PRC2 Complex Research Articles

Chromatin dynamics associated with seed desiccation tolerance/sensitivity at early germination in Medicago truncatula.

Desiccation tolerance (DT) has contributed greatly to the adaptation of land plants to severe water-deficient conditions. DT is mostly observed in reproductive parts in flowering plants such as seeds. The seed DT is lost at early post germination stage but is temporally re-inducible in 1mm radicles during the so-called DT window following a PEG treatment before being permanently silenced in 5mm radicles of germinating seeds. The molecular mechanisms that activate/reactivate/silence DT in developing and germinating seeds have not yet been elucidated. Here, we analyzed chromatin dynamics related to re-inducibility of DT before and after the DT window at early germination in Medicago truncatula radicles to determine if DT-associated genes were transcriptionally regulated at the chromatin levels. Comparative transcriptome analysis of these radicles identified 948 genes as DT re-induction-related genes, positively correlated with DT re-induction. ATAC-Seq analyses revealed that the chromatin state of genomic regions containing these genes was clearly modulated by PEG treatment and affected by growth stages with opened chromatin in 1mm radicles with PEG (R1P); intermediate openness in 1mm radicles without PEG (R1); and condensed chromatin in 5mm radicles without PEG (R5). In contrast, we also showed that the 103 genes negatively correlated with the re-induction of DT did not show any transcriptional regulation at the chromatin level. Additionally, ChIP-Seq analyses for repressive marks H2AK119ub and H3K27me3 detected a prominent signal of H3K27me3 on the DT re-induction-related gene sequences at R5 but not in R1 and R1P. Moreover, no clear H2AK119ub marks was observed on the DT re-induction-related gene sequences at both developmental radicle stages, suggesting that silencing of DT process after germination will be mainly due to H3K27me3 marks by the action of the PRC2 complex, without involvement of PRC1 complex. The dynamic of chromatin changes associated with H3K27me3 were also confirmed on seed-specific genes encoding potential DT-related proteins such as LEAs, oleosins and transcriptional factors. However, several transcriptional factors did not show a clear link between their decrease of chromatin openness and H3K27me3 levels, suggesting that their accessibility may also be regulated by additional factors, such as other histone modifications. Finally, in order to make these comprehensive genome-wide analyses of transcript and chromatin dynamics useful to the scientific community working on early germination and DT, we generated a dedicated genome browser containing all these data and publicly available at https://iris.angers.inrae.fr/mtseedepiatlas/jbrowse/?data=Mtruncatula.

EZH2 Targeting Induces CD38 Upregulation and Response to Anti-CD38 Antibodies in Multiple Myeloma

Multiple myeloma (MM) is the second most common hematological malignancy characterized by the accumulation of tumor plasma cells within the bone marrow. MM is characterized by high molecular and clinical heterogeneity. During the last 10 years new therapeutic classes including targeted immunotherapies significantly improved the overall survival of MM patients. However, drug resistance and relapse remain major challenges in MM. In MM, marked reduction of CD38 expression via exocytosis, endocytosis, degradation of the antigen-antibody complex and trogocytosis has been associated with resistance to CD38 targeted immunotherapies. Our group previously described a link and a negative correlation between CD38 and EZH2 (Enhancer of Zeste homolog 2) expression during normal B to plasma cell differentiation in association with a transcriptional control of CD38 expression involving the polycomb PRC2 complex (Herviou et al. Leukemia. 2019). Here we identified a significant negative correlation between CD38 expression and EZH2 expression in 2 independent cohorts of MM patients (p<0.05; Montpellier cohort n=198; Compass cohort n=631). Thus, we hypothesized that PRC2 targeting using EZH2 inhibitor could result in CD38 re-expression to overcome daratumumab resistance. We first characterized the membrane CD38 expression by calculating the Antibody Binding Capacity in our unique panel of 40 Human Myeloma Cell lines (HMCL) representative of MM heterogeneity. We observed a heterogeneous expression of the membrane CD38 expression on MM cell lines with cell lines demonstrating no membrane expression and cell lines demonstrating high membrane expression. Next, we selected 3 HMCLs with low (JJN3), intermediate (XG2), and high (XG7) CD38 expression, and treated these cell lines with sublethal concentration of EZH2 inhibitor (EPZ-6438) for 9 days. CD38 membrane expression was characterized at Day 3, Day 6 and Day 9. Interestingly, EZP-6438 treatment induced upregulation of cell surface CD38 expression on all the HMCLs tested with the highest effect measured at day 9. Similar re-expression pattern was observed by western blot at these timing in the JJN3 CD38low cell line. Remarkably, ChIPseq analyses underlined a significant association of H3K27me3 mark at CD38 promoter in CD38neg and CD38low cell lines compared to CD38highcells. In addition, we investigated the efficiency of CD38 monoclonal antibodies (Daratumumab and Isatuximab) lysis through ADCC (Antibody dependent cellular cytotoxicity) assay after 9 days of EPZ-6438 pretreatment. Using JJN3 CD38low HMCL, EPZ-6438 pretreatment significantly improved the ADCC lysis induced by low doses of Daratumumab and Isatuximab treatment, in presence of purified Natural Killer (NK) from healthy donor, together with CD38 re-expression (p<0.05; n=3). Finally, we searched to validate these results using primary MM cells from patients at relapse after Daratumumab treatment. EPZ-6438 pretreatment resulted in a significant CD38 membrane expression upregulation with a 2 fold difference compared to the control condition together with improved ADCC toxicity of anti-CD38 antibodies in vitro. Altogether we demonstrated that treatment of MM cells with EZH2 inhibitor leads to significant upregulation of membrane CD38 expression in cell lines and primary MM cells from patients. CD38 re-expression was linked to an improvement of Daratumumab and Isatuximab ADCC efficiency. Thus, EZH2 targeting may be of therapeutic interest to overcome resistance to anti-CD38 targeted immunotherapies in Multiple Myeloma.

Preclinical Validation of EZH2 and HDAC I Dual Inhibition As a Potent Therapy for Refractory Myeloid Leukemia Associated with Down Syndrome

Children with Down Syndrome who are less than 5 years old have approximately 400-fold increased risk of developing myeloid leukemia. Even though children with myeloid leukemia associated with Down syndrome (DS-ML) are more sensitive to chemotherapy than their non-DS-ML counterpart, relapse and treatment-related toxicity needs to be eliminated to achieve better outcome for DS-ML children. Epigenetic alterations including DNA methylation and histone modifications play a key role in the pathogenesis of DS-ML. Enhancer of zeste homolog 2 (EZH2) is one of the epigenetic regulators that harbors high frequency of mutations in DS-ML. EZH2 is a catalytic subunit of PRC2 complex which suppresses gene expression by di- and tri-methylation of lysine27 on histone 3 (H3K27me2/3). EZH2 recruits class I histone deacetylases (HDAC, which remove acetyl groups from histones), on target genes for further suppression of gene expression. Using patient-derived xenograft models of DS-ML, we showed previously that combination of DNA hypomethylating agent azacitidine with HDAC inhibitor panobinostat or BCL2 inhibitor venetoclax showed significant improvement in median survival. Here we aimed to show synergistic effect of EZH2 inhibitor, GSK126, and class I HDAC inhibitor, romidepsin. The viability of CMK, a DS-ML cell line, and NTPL-60 and NTPL-386, in house generated DS-ML PDX lines, was determined by exclusion of propidium iodide-stained cells by flow cytometry. IC50 values for GSK126 and romidepsin were calculated using GraphPad Prism (dose-response curve). The IC50 of GSK126 was 40.5, 18.0, and 17.2 mM, while that of romidepsin was 10.2, 9.2, and 3.0 nM respectively. To determine synergy, the viability of cells exposed to a variety of concentrations of GSK126 and romidepsin alone or in combination was measured. Synergy index was calculated using relative risk ratio (RRR) formula as described previously (Quagliano et al, 56:36, Leuk Res, 2017). All the 3 DS-ML lines showed RRR value of less than 1 indicating synergistic inhibition of cell death by GSK126 and romidepsin treatment in ex vivo culture (Figure 1A). NTPL-386, a refractory DS-ML PDX line, which showed minimal response to chemotherapy (11-day improvement in median survival, Barwe et al., 134:2683, Blood, 2019), was chosen to determine the in vivo efficacy of GSK126 and romidepsin combination. NTPL-386 engrafted mice (n=5 each) were treated with GSK126 (100 mg/Kg) and/or romidepsin (2 mg/Kg) twice weekly for three weeks. Thus, one treatment cycle consisted of six doses. A separate cohort of mice were treated with four cycles of the combination, with two weeks rest between cycles. Disease progression was monitored by percentage of human cells in peripheral blood by flow cytometry. Kaplan-Meier survival estimates were plotted based on the time when mice were euthanized because they met pre-determined experimental endpoints. Compared to untreated mice, GSK126 or romidepsin treatment showed an increase in median survival by 8 days and 11 days respectively (Figure 1B, P <0.005). The combination of these drugs showed a remarkable improvement in survival (41 days). The difference in the median survival of the mice treated with the combination was statistically significant when compared to GSK126 or romidepsin (P <0.005). Further treatment for three additional cycles showed sustained remission upto 150 days post cell injection, when the study was terminated. Prolonged treatment showed a statistically significant improvement in survival compared to one treatment cycle (P <0.05). Further studies to characterize the mechanism of synergy are in progress. In summary, our data show a synergistic effect of EZH2 and HDAC class I inhibition on DS-AML growth and pave the path for clinical evaluation of this combination. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

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.

CSIG-15. DEFINING THE ROLE OF HISTONE H3 K27 METHYLATION AND H3.3 S31 PHOSPHORYLATION IN H3.3K27M MUTANT DIFFUSE MIDLINE GLIOMAS

Abstract Diffuse midline gliomas (DMG) are aggressive high-grade gliomas that characteristically occurs in children. Heterozygous K27M mutation occurs in histone H3.3 as early events in DMG and are sufficient to drive a global reduction of H3K27 methylation (H3K27me) on all histone H3 variants. Histone H3K27me is responsible for epigenetic gene repression, and loss of K27me is associated with epigenetic dysregulation. However, histone H3.3 has a unique Chk1 phosphorylation site, Serine 31, critical for mitotic checkpoint regulation. Masking of S31 phosphorylation results in the abrogation of cell cycle checkpoints that monitor the alignment and segregation of chromosomes. DMG cells have decreased levels of mitotic S31P and are chromosomally unstable and aneuploid. Genomic-editing of H3.3 to revert the K27M mutation restores mitotic S31P and K27me and significantly reduces the rate of chromosome instability (CIN). Expression of H3.3 K27M or a non-phosphorylatable S31A mutant in normal, diploid cells results in chromosome missegregation and cell cycle checkpoint defects; and cells fail to undergo G1 cycle arrest leading to aneuploidy. H3.3 K27M and S31A inhibit p53 accumulation. In a mouse model of DMG, H3.3K27M and H3.3S31A promoted the development of high-grade gliomas, whereas H3.3WT controls did not. H3.3S31A is WT for K27me, demonstrating that loss of S31P is oncogenic. It remains unclear if it is the H3.3 K27M mutations or the associated loss of H3K27me3 that drives the reduction of S31P. The PRC2 complex is responsible for H3K27me, catalyzed by methyltransferases subunits EZH1 and EZH2. We used genome editing to knockout EZH1 and EZH2 in DMG cells and untransformed human diploid cell lines. Deletion of EZH2 causes a loss in K27 tri-methylation, and EZH1/2 knockouts causes a global loss of K27me. Ongoing studies examine how the loss of K27me affects gene expression, proliferation, H3.3S31P, chromosome missegregation, and aneuploid.

Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding.

The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. We therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. We identified three distinct low-complexity regions in Ph based on sequence composition. We systematically tested the role of each of these sequences in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. Our data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. Our results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. Our observations highlight the complexity of regulation of biological condensates housed in single proteins.

MiRNA profile at diagnosis predicts treatment outcome in patients with B-chronic lymphocytic leukemia: A FILO study.

ClinicalTrials.gov, identifier NCT01370772.

A transcriptional network of cell cycle dysregulation in noninvasive papillary urothelial carcinoma

Human cancers display a restricted set of expression profiles, despite diverse mutational drivers. This has led to the hypothesis that select sets of transcription factors act on similar target genes as an integrated network, buffering a tumor’s transcriptional state. Noninvasive papillary urothelial carcinoma (NIPUC) with higher cell cycle activity has higher risk of recurrence and progression. In this paper, we describe a transcriptional network of cell cycle dysregulation in NIPUC, which was delineated using the ARACNe algorithm applied to expression data from a new cohort (n = 81, RNA sequencing), and two previously published cohorts. The transcriptional network comprised 121 transcription factors, including the pluripotency factors SOX2 and SALL4, the sex hormone binding receptors ESR1 and PGR, and multiple homeobox factors. Of these 121 transcription factors, 65 and 56 were more active in tumors with greater and less cell cycle activity, respectively. When clustered by activity of these transcription factors, tumors divided into High Cell Cycle versus Low Cell Cycle groups. Tumors in the High Cell Cycle group demonstrated greater mutational burden and copy number instability. A putative mutational driver of cell cycle dysregulation, such as homozygous loss of CDKN2A, was found in only 50% of High Cell Cycle NIPUC, suggesting a prominent role of transcription factor activity in driving cell cycle dysregulation. Activity of the 121 transcription factors strongly associated with expression of EZH2 and other members of the PRC2 complex, suggesting regulation by this complex influences expression of the transcription factors in this network. Activity of transcription factors in this network also associated with signatures of pluripotency and epithelial-to-mesenchymal transition (EMT), suggesting they play a role in driving evolution to invasive carcinoma. Consistent with this, these transcription factors differed in activity between NIPUC and invasive urothelial carcinoma.

Distinct roles for CKM–Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the cyclin-dependent kinase module Mediator complex (CKM–Mediator) has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here, we show that CKM–Mediator contributes little to three-dimensional genome organization in ESCs, but it has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CKM–Mediator, which facilitates binding of cPRC1 to its target sites. Importantly, through separation-of-function experiments, we reveal that this collaboration between CKM–Mediator and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CKM supports core Mediator engagement with gene promoters during differentiation to enable gene activation.

PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction.

The Polycomb repressive complexes PRC1, PRC2, and PR-DUB repress target genes by modifying their chromatin. In Drosophila, PRC1 compacts chromatin and monoubiquitinates histone H2A at lysine 118 (H2Aub1), whereas PR-DUB is a major H2Aub1 deubiquitinase, but how H2Aub1 levels must be balanced for Polycomb repression remains unclear. We show that in early embryos, H2Aub1 is enriched at Polycomb target genes, where it facilitates H3K27me3 deposition by PRC2 to mark genes for repression. During subsequent stages of development, H2Aub1 becomes depleted from these genes and is no longer enriched when Polycomb maintains them repressed. Accordingly, Polycomb targets remain repressed in H2Aub1-deficient animals. In PR-DUB catalytic mutants, high levels of H2Aub1 accumulate at Polycomb target genes, and Polycomb repression breaks down. These high H2Aub1 levels do not diminish Polycomb protein complex binding or H3K27 trimethylation but increase DNA accessibility. We show that H2Aub1 interferes with nucleosome stacking and chromatin fiber folding in vitro. Consistent with this, Polycomb repression defects in PR-DUB mutants are exacerbated by reducing PRC1 chromatin compaction activity, but Polycomb repression is restored if PRC1 E3 ligase activity is removed. PR-DUB therefore acts as a rheostat that removes excessive H2Aub1 that, although deposited by PRC1, antagonizes PRC1-mediated chromatin compaction.

LncRNA HOTAIR functions and therapeutic perspectives.

Long non-coding RNAs (lncRNAs) exert central pathophysiological roles through the regulation of gene expression both at transcriptional and post-transcriptional levels. The characterization of lncRNAs’ interactome is disclosing several new mechanisms that control disease onset and progression thus opening the way to the development of new pioneering therapeutic approaches. Regarding the lncRNA HOTAIR, found upregulated in several cancers and in liver fibrosis, it has been proved as a potential therapeutic target. HOTAIR acts as a ceRNA for several miRNAs and it directly interacts with chromatin remodelling complexes (e.g. PRC2 and LSD1/NuRD complexes). In this regard, we recently reported the transcription factor SNAIL-mediated recruitment of HOTAIR/PRC2 complex on specific chromatin sites causing epithelial genes’ repression through epigenetic chromatin modifications. Conversely, HOTAIR is repressed by the liver-enriched transcriptional factor HNF4a that binds to both HOTAIR promoter and distant enhancer and impairs the formation of a chromatin loop between these genomic regions.In a therapeutic perspective, we design and validated the first example of a dominant negative lncRNA molecule (HOTAIR-sbid) that covers the HOTAIR portion involved in the interaction with SNAIL while is devoid of the domain of interaction with EZH2. Functionally, HOTAIR-sbid expression impairs SNAIL/EZH2/endogenous HOTAIR interaction; thus, PRC2 complex is not recruited on SNAIL-target chromatin sites (i.e. epithelial genes’ promoters). Accordingly, the cells rescue an epithelial phenotype, reduce EMT and, in turn, migratory, invasive and anchorage independent growth abilities. This approach promises high level of specificity and limited off-target effects. Future investigations should enhance RNAs’ stability and should design strategies for the delivery of these molecules to specific target cells.

Variant Polycomb complexes in Drosophila consistent with ancient functional diversity.

Polycomb group (PcG) mutants were first identified in Drosophila on the basis of their failure to maintain proper Hox gene repression during development. The proteins encoded by the corresponding fly genes mainly assemble into one of two discrete Polycomb repressive complexes: PRC1 or PRC2. However, biochemical analyses in mammals have revealed alternative forms of PRC2 and multiple distinct types of noncanonical or variant PRC1. Through a series of proteomic analyses, we identify analogous PRC2 and variant PRC1 complexes in Drosophila, as well as a broader repertoire of interactions implicated in early development. Our data provide strong support for the ancient diversity of PcG complexes and a framework for future analysis in a longstanding and versatile genetic system.

PRC1-independent binding and activity of RYBP on the KSHV genome during de novo infection.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic virus that causes lifelong infection in humans by establishing latency after primary infection. Latent infection is a prerequisite for both persistent infection and the development of KSHV-associated cancers. While viral lytic genes are transiently expressed after primary infection, their expression is significantly restricted and concomitant with the binding of host epigenetic repressors Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2) to lytic genes. PRC1 and PRC2 mediate the repressive histone marks H2AK119ub and H3K27me3, respectively, and maintain heterochromatin structure on KSHV lytic genes to inhibit their expression. In contrast to PRC2, little is known about the recruitment and role of PRC1 factors on the KSHV genome following de novo infection. Thus, the goal of this study was to examine the function of PRC1 factors in the establishment of KSHV latency. To address this question, we performed an shRNA screen targeting 7 different components of the canonical and non-canonical PRC1 complexes during primary KSHV infection. We found that RYBP, a main subunit of the non-canonical PRC1 complexes, is a potent repressor of KSHV lytic genes that can bind to the viral genome and inhibit lytic genes as early as 4 hours post infection. Surprisingly, our ChIP analyses showed that RYBP binds to lytic viral gene promoters in a PRC1-independent manner, does not affect PRC1 activity on the KSHV genome, and can reduce the level of histone marks associated with transcription elongation. Our data also suggest that RYBP can repress the viral lytic cycle after primary infection by inhibiting the transcription elongation of the lytic cycle inducer KSHV gene RTA. Based on our results we propose that RYBP uses a PRC1-independent mechanism to block KSHV RTA expression thereby promoting the establishment of KSHV latency following de novo infection.

Rif1 interacts with non-canonical polycomb repressive complex PRC1.6 to regulate mouse embryonic stem cells fate potential

Mouse embryonic stem cells (mESCs) cycle in and out of a transient 2-cell (2C)-like totipotent state, driven by a complex genetic circuit involves both the coding and repetitive sections of the genome. While a vast array of regulators, including the multi-functional protein Rif1, has been reported to influence the switch of fate potential, how they act in concert to achieve this cellular plasticity remains elusive. Here, by modularizing the known totipotency regulatory factors, we identify an unprecedented functional connection between Rif1 and the non-canonical polycomb repressive complex PRC1.6. Downregulation of the expression of either Rif1 or PRC1.6 subunits imposes similar impacts on the transcriptome of mESCs. The LacO-LacI induced ectopic colocalization assay detects a specific interaction between Rif1 and Pcgf6, bolstering the intactness of the PRC1.6 complex. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) analysis further reveals that Rif1 is required for the accurate targeting of Pcgf6 to a group of genomic loci encompassing many genes involved in the regulation of the 2C-like state. Depletion of Rif1 or Pcgf6 not only activates 2C genes such as Zscan4 and Zfp352, but also derepresses a group of the endogenous retroviral element MERVL, a key marker for totipotency. Collectively, our findings discover that Rif1 can serve as a novel auxiliary component in the PRC1.6 complex to restrain the genetic circuit underlying totipotent fate potential, shedding new mechanistic insights into its function in regulating the cellular plasticity of embryonic stem cells.

Molecular mechanisms of the Polycomblike family proteins in epigenetic regulation and their biological functions

<p indent="0mm">The PRC2 complex is a major complex in the Polycomb group of proteins. It plays an essential role in cell proliferation, differentiation, and the maintenance of cellular identity. PRC2 functions in gene repression mainly by modifying chromatin and regulating chromatin structure, among which the tri-methylation mark on histone H3 at Lys27 (H3K27me3) added by EZH2, the core PRC2 component, is a hallmark of gene repression. PRC2 core components must bind PRC2-associated proteins to form a holo-enzyme and become fully active. PRC2-associated proteins, such as Polycomblike (PCL) proteins, are important in regulating the function of PRC2. Recent studies have revealed that human PCL proteins play key roles in regulating the activity and the recruitment of chromatin by PRC2. In addition, PCL proteins are closely related to several human cancers. Thus, advances in the structure and function of PCL proteins would facilitate our understanding of their roles in PRC2 regulation and would expedite the development of strategies for therapeutically targeting PCL-related cancers. In this review, we summarize recent progress on the structure and function of PCL proteins, with an emphasis on the molecular mechanisms and biological functions of human PCL proteins.

Identification of Two Non-Peptidergic Small Molecule Inhibitors of CBX2 Binding to K27 Trimethylated Oligonucleosomes.

The dysregulation of the PRC1/2 complex plays a key role in lineage plasticity in prostate cancer and may be required to maintain neuroendocrine phenotype. [1] CBX2, a key component of the canonical PRC1 complex, is an epigenetic reader, recognizing trimethylated lysine on histone 3 (H3K27me3) [2] and is overexpressed in metastatic neuroendocrine prostate cancer. [3,4] We implemented a screening strategy using nucleosome substrates to identify inhibitors of CBX2 binding to chromatin. Construct design and phosphorylation state of CBX2 were critical for successful implementation and execution of an HTS library screen. A rigorous screening funnel including counter and selectivity assays allowed us to quickly focus on true positive hit matter. Two distinct non-peptide-like chemotypes were identified and confirmed in orthogonal biochemical and biophysical assays demonstrating disruption of CBX2 binding to nucleosomes and direct binding to purified CBX2, respectively.

Replication protein RPA2A regulates floral transition by cooperating with PRC2 in Arabidopsis.

RPA2A is a subunit of the conserved heterotrimeric replication protein A (RPA) in Arabidopsis, which is an essential replisome component that binds to single-stranded DNA during DNA replication. RPA2A controls a set of developmental processes, but the underlying mechanism is largely unknown. Here we show that RPA2A represses key flowering genes including FLOWERING LOCUS T (FT), AGAMOUS (AG) and AGAMOUS LIKE 71 (AGL71) to suppress floral transition by cooperating with the PRC2 complex. RPA2A is vigorously expressed in dividing cells and required for correct DNA replication. Mutation of RPA2A leads to early flowering, which is dependent on ectopic expression of key flowering genes including FT molecularly and genetically. RPA2A and PRC2 have common target genes including FT, AG and AGL71 supported using genetic analysis, transcriptome profiling and H3K27me3 ChIP-seq analysis. Furthermore, RPA2A physically interacts with PRC2 components CLF, EMF2 and MSI1, which recruits CLF to the chromatin loci of FT, AG and AGL71. Together, our results show that the replication protein RPA2A recruits PRC2 to key flowering genes through physical protein interaction, thereby repressing the expression of these genes to suppress floral transition in Arabidopsis.

CUL4B Upregulates RUNX2 to Promote the Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Epigenetically Repressing the Expression of miR-320c and miR-372/373-3p.

Mesenchymal stem cells (MSCs) within the periodontal ligament (PDL), termed periodontal ligament stem cells (PDLSCs), have a self-renewing capability and a multidirectional differentiation potential. The molecular mechanisms that regulate multidirectional differentiation, such as the osteogenic differentiation of PDLSCs, remain to be elucidated. Cullin 4B (CUL4B), which assembles the CUL4B-RING ubiquitin ligase (CRL4B) complex, is involved in regulating a variety of developmental and physiological processes including the skeletal development and stemness of cancer stem cells. However, nothing is known about the possible role of CUL4B in the osteogenic differentiation of PDLSCs. Here, we found that knockdown of CUL4B decreased the proliferation, migration, stemness and osteogenic differentiation ability of PDLSCs. Mechanistically, we demonstrate that CUL4B cooperates with the PRC2 complex to repress the expression of miR-320c and miR-372/373-3p, which results in the upregulation of RUNX2, a master transcription factor (TF) that regulates osteogenic differentiation. In brief, the present study reveals the role of CUL4B as a new regulator of osteogenic differentiation in PDLSCs.

Abstract 5435: CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine

Abstract INTRODUCTION: We recently found that the atypical chemokine receptor, CCRL2 promotes the growth of MDS and secondary AML (sAML) while CCRL2 knockdown inhibits their growth. The aim of the current study is to investigate if CCRL2 regulates pathways associated with the development of resistance to hypomethylating agents (HMA), commonly used drugs in MDS and occasionally sAML. MATERIALS AND METHODS: We used lentivirus-mediated transduction of MDS92, MDS-L and TF-1 MDS/sAML cells to suppress CCRL2 expression. Two different shRNA constructs were used. We performed RNA sequencing and gene-set enrichment analysis of CCRL2 knocked down (KD) and wild-type (WT) TF-1 cells. We measured DNA methyl-transferases’ expression by western blot, CD11b and CD71 expression was measured in MDS and sAML cells to assess cell differentiation. Apoptosis was measured by Annexin V/PI staining, and clonogenicity by methylcellulose assays. CD34+ cells were sorted from bone marrow aspirates of MDS patients before the initiation of treatment with HMA by using magnetic beads and measurement of CCRL2 was performed by flow cytometry. Response to HMA in MDS patients was assessed by 6 months of treatment based on the International Working Group response criteria. RESULTS: CCRL2 KD cells demonstrated suppression of pathways associated with PRC2 complex activity, histone modification, and DNA methylation. CCRL2 KD also lead to a more prominent degradation of DNMT1, DNMT3A and DNTM3B under azacitidine treatment. CCRL2 increased apoptosis in response to 0.5 and 1 μM azacitidine (P&amp;lt;0.010) and MDS-L cells (P&amp;lt;0.010 with both sh1 and sh2). Similarly, CCRL2 knockdown increased morphologic differentiation with both 0.5 and 1 μΜ azacitidine (P&amp;lt;0.010) as well as increased the clonogenic inhibition caused by azacitidine (P&amp;lt;0.05). In order to analyze the effect CCRL2 clinically, we analyzed CCRL2 expression in CD34+ cells from patients undergoing HMA treatment. Non-responders to HMA (progressive disease) express higher levels of CCRL2 compared to CD34+ cells from responders (complete remission, partial remission or stable disease) (P=0.020). DISCUSSION: Our analysis suggests that CCRL2 regulates the expression of genes associated with induction of PRC2-mediated histone modification and DNA methylation in sAML cells. CCRL2 suppression also increased the sensitivity of MDS and sAML cells to azacitidine. In addition, increased CCRL2 expression in MDS cells is associated with worse response to HMA. These data suggest that targeting CCRL2 has therapeutic potential in MDS and sAML. Citation Format: Theodoros Karantanos, Patric Teodorescu, Brandy Perkins, Marios Arvanitis, Ilias Christodolou, Christopher Esteb, W. Brian Dalton, Tania Jain, Amy E. DeZern, Lukasz P. Gondek, Mark J. Levis, Gabriel Ghiaur, Richard J. Jones. CCRL2 affects the sensitivity of MDS and secondary AML to azacitidine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5435.

Abstract 6084: Recurrent genomic patterns of MPNST evolution correlate with clinical outcome

Abstract Neurofibromatosis type 1 (NF1) is the most common tumor predisposition syndrome, and is associated with an aggressive soft-tissue sarcoma, malignant peripheral nerve sheath tumours (MPNSTs), the greatest cause of mortality in people with NF1. The only potentially curative therapy involves en bloc resection with negative margins, which is not always appropriate. Even therapy with curative intent is associated with poor overall survival for both sporadic and NF1-related MPNSTs. The development of novel therapies has been largely hindered by a poor understanding of the molecular events underpinning MPNST pathogenesis. We report a comprehensive multi-omic study of MPNST evolution based on whole genome sequencing, transcriptomic and methylation profiling data on 95 tumors (64 NF1-related; 31 sporadic). In all cases, the early events in MPNST evolution involve biallelic inactivation of NF1 followed by inactivation of CDKN2A, as well as mutations in TP53 or PRC2 complex genes in a subset of cases. Analysis of the genomic architecture revealed distinct pathways of tumor evolution that can be identified through H3K27 trimethylation (H3K27me3) status. Integration of these data allows us to propose several mechanistic tumor evolution models. Tumors with H3K27me3 loss evolve through extensive copy number aberrations (CNAs) including haploidization followed by whole genome doubling and chromosome 8 amplifications, whereas tumors with H3K27me3 retention evolve through extensive chromosome instability and chromothripsis. Taken together, these genome-wide CNA profiles act as a surrogate for the loss of H3K27me3 status and correlate with prognosis, suggesting that CNA profiling of cell-free DNA could be incorporated in clinical decision-making. Citation Format: Isidro Cortes Ciriano, Chris D. Steele, Katherine Piculell, Alyaa Al-Ibraheemi, Vanessa Eulo, Marilyn M. Bui, Aikaterini Chatzipli, Brendan C. Dickson, Dana C. Borcherding, Alon Galor, Jesse Hart, Andrew Feber, Kevin B. Jones, Justin T. Jordan, Raymond H. Kim, Daniel Lindsay, Colin Miller, Yoshihiro Nishida, Jonathan Serrano, Nicole J. Ullrich, David Viskochil, Xia Wang, Matija Snuderl, Paula Proszek, Peter J. Park, Adrienne M. Flanagan, Angela C. Hirbe, Nischalan Pillay, David T. Miller, The Genomics of MPNST (GeM) Consortium. Recurrent genomic patterns of MPNST evolution correlate with clinical outcome [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6084.