGF2BP1, also contributes to the effects of BMI1 depletion. Together, these data indicate that IGF2BP1, IGF2BP3, and LIN28B are the critical BMI1 targets in human erythroid cells. Since BMI1 functions in two distinct PRC1 complexes (cPRC1 and ncPRC1), we aimed to define which of these is critical for HbF regulation. cPRC1 is defined by the presence of CBX proteins 2, 4, 6, 7, and 8, while ncPRC1 contains RYBP or its homolog YAF2. ncPRC1 and cPRC1 deposit the histone mark H2AK119ub1, and facilitate chromatin compaction, respectively. Disruption of CBX2/4/7/8 proteins but not RYAP/YAF2 in HUDEP2 cells activated HbF to levels similar to those observed upon BMI1 depletion, implicating cPRC1 in HbF control. Accordingly, CUT&RUN analysis in adult erythroid cells revealed a strong genome-wide co-localization of BMI1 and CBX2/4/7/8, including at the IGF2BP1, IGF2BP3, and LIN28B genes. Finally, BMI1 depletion does not lead to significant changes in H2AK119ub1 at these genes, in line with the fact that the repressive activity of the cPRC1 complex does not rely on its H2A ubiquitination activity. Together these results indicate that BMI1 represses HbF via the cPRC1 complex in adult erythroid cells. The BMI1-containing cPRC1 complex is known to repress transcription in conjunction with the PRC2 complex which deposits the histone mark H3K27me3. CUT&RUN profiling studies revealed focal loss of H3K27me3 in BMI1-deficient HUDEP2 cells, including at the CG-rich regions near the promoters of the IGF2BP1 and LIN28B genes. Therefore, we tested whether cPRC1 and PRC2 complexes converge on HbF regulation by regulating IGF2BP1 and LIN28B. Disruption in HUDEP2 cells of EZH2, the catalytic component of PRC2, led to global depletion of H3K27me3 along with upregulation of IGF2BP1 and LIN28B. Co-depletion of IGF2BP1 and LIN28B largely restored the EZH2 depletion-induced HbF upregulation. In summary, our studies further define the nature of PRC complexes involved in HbF regulation and identify the IGF2BP1, IGF2BP3, and LIN28B genes as critical links between PcG activity and HbF silencing. Our results may guide the development of improved PRC inhibitors for the treatment of β-globin disorders.

DDB1 and CUL4 Associated Factor 7 (DCAF7) Is Essential for Hematopoiesis

Mutations in DDB1 And CUL4 Associated Factor 7 (DCAF7) have been detected in a subset of patients with Myeloid Leukemia in Down syndrome. DCAF7 is a highly conserved WD40 domain protein with 100% homology among vertebrates and is a prominent binding partner of DYRK1A, a kinase located on chromosome 21. Analysis of the DepMap database revealed that hematopoietic cell lines are highly dependent on DCAF7 compared to cell lines from other tumors. However, the function of DCAF7 during normal hematopoiesis is currently unknown. Thus, we aimed to study its function in hematopoietic cell lines and in a conditional knockout mouse model. We first deleted DCAF7 from a panel of hematopoietic cell lines by CRISPR/Cas9 editing and observed its absence led to cell cycle arrest and decreased proliferation. Rescue experiments in which a CRISPR resistant DCAF7 allele was expressed in the targeted clones restored proliferation and cell cycling. A comparison of RNA-seq data between parental HEL cells and those without DCAF7 showed decreased expression of genes involved in differentiation, TNF signaling and the IFN pathway, indicating that DCAF7 is important for cell differentiation. Next, we created a Dcaf7 conditionally targeted mouse strain (Dcaf7F/F Mx-1 Cre) and assessed its role in hematopoiesis in vivo. Following Cre induction by pIpC, we observed a decrease in white blood cells, hemoglobin and platelet counts in KO mice. Analysis of the bone marrow showed an accumulation of LT-HSCs and multipotent progenitors. In addition, we observed a defect in the generation of B-cells, reminiscent of the phenotype observed upon Dyr1ka deletion. We then tested the recovery of Dcaf7 KO hematopoietic cells after different types of stress, which included ex-vivo culture, transplantation, and myelosuppression. Dcaf7 deficient HSCs showed defective expansion in PVA-containing media and poor differentiation in single cell colony assays in methylcellulose. In non-competitive transplants, KO cells reconstituted hematopoiesis although the number of mature myeloid and lymphoid cells was dramatically decreased. In competitive transplants, Dcaf7 KO cells were outcompeted by wild-type cells. Lastly, upon myelosuppression with 5-FU, Dcaf7 deficient mice were not able to recover and died acutely. Overall, these results indicate that DCAF7 plays a critical role in HSCs during homeostasis and regeneration. To uncover the mechanisms by which DCAF7 contributes to hematopoiesis, we performed IP-MS using tagged DCAF7 as bait in a hematopoietic cell line. In addition to DYRK1A, we found that DCAF7 interacts with multiple components of the polycomb repressive complex1 (PRC1), specifically the non-canonical PRC1.5 complex, which has been previously implicated in gene activation and transcription. We confirmed that DCAF7 engages in the PRC1.5 complex by glycerol gradient centrifugation. Upon DCAF7 absence the size the PRC1 complex shifts towards a lower molecular weight, indicating that DCAF7 is a core subunit of the complex. We then performed RNASeq analysis in sorted LT-HSCs (Lin-cKit+Sca1+CD48-CD150+) and found that 70% of the differentially expressed genes were downregulated in KO cells. These downregulated genes were associated with diminished activation of GATA1, GATA2, RUNX1 and SP1 target genes, in agreement with the functional role of the PRC1.5 complex in gene activation and cell differentiation. Together our results reveal that DCAF7 regulates hematopoiesis by participating in transcriptional regulatory complexes, and we predict that DCAF7 mutations contribute to leukemia by cooperating with DYRK1A and altering the activity of the PRC1 complex.

Polycomb repressive complex 2 controls cardiac cell fate decision via interacting with RNA: Promiscuously or well-ordered.

The epigenetic landscape determines cell fate during heart development. Polycomb repressive complex 2 (PRC2) mediates histone methyltransferase activity during cardiac cell differentiation. The PRC2 complex contains the proteins embryonic ectoderm development (EED), suppressor of zeste (SUZ12), the chromatin assembly factor 1 (CAF1) histone-binding proteins RBBP4 and RBBP7, and the histone methyltransferase called enhancer of zeste (EZH2 or EZH1), which incorporates the Su(var)3-9, Enhancer-of-zeste, Trithorax (SET) domain. Cardiac PRC2-deficient mice display lethal congenital heart malformations. The dynamic process of cardiac cell fate decisions is controlled by PRC2 and the PRC2-mediated epigenetic landscape. Although specific individual long noncoding RNAs (lncRNAs) including Braveheart were widely reported to regulate the recruitments of PRC2 to their specific targets, a promiscuous RNA binding profile by PRC2 was also identified to play an essential role in cardiac cell fate decision. In this review, we focus on RNA-mediated PRC2 recruitment machinery in the process of cardiac cell fate decisions. The roles of individual lncRNAs which recruit PRC2, as well as promiscuous RNA binding by PRC2 in heart development are summarized. Since the binding priority of RNAs with different primary and secondary structures differs in its affinity to PRC2, the competitive relationship between individual lncRNAs binding and promiscuous RNA binding by PRC2 may be important for understanding the machinery by which biding of individual lncRNA and promiscuous RNA by PRC2 coordinately control the well-ordered dynamic cardiac cell lineage differentiation process.

1258P HJM-353: A potent, selective and orally bioavailable EED inhibitor with robust anti-tumor activities

Polycomb Repressive Complex 2 (PRC2), a multimeric complex that catalyzes the methylation of histone H3 at lysine 27 with core subunits EZH2, EED and SUZ12, functions as an epigenetic modulator in regulating cell proliferation and development. Dysregulation of the PRC2 complex is implicated in hematological and solid malignancies and shown to correlate with poor prognosis in cancers. Recently EZH2 inhibitor Tazemetostat has been approved by FDA for the treatment of advanced epithelioid sarcoma and follicular lymphoma. However, acquired resistant mutations in EZH2 and complementary activation of EZH1 might impair the effectiveness of this class of PRC2 inhibitors. Targeting the allosteric subunit EED represents a new strategy to fully inhibit PRC2 activity and address the limitations of EZH2 inhibition. Herein, we report HJM-353 as an efficacious EED inhibitor for cancer therapy. In vitro activities of HJM-353 were assessed by CTG assay and Western blot in EZH2 mutant human cell lines.In vivo anti-tumor activities of HJM-353 were evaluated in DLBCL and gastric cancer xenograft models.Pharmacokinetic/pharmacodynamics correlation of HJM-353 was determined by LC-MS/MS, Western blot and Q-PCR. HJM-353 effectively reduced H3K27me3 level and inhibited cell proliferation with an IC50 of 33.8 nM in WSU-DLCL2 cells. It displayed favorable PK properties and induced complete tumor regression in KARPAS-422 derived DLBCL xenograft model. Plasma concentration of HJM-353 correlated with the inhibition of H3K27me3 and upregulation of PRC2 target genes in tumors, indicating a clear PK/PD relationship in the xenografts. In addition, HJM-353 caused tumor stasis as MAK683 did in a xKATO-III gastric xenograft model. In summary, HJM-353 is a potent, selective and orally bioavailable EED inhibitor with robust anti-tumor activities. We are developing HJM-353 for the treatment of hematological malignancies and solid tumors and expect to file an IND in the second half of 2022.

The Polycomb group gene rnf2 is essential for central and enteric neural system development in zebrafish.

The development of central nervous system (CNS) and enteric nervous system (ENS) is under precise and strict control in vertebrates. Whether and how the Polycomb repressive complex 1 (PRC1) is involved in it remain unclear. To investigate the role of PRC1 in the nervous system development, using CRISPR/Cas9 technology, we have generated mutant zebrafish lines for the rnf2 gene which encodes Ring1b, the enzymatic component of the PRC1 complex. We show that rnf2 loss of function leads to abnormal migration and differentiation of neural crest and neural precursor cells. rnf2 mutant embryos exhibit aganglionosis, in which the hindgut is devoid of neurons. In particular, the formation of 5-HT serotonin neurons and myelinating glial cells is defective. Furthermore, ectopic expression of ENS marker genes is observed in forebrain of rnf2 mutant embryos. These findings suggest that the rnf2 gene plays an important role in the migration and differentiation of neural precursor cells, and its absence leads to abnormal development of ENS and CNS in zebrafish.

A small RNA-guided PRC2 complex eliminates DNA as an extreme form of transposon silencing.

In animal germlines, transposons are silenced at the transcriptional or post-transcriptional level to prevent deleterious expression. Ciliates employ a more direct approach by physically eliminating transposons from their soma, utilizing piRNAs to recognize transposons and imprecisely excise them. Ancient, mutated transposons often do not require piRNAs and are precisely eliminated. Here, we characterize the Polycomb Repressive Complex 2 (PRC2) in Paramecium and demonstrate its involvement in the removal of transposons and transposon-derived DNA. Our results reveal a striking difference between the elimination of new and ancient transposons at the chromatin level and show that the complex may be guided by Piwi-bound small RNAs (sRNAs). We propose that imprecise elimination in ciliates originates from an ancient transposon silencing mechanism, much like in plants and metazoans, through sRNAs, repressive methylation marks, and heterochromatin formation. However, it is taken a step further by eliminating DNA as an extreme form of transposon silencing.

HDAC1 and PRC2 mediate combinatorial control in SPI1/PU.1-dependent gene repression in murine erythroleukaemia.

Although originally described as transcriptional activator, SPI1/PU.1, a major player in haematopoiesis whose alterations are associated with haematological malignancies, has the ability to repress transcription. Here, we investigated the mechanisms underlying gene repression in the erythroid lineage, in which SPI1 exerts an oncogenic function by blocking differentiation. We show that SPI1 represses genes by binding active enhancers that are located in intergenic or gene body regions. HDAC1 acts as a cooperative mediator of SPI1-induced transcriptional repression by deacetylating SPI1-bound enhancers in a subset of genes, including those involved in erythroid differentiation. Enhancer deacetylation impacts on promoter acetylation, chromatin accessibility and RNA pol II occupancy. In addition to the activities of HDAC1, polycomb repressive complex 2 (PRC2) reinforces gene repression by depositing H3K27me3 at promoter sequences when SPI1 is located at enhancer sequences. Moreover, our study identified a synergistic relationship between PRC2 and HDAC1 complexes in mediating the transcriptional repression activity of SPI1, ultimately inducing synergistic adverse effects on leukaemic cell survival. Our results highlight the importance of the mechanism underlying transcriptional repression in leukemic cells, involving complex functional connections between SPI1 and the epigenetic regulators PRC2 and HDAC1.

HOTAIR interacts with PRC2 complex regulating the regional preadipocyte transcriptome and human fat distribution.

Mechanisms governing regional human adipose tissue (AT) development remain undefined. Here, we show that the long non-coding RNA HOTAIR (HOX transcript antisense RNA) is exclusively expressed in gluteofemoral AT, where it is essential for adipocyte development. We find that HOTAIR interacts with polycomb repressive complex 2 (PRC2) and we identify core HOTAIR-PRC2 target genes involved in adipocyte lineage determination. Repression of target genes coincides with PRC2 promoter occupancy and H3K27 trimethylation. HOTAIR is also involved in modifying the gluteal adipocyte transcriptome through alternative splicing. Gluteal-specific expression of HOTAIR is maintained by defined regions of open chromatin across the HOTAIR promoter. HOTAIR expression levels can be modified by hormonal (estrogen, glucocorticoids) and genetic variation (rs1443512 is a HOTAIR eQTL associated with reduced gynoid fat mass). These data identify HOTAIR as a dynamic regulator of the gluteal adipocyte transcriptome and epigenome with functional importance for human regional AT development.