Component Of Polycomb Repressive Complex Research Articles

Tazemetostat for Advanced Epithelioid Sarcoma: Current Status and Future Perspectives

Epithelioid sarcoma (ES) is an aggressive ultra-rare soft-tissue sarcoma marked by SMARCB1/INI1 deficiency. SMARCB1/INI1 deficiency leads to elevated expression of EZH2, a component of polycomb repressive complex 2, which mediates gene silencing by catalyzing H3K27me3. Tazemetostat is an oral, SAM-competitive inhibitor of EZH2, whose blockade prevents the methylation of histone H3K27, thus decreasing the growth of EZH2 mutated or over-expressing cancer cells. Tazemetostat has been approved for the treatment of patients aged 16 years and older with metastatic or advanced ES not eligible for complete resection, based on the positive results of a single-arm Phase II basket study. Tazemetostat though represents a new treatment option for ES patients, although clinical/molecular predictors of response are still to be identified. The combination of tazemetostat with other drugs like doxorubicin and immunotherapeutic agents is currently under investigation in ES patients.

MODL-04. MODELING CNS HGNET-BCOR PATHOGENESIS USING NEURAL STEM CELLS

Central nervous system high-grade neuroepithelial tumor with BCL6-corepressor alteration (CNS HGNET-BCOR) is a recently identified entity characterized by internal tandem duplication (ITD) of BCOR, a core component of polycomb repressive complex (PRC) 1.1. BCOR-ITD exclusively occurs within an essential binding domain, suggesting aberrant epigenetic activities as a possible mechanism of gliomagenesis; however, the effect of this alteration on the transcriptome and DNA methylation are poorly understood. We have generated new CNS HGNET-BCOR models by lentiviral transduction of the BCOR-ITD into human and murine neural stem cells. In the human model, qRT-PCR and subsequent RNA-seq identified a transient derepression of PRC2-target genes comparing to an isogenic model with overexpression of wildtype-BCOR. A similar effect was found in clinical specimens from previous studies. In the murine-cell model, we confirmed increased clonogenicity in soft-agar assays, and tumors developed in mice flanks. Global DNA methylation levels evaluated by ELISA were significantly lower than those of parent cells, and 177 genes were differentially expressed on RNA-seq analysis comparing to BCOR-overexpressing control cells, including upregulation of known oncogenes. These results suggest that BCOR-ITD and associated alterations in the function of PRC1.1 affect methylation patterns in neural stem cells, driving transcriptional changes and oncogenic transformation into CNS HGNET-BCOR. More detailed analyses, including methylation arrays comparisons with clinical samples and in-silico drug sensitivity testing, are being performed.

Mutation of the imprinted gene OsEMF2a induces autonomous endosperm development and delayed cellularization in rice.

In angiosperms, endosperm development comprises a series of developmental transitions controlled by genetic and epigenetic mechanisms that are initiated after double fertilization. Polycomb repressive complex 2 (PRC2) is a key component of these mechanisms that mediate histone H3 lysine 27 trimethylation (H3K27me3); the action of PRC2 is well described in Arabidopsis thaliana but remains uncertain in cereals. In this study, we demonstrate that mutation of the rice (Oryza sativa) gene EMBRYONIC FLOWER2a (OsEMF2a), encoding a zinc-finger containing component of PRC2, causes an autonomous endosperm phenotype involving proliferation of the central cell nuclei with separate cytoplasmic domains, even in the absence of fertilization. Detailed cytological and transcriptomic analyses revealed that the autonomous endosperm can produce storage compounds, starch granules, and protein bodies specific to the endosperm. These events have not been reported in Arabidopsis. After fertilization, we observed an abnormally delayed developmental transition in the endosperm. Transcriptome and H3K27me3 ChIP-seq analyses using endosperm from the emf2a mutant identified downstream targets of PRC2. These included >100 transcription factor genes such as type-I MADS-box genes, which are likely required for endosperm development. Our results demonstrate that OsEMF2a-containing PRC2 controls endosperm developmental programs before and after fertilization.

Morphine leads to global genome changes in H3K27me3 levels via a Polycomb Repressive Complex 2 (PRC2) self-regulatory mechanism in mESCs

BackgroundEnvironmentally induced epigenetic changes can lead to health problems or disease, but the mechanisms involved remain unclear. Morphine can pass through the placental barrier leading to abnormal embryo development. However, the mechanism by which morphine causes these effects and how they sometimes persist into adulthood is not well known. To unravel the morphine-induced chromatin alterations involved in aberrant embryo development, we explored the role of the H3K27me3/PRC2 repressive complex in gene expression and its transmission across cellular generations in response to morphine.ResultsUsing mouse embryonic stem cells as a model system, we found that chronic morphine treatment induces a global downregulation of the histone modification H3K27me3. Conversely, ChIP-Seq showed a remarkable increase in H3K27me3 levels at specific genomic sites, particularly promoters, disrupting selective target genes related to embryo development, cell cycle and metabolism. Through a self-regulatory mechanism, morphine downregulated the transcription of PRC2 components responsible for H3K27me3 by enriching high H3K27me3 levels at the promoter region. Downregulation of PRC2 components persisted for at least 48 h (4 cell cycles) following morphine removal, though promoter H3K27me3 levels returned to control levels.ConclusionsMorphine induces targeting of the PRC2 complex to selected promoters, including those of PRC2 components, leading to characteristic changes in gene expression and a global reduction in H3K27me3. Following morphine removal, enhanced promoter H3K27me3 levels revert to normal sooner than global H3K27me3 or PRC2 component transcript levels. We suggest that H3K27me3 is involved in initiating morphine-induced changes in gene expression, but not in their maintenance.Graphic abstractModel of Polycomb repressive complex 2 (PRC2) and H3K27me3 alterations induced by chronic morphine exposure. Morphine induces H3K27me3 enrichment at promoters of genes encoding core members of the PRC2 complex and is associated with their transcriptional downregulation.

Critical regulation of a NDIME/MEF2C axis in embryonic stem cell neural differentiation and autism.

A microdeletion within human chromosome 5q14.3 has been associated with the occurrence of neurodevelopmental disorders, such as autism and intellectual disability, and MEF2C haploinsufficiency was identified as main cause. Here, we report that a brain-enriched long non-coding RNA,NDIME, is located near the MEF2C locus and is required for normal neural differentiation of mouse embryonic stem cells (mESCs). NDIME interacts with EZH2, the major component of polycomb repressive complex 2 (PRC2), and blocks EZH2-mediated trimethylation of histone H3 lysine 27 (H3K27me3) at the Mef2c promoter, promoting MEF2C transcription. Moreover, the expression levels of both NDIME and MEF2C were strongly downregulated in the hippocampus of a mouse model of autism, and the adeno-associated virus (AAV)-mediated expression of NDIME in the hippocampus of these mice significantly increased MEF2C expression and ameliorated autism-like behaviors. The results of this study reveal an epigenetic mechanism by which NDIME regulates MEF2C transcription and neural differentiation and suggest potential effects and therapeutic approaches of the NDIME/MEF2C axis in autism.

Antihistamine Drug Ebastine Inhibits Cancer Growth by Targeting Polycomb Group Protein EZH2.

Enhancer of zester homolog 2 (EZH2), a histone lysine methyltransferase and the catalytic component of polycomb repressive complex 2, has been extensively investigated as a chromatin regulator and a transcriptional suppressor by methylating H3 at lysine 27 (H3K27). EZH2 is upregulated or mutated in most cancers, and its expression levels are negatively associated with clinical outcomes. However, the current developed small-molecule inhibitors targeting EZH2 enzymatic activities could not inhibit the growth and progression of solid tumors. Here, we discovered an antihistamine drug, ebastine, as a novel EZH2 inhibitor by targeting EZH2 transcription and subsequently downregulating EZH2 protein level and H3K27 trimethylation in multiple cancer cell lines at concentrations below 10 μmol/L. The inhibition of EZH2 by ebastine further impaired the progression, migration, and invasiveness of these cancer cells. Overexpression of Ezh2 wild-type and its mutant, H689A (lacking methyltransferase activity), rescued the neoplastic properties of these cancer cells after ebastine treatment, suggesting that EZH2 targeted by ebastine is independent of its enzymatic function. Next-generation RNA-sequencing analysis also revealed that C4-2 cells treated with 8 μmol/L ebastine showed a gene profiling pattern similar to EZH2-knockdown C4-2 cells, which was distinctively different from cells treated with GSK126, an EZH2 enzyme inhibitor. In addition, ebastine treatment effectively reduced tumor growth and progression, and enhanced progression-free survival in triple-negative breast cancer and drug-resistant castration-resistant prostate cancer patient-derived xenograft mice. Our data demonstrated that ebastine is a novel, safe, and potent anticancer agent for patients with advanced cancer by targeting the oncoprotein EZH2.

Polycomb Repressive Complex 2-mediated histone modification H3K27me3 is associated with embryogenic potential in Norway spruce.

Epigenetic reprogramming during germ cell formation is essential to gain pluripotency and thus embryogenic potential. The histone modification H3K27me3, which is catalysed by the Polycomb repressive complex 2 (PRC2), regulates important developmental processes in both plants and animals, and defects in PRC2 components cause pleiotropic developmental abnormalities. Nevertheless, the role of H3K27me3 in determining embryogenic potential in gymnosperms is still elusive. To address this, we generated H3K27me3 profiles of Norway spruce (Picea abies) embryonic callus and non-embryogenic callus using CUT&RUN, which is a powerful method for chromatin profiling. Here, we show that H3K27me3 mainly accumulated in genic regions in the Norway spruce genome, similarly to what is observed in other plant species. Interestingly, H3K27me3 levels in embryonic callus were much lower than those in the other examined tissues, but markedly increased upon embryo induction. These results show that H3K27me3 levels are associated with the embryogenic potential of a given tissue, and that the early phase of somatic embryogenesis is accompanied by changes in H3K27me3 levels. Thus, our study provides novel insights into the role of this epigenetic mark in spruce embryogenesis and reinforces the importance of PRC2 as a key regulator of cell fate determination across different plant species.

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

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

PRC2 insufficiency causes p53-dependent dyserythropoiesis in myelodysplastic syndrome

EZH1 and EZH2 are enzymatic components of polycomb repressive complex (PRC) 2, which catalyzes histone H3K27 tri-methylation (H3K27me3) to repress the transcription of PRC2 target genes. We previously reported that the hematopoietic cell-specific Ezh2 deletion (Ezh2Δ/Δ) induced a myelodysplastic syndrome (MDS)-like disease in mice. We herein demonstrated that severe PRC2 insufficiency induced by the deletion of one allele Ezh1 in Ezh2-deficient mice (Ezh1+/-Ezh2Δ/Δ) caused advanced dyserythropoiesis accompanied by a differentiation block and enhanced apoptosis in erythroblasts. p53, which is activated by impaired ribosome biogenesis in del(5q) MDS, was specifically activated in erythroblasts, but not in hematopoietic stem or progenitor cells in Ezh1+/-Ezh2Δ/Δ mice. Cdkn2a, a major PRC2 target encoding p19Arf, which activates p53 by inhibiting MDM2 E3 ubiquitin ligase, was de-repressed in Ezh1+/-Ezh2Δ/Δ erythroblasts. The deletion of Cdkn2a as well as p53 rescued dyserythropoiesis in Ezh1+/-Ezh2Δ/Δ mice, indicating that PRC2 insufficiency caused p53-dependent dyserythropoiesis via the de-repression of Cdkn2a. Since PRC2 insufficiency is often involved in the pathogenesis of MDS, the present results suggest that p53-dependent dyserythropoiesis manifests in MDS in the setting of PRC2 insufficiency.

Abstract 2415: H2A monoubiquitination links glucose availability to epigenetic regulation of the endoplasmic reticulum stress induced cell death in cancer

Abstract Introduction Epigenetic regulation of gene transcription coordinates with nutrient availability, although the underlying mechanisms remain incompletely understood. Histone 2A monoubiquitination at lysine 119 (H2Aub), catalyzed mainly by its writer polycomb repressive complex 1 (PRC1), is a histone modification that has been associated with transcriptional repression. Initially, we found glucose withdrawal dramatically decreased H2Aub levels in multiple cancer cell lines. Next, we sought to study the role of H2Aub and H2Aub-associated transcriptional network in tumor cells while modulating glucose availability. Experimental design To determine the genes regulated by H2Aub upon glucose deprivation, H2Aub ChIP-seq and RNA-seq analyses were performed in UMRC6 kidney cancer cells with or without glucose. To identify the biological processes associated with the genes identified in our genome-wide analyses, we used Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. Results We found that glucose starvation suppresses histone 2A K119 monoubiquitination (H2Aub), a histone modification that correlates with gene repression. Mechanistically, glucose starvation suppressed H2Aub levels independently of energy stress-mediated AMPK activation and likely acts through reactive oxygen species (ROS)-mediated inhibition of BMI1, an integral component of polycomb repressive complex 1 (PRC1) that catalyzes H2Aub on chromatin. Integrated transcriptomic and epigenomic analyses linked glucose starvation-mediated H2Aub repression to the activation of genes involved in the endoplasmic reticulum (ER) stress response. We further showed that this epigenetic mechanism has a role in glucose starvation-induced cell death. In addition, pharmacologic inhibition of glucose transporter 1 (GLUT1) and PRC1 synergistically promoted ER stress and suppressed tumor growth in vitro and in vivo. Conclusion Our study suggests a model in which glucose starvation inhibits PRC1 function potentially through glucose starvation-induced BMI phosphorylation and thereby decreases H2Aub levels, which leads to the de-repression of ER stress gene expression and contributes to the ER stress response and glucose starvation-induced cell death. Together, our results reveal a hitherto unrecognized epigenetic mechanism coupling glucose availability to the ER stress response. Citation Format: Yilei Zhang, Jiejun Shi, Zhenna Xiao, Guang Lei, Xiaoguang Liu, Hyemin Lee, Pranavi Koppula, Weijie Cheng, Chao Mao, Li Zhuang, Li Ma, Wei Li, Boyi Gan. H2A monoubiquitination links glucose availability to epigenetic regulation of the endoplasmic reticulum stress induced cell death in cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2415.

Dual Regulation of Histone Methylation by mTOR Complexes Controls Glioblastoma Tumor Cell Growth via EZH2 and SAM.

Epigenetic regulation known for DNA methylation and histone modification is critical for securing proper gene expression and chromosomal function, and its aberration induces various pathologic conditions including cancer. Trimethylation of histone H3 on lysine 27 (H3K27me3) is known to suppress various genes related to cancer cell survival and the level of H3K27me3 may have an influence on tumor progression and malignancy. However, it remains unclear how histone methylation is regulated in response to genetic mutation and microenvironmental cues to facilitate the cancer cell survival. Here, we report a novel mechanism of the specific regulation of H3K27me3 by cooperatively two mTOR complexes, mTORC1 and mTORC2 in human glioblastoma (GBM). Integrated analyses revealed that mTORC1 upregulates the protein expression of enhancer of zeste homolog 2, a main component of polycomb repressive complex 2 which is known as H3K27-specific methyltransferase. The other mTOR complex, mTORC2, regulates production of S-adenosylmethionine, an essential substrate for histone methylation. This cooperative regulation causes H3K27 hypermethylation which subsequently promotes tumor cell survival both in vitro and in vivo xenografted mouse tumor model. These results indicate that activated mTORC1 and mTORC2 complexes cooperatively contribute to tumor progression through specific epigenetic regulation, nominating them as an exploitable therapeutic target against cancer. IMPLICATIONS: A dynamic regulation of histone methylation by mTOR complexes promotes tumor growth in human GBM, but at the same time could be exploitable as a novel therapeutic target against this deadly tumor.

Deregulation of Polycomb Repressive Complex-2 in Mantle Cell Lymphoma Confers Growth Advantage by Epigenetic Suppression of cdkn2b.

The polycomb repressive complex 2 (PRC2) maintains the transcriptional repression of target genes through its catalytic component enhancer of zeste homolog 2 (EZH2). Through modulating critical gene expression, EZH2 also plays a role in cancer development and progression by promoting cancer cell survival and invasion. Mutations in EZH2 are prevalent in certain B-cell lymphoma subtypes such as diffuse large cell lymphoma and follicular lymphoma; while no EZH2 mutation has been reported in the mantle cell lymphoma (MCL). Here we demonstrate that the PRC2 components EZH2, EED and SUZ12 are upregulated in the MCL cells as compared to normal B-cells. Moreover, stably transfected cells with wild-type EZH2 or-EED showed increased cell growth and H3K27-trimehtylation. However, unlike wild-type EZH2, ectopic expression of a deletion construct of EZH2 (EZH2Δ550−738 lacking SET domain) had no growth advantage over control cells. Pharmacological inhibition of EZH2 suppressed H3K27me3 and had significant inhibitory effect on cell growth and colony forming capacity (p < 0.05) of MCL cells, and this effect was more or less comparable to the anti-proliferative effects of EZH2 inhibition in cells harboring EZH2-mutation. Mechanistically, EZH2 appears to downregulate expression of cdkn2b gene via enhanced H3K27me3, a well-known suppressive epigenetic mark, at the cdkn2b promoter region. Overall, these results highlight that deregulation of PRC2/EZH2 is associated with epigenetic suppression of cdkn2b in MCL, and in part responsible for increased cell growth, thus the EZH2 inhibitors may have therapeutic potential in the patients with MCL.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

Polycomb-like 2 regulates PRC2 components to affect proliferation in glioma cells

IntroductionThe Polycomb group (PcG) is an important family of transcriptional regulators that controls growth and tumorigenesis. The PcG mainly consists of two complexes, PRC1 and Polycomb Repressive Complex 2 (PRC2). Polycomb-like 2 (PCL2) is known to interact with the PRC2 protein. The role of PCL2 in the development and progression of glioma is unclear.MethodsWe use The Cancer Genome Atlas (TCGA) database to detect the expression of PCL2 in various tumors. 117 cases of clinical glioma (WHOI–IV) were collected, and PCL2 expression and localization were detected by immunohistochemical staining. Glioma cells U87/U251 were infected with overexpressed and interfered PCL2. CCK8 assay, colony formation assay, EdU method, cell cycle and apoptosis were used to detect cell proliferation and apoptosis. Western blot was used to detect the expression of PRC2-related core proteins. After DZNeP intervention, PRC2 protein expression was again measured to discuss the mechanism of PCL2 action.ResultsTCGA database results and immunohistochemical staining results suggest that PCL2 is highly expressed in gliomas. We found that the PCL2 gene promoted tumor cell proliferation, enhanced the colony formation ability, and increased S phase in the cell cycle. The overexpression of PCL2 upregulated the expression levels of EZH2 and EED (two core members of PRC2), decreased the expression of SUZ12, increased the level of H3K27 trimethylation (H3K27me3), H3K4 dimethylation (H3K4me2), and decreased H3K9 dimethylation (H3K9me2). The result after interfering with PCL2 was the opposite.ConclusionsAs an important accessory protein of PRC2, PCL2 can not only change the expression of PRC2 components, but also affect the expression level of Histone methylation. Therefore, PCL2 may be an important hub for regulating the synergy among PRC2 members. This study revealed PCL2 as a new target for tumor research and open up a new avenue for future research in glioma.

SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin

Chromatin integrity is essential for cellular homeostasis. Polycomb group proteins modulate chromatin states and transcriptionally repress developmental genes to maintain cell identity. They also repress repetitive sequences such as major satellites and constitute an alternative state of pericentromeric constitutive heterochromatin at paternal chromosomes (pat‐PCH) in mouse pre‐implantation embryos. Remarkably, pat‐PCH contains the histone H3.3 variant, which is absent from canonical PCH at maternal chromosomes, which is marked by histone H3 lysine 9 trimethylation (H3K9me3), HP1, and ATRX proteins. Here, we show that SUMO2‐modified CBX2‐containing Polycomb Repressive Complex 1 (PRC1) recruits the H3.3‐specific chaperone DAXX to pat‐PCH, enabling H3.3 incorporation at these loci. Deficiency of Daxx or PRC1 components Ring1 and Rnf2 abrogates H3.3 incorporation, induces chromatin decompaction and breakage at PCH of exclusively paternal chromosomes, and causes their mis‐segregation. Complementation assays show that DAXX‐mediated H3.3 deposition is required for chromosome stability in early embryos. DAXX also regulates repression of PRC1 target genes during oogenesis and early embryogenesis. The study identifies a novel critical role for Polycomb in ensuring heterochromatin integrity and chromosome stability in mouse early development.

DNA Methylation Signature for EZH2 Functionally Classifies Sequence Variants in Three PRC2 Complex Genes

Weaver syndrome (WS), an overgrowth/intellectual disability syndrome (OGID), is caused by pathogenic variants in the histone methyltransferase EZH2, which encodes a core component of the Polycomb repressive complex-2 (PRC2). Using genome-wide DNA methylation (DNAm) data for 187 individuals with OGID and 969 control subjects, we show that pathogenic variants in EZH2 generate a highly specific and sensitive DNAm signature reflecting the phenotype of WS. This signature can be used to distinguish loss-of-function from gain-of-function missense variants and to detect somatic mosaicism. We also show that the signature can accurately classify sequence variants in EED and SUZ12, which encode two other core components of PRC2, and predict the presence of pathogenic variants in undiagnosed individuals with OGID. The discovery of a functionally relevant signature with utility for diagnostic classification of sequence variants in EZH2, EED, and SUZ12 supports the emerging paradigm shift for implementation of DNAm signatures into diagnostics and translational research.

PRC2 Components Maintain DNA Hypermethylation of the Upstream Promoter and Regulate Robo4 Expression in Endothelial Cells.

Roundabout4 (Robo4) is an endothelial cell-specific protein that stabilizes the vasculature in pathological angiogenesis and inflammation. We previously determined a 3-kb Robo4 promoter and demonstrated the importance of the upstream region for nuclear factor-kappaB (NF-κB)-mediated promoter activation induced by tumor necrosis factor α (TNFα). This region contains unique genomic features, including promoter region-specific DNA hypermethylation and chromatin condensation; however, the function of the region remains poorly understood. In this study, we analyzed the DNA sequences of the region and identified a motif for polycomb repressive complex 2 (PRC2). Chromatin immunoprecipitation assay indicates the binding of the PRC2 component, SUZ12, to the motif. A mutation in the motif decreased DNA methylation in embryonic stem cells and increased Robo4 promoter activity in endothelial cells. An inhibitor for the PRC2 component, EZH2, induced the promoter activity and expression of Robo4 in endothelial cells treated with or without TNFα. Taken together, these results indicate that the PRC2 components maintain DNA hypermethylation and suppress Robo4 expression via the PRC2 binding motif in the upstream promoter.

Memory of Divisional History Directs the Continuous Process of Primitive Hematopoietic Lineage Commitment.

SummaryHematopoietic stem cells (HSCs) exist in a dormant state and progressively lose regenerative potency as they undergo successive divisions. Why this functional decline occurs and how this information is encoded is unclear. To better understand how this information is stored, we performed RNA sequencing on HSC populations differing only in their divisional history. Comparative analysis revealed that genes upregulated with divisions are enriched for lineage genes and regulated by cell-cycle-associated transcription factors, suggesting that proliferation itself drives lineage priming. Downregulated genes are, however, associated with an HSC signature and targeted by the Polycomb Repressive Complex 2 (PRC2). The PRC2 catalytic subunits Ezh1 and Ezh2 promote and suppress the HSC state, respectively, and successive divisions cause a switch from Ezh1 to Ezh2 dominance. We propose that cell divisions drive lineage priming and Ezh2 accumulation, which represses HSC signature genes to consolidate information on divisional history into memory.

The Effects of Ezh2 Mutation on Kras‐Driven Adenocarcinoma

Lung cancer is the deadliest form of cancer worldwide. Non‐small cell lung cancer (NSCLC) is the most common type of lung cancer, constituting an estimated 84% of diagnoses. Conducting research regarding the metastasis of lung adenocarcinomas is especially pertinent, as the 5‐year survival rate for localized NSCLC is an estimated 50% versus 6% in metastatic lung cancer. Here we investigate the activation effects of Polycomb Repressive Complex 2 (PRC2), an epigenetic regulator commonly found to be dysregulated in human lung cancer, as a driver of metastasis. We employed genetically engineered mouse models harboring activating point mutations in Kras and inactivating mutations in the p53 pathway (KP model) which develop lung adenocarcinomas but exhibit low penetrance and long latency for metastasis. As a resolution, our lab has generated a KPE mouse model which carries a mutation that induces Ezh2 activation, a core component of PRC2. We hypothesize that KPE is an effective and robust metastatic model which displays a shorter latency period than previously established lung adenocarcinoma models. In this project we used a histological approach to characterize morphological properties of primary tumors as well as distinct lymph node metastases in KPE mouse models. By visualizing cellular phenotypes through Hematoxylin &amp; Eosin staining, we intended to quantify tumor area and composition of lymphatic micrometastases. The results illustrated that mice with the Ezh2 mutation in addition to the Kras and p53 mutations experienced a shorter latency period as well as a higher occurrence of lymph node metastases as opposed to the KP model.Support or Funding InformationThis project is supported in part by National Science Foundation Grant 1758007.

EZH2 function in immune cell development.

The polycomb repressive complex 2 (PRC2) consists of three core components EZH2, SUZ12 and EED. EZH2 catalyzes the methylation of lysine 27 of histone H3, a modification associated with gene silencing. Through gene duplication higher vertebrate genomes also encode a second partially redundant methyltransferase, EZH1. Within the mammalian immune system most research has concentrated on EZH2 which is expressed predominantly in proliferating cells. EZH2 and other PRC2 components are required for hematopoietic stem cell function and lymphocyte development, at least in part by repressing cell cycle inhibitors. At later stages of immune cell differentiation, EZH2 plays essential roles in humoral and cell-mediated adaptive immunity, as well as the maintenance of immune homeostasis. EZH2 is often overactive in cancers, through both gain-of-function mutations and over-expression, an observation that has led to the development and clinical testing of specific EZH2 inhibitors. Such inhibitors may also be of use in inflammatory and autoimmune settings, as EZH2 inhibition dampens the immune response. Here, we will review the current state of understanding of the roles for EZH2, and PRC2 more generally, in the development and function of the immune system.