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The characteristic features of diabetic nephropathy include thickening of the glomerular basement membranes, expansion of mesangium, and appearance of albumin in the urine (microalbuminuria and macroalbuminuria). Experimental studies have documented that 12-lipoxygenase (12-LOX) and its metabolite 12(S)-hydroxyeicosatetraenoic acid (HETE) play an important role in the pathogenesis of diabetic nephropathy. 12(S)-HETE may work in association with angiotensin II and transforming growth factor- β (TGF-β) reciprocally to induce fibrotic changes in the diabetic kidneys. The fibrotic actions of angiotensin II on the kidneys are mediated indirectly through an increase in the synthesis of 12(S)-HETE. Conversely, 12(S)-HETE may also enhance the actions of angiotensin II by upregulating the expression of AT1 receptors on the glomerulus, mesangium, and podocytes. 12(S)-HETE may also cross-talk with TGF-β in a reciprocal manner to induce the fibrotic changes in the diabetic kidney. 12(S)-HETE-triggered signaling pathways may involve activation of p38 mitogen-activated protein (p38MAP) kinase, increase in cAMP-responsive element-binding protein (CREB) transcriptional activity, epigenetic changes involving histone methylation through an increase in histone methyltransferase activity along with an upregulation of cyclin-kinase inhibitors including, p16, p21, and p27. The present review discusses the role of 12-LOX in the pathogenesis of diabetic nephropathy along with the possible mechanisms.

SUMMARYProtein degradation via the use of bivalent chemical degraders provides an alternative strategy to block protein function and assess the biological roles of putative drug targets. This approach capitalizes on the advantages of small molecule inhibitors while moving beyond the restrictions of traditional pharmacology. Herein we report a first-in-class chemical degrader (UNC6852) that targets Polycomb Repressive Complex 2 (PRC2). UNC6852 contains an EED226 derived ligand and a ligand for VHL which bind to the WD40 aromatic cage of EED and CRL2VHL, respectively, to induce proteasomal degradation of PRC2 components, EED, EZH2, and SUZ12. Degradation of PRC2 with UNC6852 blocks the histone methyltransferase activity of EZH2, decreasing H3K27me3 levels in HeLa cells and diffuse large B-cell lymphoma (DLBCL) cells containing an EZH2Y641N gain-of-function mutation. UNC6852 degrades both wild type EZH2 and EZH2Y641N, and additionally displays anti-proliferative effects in this cancer model system.Abstract Figure

Polycomb Repressive Complex 2 (PRC2) is a histone methyltransferase whose function is critical for regulating transcriptional repression in many eukaryotes including humans. Its catalytic moiety EZH2 is responsible for the tri-methylation of H3K27 and also undergoes automethylation. Using mass spectroscopic analysis of recombinant human PRC2, we identified three methylated lysine residues (K510, K514, K515) on a disordered but highly conserved loop of EZH2. These lysines were mostly mono- and di-methylated. Either mutation of these lysines or their methylation increases PRC2 histone methyltransferase activity. In addition, mutation of these three lysines in HEK293T cells using CRISPR genome-editing increases global H3K27 methylation levels. EZH2 automethylation occurs intramolecularly (in cis) by methylation of a pseudosubstrate sequence on the flexible loop. This post-translational modification andcis-regulation of PRC2 are analogous to the activation of many protein kinases by autophosphorylation. We therefore propose that EZH2 automethylation provides a way for PRC2 to modulate its histone methyltransferase activity by sensing histone H3 tails, SAM concentration, and perhaps other effectors.

Shuang-xiang Tan,1,2 Rui-cheng Hu,1,2 Qian Xia,2 Yong-li Tan,1 Jing-jing Liu,1 Gui-xiang Gan,1 Li-le Wang1 1Hunan Province Institute of Gerontology, Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University, Changsha, China; 2Department of Respiratory Medicine, Hunan Provincial People’s Hospital/The First Affiliated Hospital of Hunan Normal University, Changsha, China Background: Non–small cell lung cancer (NSCLC) is one of the leading malignant tumors worldwide. Aberrant gene promoter methylation contributes to NSCLC, and PRDM is a tumor suppressor gene family that possesses histone methyltransferase activity. This study aimed to investigate whether aberrant methylation of PRDM promoter is involved in NSCLC. Materials and methods: Primary tumor tissues, adjacent nontumorous tissues, and distant lung tissues were collected from 75 NSCLC patients including 52 lung squamous cell carcinoma (LSCC) patients and 23 lung adenocarcinoma patients. The expression of PRDMs was detected by polymerase chain reaction (PCR), Western blot, and immunohistochemical analysis. The methylation of PRDM promoters was detected by methylation-specific PCR. The correlation of methylation and expression of PRDMs with clinicopathological characteristics of patients were analyzed. Results: mRNA expression of PRDM2, PRDM5, and PRDM16 was low or absent in tumor tissues compared to distant lung tissues. The methylation frequencies of PRDM2, PRDM5, and PRDM16 in tumor tissues were significantly higher than those in distal lung tissues. In LSCC patients, methylation of PRDM2 and PRDM16 was correlated with smoking status and methylation of PRDM5 was correlated with tumor differentiation. Conclusion: The expression of PRDM2, PRDM5, and PRDM16 is low or absent in NSCLC, and this is mainly due to gene promoter methylation. Smoking may be an important cause of PRDM2 and PRDM16 methylation in NSCLC. Keywords: lung tumor, PR domain zinc finger protein, epigenetics, methylation, gene expression

Summary: Although EZH2 enzymatic inhibitors have shown antitumor effects in EZH2-mutated lymphoma and ARID1A-mutated ovarian cancer, many cancers do not respond because EZH2 can promote cancer independently of its histone methyltransferase activity. Here we identify ZRANB1 as the EZH2 deubiquitinase. ZRANB1 binds, deubiquitinates, and stabilizes EZH2. Depletion of ZRANB1 in breast cancer cells results in EZH2 destabilization and growth inhibition. Systemic delivery of ZRANB1 small interfering RNA (siRNA) leads to marked antitumor and antimetastatic effects in preclinical models of triple-negative breast cancer (TNBC). Intriguingly, a small-molecule inhibitor of ZRANB1 destabilizes EZH2 and inhibits the viability of TNBC cells. In patients with breast cancer, ZRANB1 levels correlate with EZH2 levels and poor survival. These findings suggest the therapeutic potential for targeting the EZH2 deubiquitinase ZRANB1. : Many cancer cells are sensitive to depletion of EZH2 but resistant to EZH2 inhibitors, due to EZH2’s enzyme-independent cancer-promoting function. Zhang et al. identify ZRANB1 as an EZH2 deubiquitinase and a potential anticancer target. Keywords: ZRANB1, EZH2, deubiquitinase, breast cancer

Previous research indicates that knocking out absent, small, or homeotic-like (Ash1l) in mice, a histone 3 lysine 4 (H3K4) trimethyltransferase, can result in arthritis with more severe cartilage and bone destruction. Research has documented the essential role of Ash1l in stem cell fate decision such as hematopoietic stem cells and the progenitors of keratinocytes. Following up on those insights, our research seeks to document the function of Ash1l in skeletal formation, specifically whether it controls the fate decision of mesenchymal progenitor cells. Our findings indicate that in osteoporotic bones, Ash1l was significantly decreased, indicating a positive correlation between bone mass and the expression of Ash1l. Silencing of Ash1l that had been markedly upregulated in differentiated C3H10T1/2 (C3) cells hampered osteogenesis and chondrogenesis but promoted adipogenesis. Consistently, overexpression of an Ash1l SET domain-containing fragment 3 rather than Ash1lΔN promoted osteogenic and chondrogenic differentiation of C3 cells and simultaneously inhibited adipogenic differentiation. This indicates that the role of Ash1l in regulating the differentiation of C3 cells is linked to its histone methyltransferase activity. Subcutaneous ex vivo transplantation experiments confirmed the role of Ash1l in the promotion of osteogenesis. Further experiments proved that Ash1l can epigenetically affect the expression of essential osteogenic and chondrogenic transcription factors. It exerts this impact via modifications in the enrichment of H3K4me3 on their promoter regions. Considering the promotional action of Ash1l on bone, it could potentially prompt new therapeutic strategy to promote osteogenesis. Stem Cells 2019;37:115–127

The enhancer of zeste homolog 2 (EZH2) is the enzymatic subunit of the polycomb repressive complex 2 (PRC2) that exerts important functions during normal development as well as disease. PRC2 through EZH2 tri-methylates histone H3 lysine tail residue 27 (H3K27me3), a modification associated with repression of gene expression programs related to stem cell self-renewal, cell cycle, cell differentiation, and cellular transformation. EZH2 is deregulated and subjected to gain of function or loss of function mutations, and hence functions as an oncogene or tumor suppressor gene in a context-dependent manner. The development of highly selective inhibitors against the histone methyltransferase activity of EZH2 has also contributed to insight into the role of EZH2 and PRC2 in tumorigenesis, and their potential as therapeutic targets in cancer. EZH2 can function as an oncogene in multiple myeloma (MM) by repressing tumor suppressor genes that control apoptosis, cell cycle control and adhesion properties. Taken together these findings have raised the possibility that EZH2 inhibitors could be a useful therapeutic modality in MM alone or in combination with other targeted agents in MM. Therefore, we review the current knowledge on the regulation of EZH2 and its biological impact in MM, the anti-myeloma activity of EZH2 inhibitors and their potential as a targeted therapy in MM.

The positive regulatory domain containing 16 (PRDM16) is commonly regarded as a "switch" controlling the transdifferentiation of myoblasts to brown adipocytes. The N-positive regulatory (PR) domain, which is highly homologous to SET domain, is a characteristic structure for the PRDM family. Many SET domain containing proteins and several PRDM members have been found to possess histone methyltransferase activity, yet the role of PRDM16 and its PR domain in the epigenetic regulation of myogenic and adipogenic genes during myoblasts/adipocytes transdifferentiation remains unexplored. In this study, we transfected C2C12 myoblasts to stably express PRDM16 and observed the repression of myogenic genes and activation of adipogenic genes at both proliferation and differentiation stages. Ectopic PRDM16-induced reprogramming of myogenic and adipogenic genes was associated with the hypermethylation on some CpG sites in the enhancer or promoter of MyoD and myogenin, but the methylation status of PPARγ promoter was not affected. C2C12 cells expressing truncated PRDM16 lacking PR domain (ΔPR-PRDM16) demonstrated attenuation of both adipogenic and myogenic potentials, indicated by PPARγ inactivation and decreased triglyceride deposition, as well as a downregulation of MyoD, MyHC and MCK genes, as compared with C2C12 cells expressing intact PRDM16. Furthermore, C2C12 cells expressing ΔPR-PRDM16 exhibited significant differences in histone modifications on the promoters of MyoD and PPARγ genes. Taken together, PRDM16-induced C2C12 transdifferentiation is associated with alterations in CpG methylation of myogenic factors, and PR domain affects both myogenesis and adipogenesis with modified histone methylation marks on MyoD and PPARγ promoters.

Malignant meningiomas are a rare meningioma subtype and tend to have post-surgical recurrence. Significant endeavors have been taken to identify functional therapeutic targets to halt the growth of this aggressive cancer. We have recently discovered that RIZ1 is downregulated in high-grade meningiomas, and RIZ1 overexpression inhibits proliferation while promoting cell apoptosis of the IOMM-Lee malignant meningioma cell line. In this report, we show that the N-terminal PR domain of RIZ1 alone possessed growth-inhibitory activity and anticancer activity in primary human meningioma cells. Interestingly, the effects seem to be dependent on differential RIZ1 protein levels. Transducible TAT-RIZ1-PR protein could also inhibit meningioma tumor growth in nude mice models. We further demonstrate that PR protein exerts histone methyltransferase activity. A microarray analysis of TAT-RIZ1-PR-treated human malignant meningioma cells reveals 969 differentially expressed genes and 848 alternative splicing exons. Moreover, c-Myc and TXNIP, two putative downstream targets of H3K9 methylation, may be involved in regulating RIZ1 tumor-suppressive effects. The reciprocal relationship between RIZ1 and c-Myc was then validated in primary meningioma cells and human tumor samples. These findings provide insights into RIZ1 tumor suppression mechanisms and suggest that TAT-RIZ1-PR protein is a potential new epigenetic therapeutic agent for advanced meningiomas.

Prostate cancer (PCa) is one of the most incident cancers worldwide but clinical and pathological parameters have limited ability to discriminate between clinically significant and indolent PCa. Altered expression of histone methyltransferases and histone methylation patterns are involved in prostate carcinogenesis. SMYD3 transcript levels have prognostic value and discriminate among PCa with different clinical aggressiveness, so we decided to investigate its putative oncogenic role on PCa.We silenced SMYD3 and assess its impact through in vitro (cell viability, cell cycle, apoptosis, migration, invasion assays) and in vivo (tumor formation, angiogenesis). We evaluated SET domain's impact in PCa cells' phenotype. Histone marks deposition on SMYD3 putative target genes was assessed by ChIP analysis.Knockdown of SMYD3 attenuated malignant phenotype of LNCaP and PC3 cell lines. Deletions affecting the SET domain showed phenotypic impact similar to SMYD3 silencing, suggesting that tumorigenic effect is mediated through its histone methyltransferase activity. Moreover, CCND2 was identified as a putative target gene for SMYD3 transcriptional regulation, through trimethylation of H4K20.Our results support a proto-oncogenic role for SMYD3 in prostate carcinogenesis, mainly due to its methyltransferase enzymatic activity. Thus, SMYD3 overexpression is a potential biomarker for clinically aggressive disease and an attractive therapeutic target in PCa., Liga Portuguesa Contra o Cancro – Núcleo Regional do Norte , Research Center of Portuguese Oncology Institute – Porto (CI-IPOP 4-2012) and European Community’s Seventh Framework Programme – Grant number FP7-HEALTH-F5-2009-241783. FQV and SS-S are supported by FCT-Fundação para a Ciência e a Tecnologia grants (SFRH/BD/70564/2010 and PTDC/ SAU-MET/113415/2009), IG is a Posdoc fellow from FCT (PEst-OE/SAU/UI0776/2014).

We described herein structure-based design, synthesis and evaluation of conformationally constrained, cyclic peptidomimetics to block the MLL1-WDR5 protein–protein interaction as inhibitors of the MLL1 histone methyltransferase activity. Our study has yielded cyclic peptidomimetics with very high binding affinities to WDR5 (Ki values

Enhancer of zeste homolog 2 (EZH2) is a key epigenetic regulator that catalyzes the trimethylation of H3K27 and is modulated by post-translational modifications (PTMs). However, the precise regulation of EZH2 PTMs remains elusive. We, herein, report that EZH2 is acetylated by acetyltransferase P300/CBP-associated factor (PCAF) and is deacetylated by deacetylase SIRT1. We identified that PCAF interacts with and acetylates EZH2 mainly at lysine 348 (K348). Mechanistically, K348 acetylation decreases EZH2 phosphorylation at T345 and T487 and increases EZH2 stability without disrupting the formation of polycomb repressive complex 2 (PRC2). Functionally, EZH2 K348 acetylation enhances its capacity in suppression of the target genes and promotes lung cancer cell migration and invasion. Further, elevated EZH2 K348 acetylation in lung adenocarcinoma patients predicts a poor prognosis. Our findings define a new mechanism underlying EZH2 modulation by linking EZH2 acetylation to its phosphorylation that stabilizes EZH2 and promotes lung adenocarcinoma progression.

PURPOSE OF REVIEW: Somatic gain-of-function mutations that drive cancer pathogenesis are well established opportunities for therapeutic intervention, as demonstrated by the clinical efficacy of kinase inhibitors in kinase-mutant malignancies. Here, we discuss the recently discovered gain-of-function mutations in chromatin-regulatory machineries that promote the pathogenesis of cancer. The current understanding of the underlying molecular mechanisms and the therapeutic potential for direct chemical inhibition will be reviewed. RECENT FINDINGS: Point mutations that increase the catalytic activity of EZH2 and NSD2 histone methyltransferases are found in distinct subsets of B-cell neoplasms, which promote cell transformation by elevating the global level of H3K27 tri-methylation or H3K36 di-methylation, respectively. In addition, mutations in histone H3 have been identified in certain pediatric cancers which cause reprogramming of H3K27 and H3K36 methylation by interfering with the histone methyltransferase activity. Finally, chromosomal translocations involving chromatin regulator genes can lead to the formation of fusion oncoproteins that directly modify chromatin as their mechanism of action. SUMMARY: Although relatively rare in aggregate, gain-of-function mutations in chromatin regulators represent compelling therapeutic targets in genetically defined subsets of cancer patients. However, a broader clinical impact for epigenetic therapies in oncology will require an increased understanding of how nonmutated chromatin regulators function as cancer-specific dependencies.

EZH2 is a subunit of polycomb repressive complex 2 (PRC2) that silences gene transcription via H3K27me3 and was shown to be essential for mammalian liver circadian regulation and hematopoiesis through gene silencing. Much less, however, is known about how Ezh2 acts in live zebrafish. Here, we show that zebrafish ezh2 is regulated directly by the circadian clock via both E-box and RORE motif, while core circadian clock genes per1a, per1b, cry1aa and cry1ab are down-regulated in ezh2 null mutant and ezh2 morphant zebrafish, and either knockdown or overexpression of ezh2 alters locomotor rhythms, indicating that Ezh2 is required for zebrafish circadian regulation. In contrast to its canonical silencing function, zebrafish Ezh2 up-regulates these key circadian clock genes independent of histone methyltransferase activity by directly binding to key circadian clock proteins. Similarly, Ezh2 contributes to hematopoiesis by enhancing expression of hematopoietic genes such as cmyb and lck. Together, our findings demonstrate for the first time that Ezh2 acts in both circadian regulation and hematopoiesis independent of silencing PRC2.

Primary cutaneous anaplastic T-cell lymphoma, characterized by the CD30+ anaplastic large T cells, comprises the second most common group of cutaneous T-cell lymphoma. Little is known about the mechanisms of disease progression. Here we report that enhancer of zeste homolog 2 (EZH2), the catalytic subunit of polycomb repressive complex 2 that mediates histone H3 lysine 27 trimethylation, is overexpressed in CD30+ anaplastic cells in primary cutaneous anaplastic T-cell lymphoma and large-cell transformed cutaneous T-cell lymphoma. Silencing EZH2 or inhibiting its histone methyltransferase activity conferred increased apoptosis and G1 cell-cycle arrest in primary cutaneous anaplastic T-cell lymphoma cells in vitro and a xenograft model in vivo. This was mediated by the de-repression of thioredoxin-interacting protein, a major redox control molecule, and consequent formation of reactive oxygen species. Silencing thioredoxin-interacting protein abrogated reactive oxygen species accumulation in EZH2 suppressed cells and rescued cell growth disadvantage. Moreover, EZH2 suppression de-repressed C-X-C motif chemokine ligand 10 and facilitated the recruitment of effector CD4+ and CD8+ T cells into the tumor microenvironment via a C-X-C motif chemokine ligand 10/receptor 3 interaction. These results demonstrate a dual role for polycomb repressive complex 2-mediated epigenetic silencing in tumor progression and antitumor immunity in primary cutaneous anaplastic T-cell lymphoma, and provide a rationale for the pharmacologic inhibition of EZH2 activity in large-cell transformed cutaneous T-cell lymphoma.

RIZ1 displays strong tumor-suppressive activities, which has a potential histone methyltransferase activity. The objective of the study was to evaluate the level and the methylation status of RIZ1 and analyze its association with clinicopathological features and the histone in the pituitary adenomas. We found that RIZ1-positive cases were 11/50 and H-Scores 22.75 ± 11.83 in invasive pituitary adenomas and 26/53 and 66.3 ± 21.7 in non-invasive pituitary adenomas (χ2 = 8.182, p = 0.004). RIZ1 and C-myc showed the opposite trend in these cases. The methylation levels of RIZ1 were more than 50% in 30.4% (7/23) CpG sites through MALDI-TOF Mass array. There was significant difference (p

Epigenetic drug discovery is an emerging strategy for the treatment of cancer and other pathologies. Here, we discuss our recent discovery of first-in-class dual reversible inhibitors of the histone methyltransferase activity of G9a/EHMT2 and DNA methyltransferases showing in vivo efficacy in human tumors. Current and future investigation lines are presented.

Spermatogenesis is crucial for male fertility and is therefore tightly controlled by a variety of epigenetic regulators. However, the function of enhancer of zeste homolog 2 (EZH2) in spermatogenesis and the molecular mechanisms underlying its activity remain poorly defined. Here, we demonstrate that deleting EZH2 promoted spermatogonial differentiation and apoptosis. EZH2 is expressed in spermatogonia, spermatocytes and round and elongated spermatids from stage 9 to 11 but not in leptotene and zygotene spermatocytes. Knocking downEzh2 in vitrousing a lentivirus impaired self-renewal in spermatogonial stem cells (SSCs), and the conditional knockout ofEzh2in spermatogonial progenitors promoted precocious spermatogonial differentiation. EZH2 functions to balance self-renewal and differentiation in spermatogonia by suppressing NEUROG3 and KIT via a direct interaction that is independent of its histone methyltransferase activity. Moreover, deletingEzh2enhanced the activation of CASP3 in spermatids, resulting in reduced spermatozoa production. Collectively, these data demonstrate that EZH2 plays a nonclassical role in the regulation of spermatogonial differentiation and apoptosis in murine spermatogenesis.

PURPOSE : Chromosomal translocation of the Mixed Lineage Leukemia (MLL) locus generates fusion proteins that drive acute myeloid leukemia (AML) resulting in atypical histone methyltransferase activity and alterations in the epigenetic regulation of gene expression. Targeting histone regulators, such as Enhancer of Zeste Homologue 2 (EZH2), has shown promise in AML. Profiling differential protein expression following inhibition of epigenetic regulators in AML may help to identify novel targets for therapeutics. EXPERIMENTAL DESIGN : Murine models of AML combined with quantitative SILAC analysis were used to identify differentially expressed proteins following inhibition of EZH2 activity using 3-Deazaneplanocin A (DZnep). Western blotting and flow cytometry were used to validate a subset of differentially expressed proteins. Gene set analysis was used to determine changes to reported EZH2 target genes. RESULTS : Our quantitative proteomic analysis and subsequent validation of protein changes identified that epigenetic therapy leads to cell death preceded by the induction of differentiation with concurrent p53 up-regulation and cell cycle arrest. Gene set analysis revealed a specific subset of EZH2 target genes that were regulated by DZnep in AML. CONCLUSIONS AND CLINICAL RELEVANCE : These discoveries highlight how this new class of drugs affects AML cell biology and cell survival, and may help identify novel targets and strategies to increase treatment efficacy. This article is protected by copyright. All rights reserved

In vitro histone modification (HM) assays are used to characterize the activity of chromatin-modifying enzymes. These assays provide information regarding the modification sites on histones, the product specificity, and the impact of other histone or nucleotide modifications on enzyme activity. In particular, histone methyltransferase (HMT) assays have been instrumental in elucidating the activity and site specificity of many plant HMT enzymes. In this chapter, we describe a general protocol that can be used to perform HMT assays using different chromatin substrates, detection methods, and enzymes directly purified from plant material or heterologous sources.

The SU(VAR)3-9-like histone methyltransferases usually catalyze repressive histone H3K9 methylation and are involved in transcriptional gene silencing in eukaryotic organisms. We identified a putative SU(VAR)3-9-like histone methyltransferase SUVR2 by a forward genetic screen and demonstrated that it is involved in transcriptional gene silencing at genomic loci targeted by RNA-directed DNA methylation (RdDM). We found that SUVR2 has no histone methyltransferase activity and the conserved catalytic sites of SUVR2 are dispensable for the function of SUVR2 in transcriptional silencing. SUVR2 forms a complex with its close homolog SUVR1 and associate with three previously uncharacterized SNF2-related chromatin-remodeling proteins CHR19, CHR27, and CHR28. SUVR2 was previously thought to be a component in the RdDM pathway. We demonstrated that SUVR2 contributes to transcriptional gene silencing not only at a subset of RdDM target loci but also at many RdDM-independent target loci. Our study suggests that the involvement of SUVR2 in transcriptional gene silencing is related to nucleosome positioning mediated by its associated chromatin-remodeling proteins.

CDKN2A (p16(INK4a)) is a crucial tumor suppressor involved in many cancers. Our recent investigations revealed that FOXA1 as a forkhead transcription factor mediates CDKN2A activation in cellular senescence. However, the contribution of this axis in carcinogenesis remains unclear. Here, using a comprehensive collection of cancer microarray data, we found FOXA1 is down-regulated in many cancers compared to their normal counterparts and the positive correlation between FOXA1 and CDKN2A could be observed in prostate and breast cancers with lower EZH2 (epigenetic repressor for CDKN2A) expression. Experimentally, epistasis analysis in prostate and breast cancer cells indicated that higher expression of FOXA1 opposes EZH2-mediated CDKN2A repression, as further depletion of FOXA1 reverts the de-silencing of CDKN2A caused by EZH2 inhibition. Concomitantly, EZH2-depletion suppresses cancer cell cycle progression and this regulation is optimized in the presence of FOXA1 and CDKN2A. A further oncogenic transformation assay suggested that overexpression of EZH2 is insufficient to block RAS-induced CDKN2A activation and loss of FOXA1 is mandatory to potentiate EZH2-mediated CDKN2A silencing and to bypass the senescence barrier. Importantly, using an in vitro histone methyltransferase (HMTase) system, we found FOXA1 directly inhibits EZH2's histone methyltransferase activity through its C-terminal histone binding motif. These data support that positive regulation of CDKN2A by FOXA1 counteracts its tumorigenic repression of by EZH2 in cancers.

SUMMARY Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here, we show that the SET domain, and thus HMT activity of MLL1, is dispensable for maintaining HSCs and supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation is selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 is sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function.

During aging, a decline in stem cell function is observed in many tissues. This decline is accompanied by complex changes of the chromatin structure among them changes in histone modifications and DNA methylation which both affect transcription of a tissue-specific subset of genes. A mechanistic understanding of these age-associated processes, their interrelations and environmental dependence is currently lacking. Here, we discuss related questions on the molecular, cellular, and population level. We combine an individual cell-based model of stem cell populations with a model of epigenetic regulation of transcription. The novel model enables to simulate age-related changes of trimethylation of lysine 4 at histone H3 and of DNA methylation. These changes entail expression changes of genes that induce age-related phenotypes (ARPs) of cells. We compare age-related changes of regulatory states in quiescent stem cells occupying a niche with those observed in proliferating cells. Moreover, we analyze the impact of the activity of the involved epigenetic modifiers on these changes. We find that epigenetic aging strongly affects stem cell heterogeneity and that homing at stem cell niches retards epigenetic aging. Our model provides a mechanistic explanation how increased stem cell proliferation can lead to progeroid phenotypes. Adapting our model to properties observed for aged hematopoietic stem cell (HSC) clones, we predict that the hematopoietic ARP activates young HSCs and thereby retards aging of the entire HSC population. In addition, our model suggests that the experimentally observed high interindividual variance in HSC numbers originates in a variance of histone methyltransferase activity.

Backgrounds: Monosomy of chromosome 7 (monosomy 7) is observed in about 9% of acute myeloid leukemia (AML) patients and related to resistance to conventional chemotherapy and poor prognosis. It is required to establish an effective treatment strategy based on the specific molecular pathogenesis associated with the chromosomal abnormality. In this study, we aimed to find a novel therapeutic target in AML with monosomy 7. Methods and Results: Chromosome 7 encodes multiple genes regulating histone methylation such as EZH2, KTM2C and KTM2E. Although loss of these genes is considered to contribute to the development of AML with monosomy 7, it may also be required for some leukemia cell functions. We hypothesized that haploinsufficiency of the epigenetic modifiers in the chromosome 7 is compensated by other epigenetic molecules in different chromosomes, and the AML cells with monosomy 7 are vulnerable to inhibition of those molecules. Based on this notion, we performed an RNA-interference (RNAi)-based screening by targeting 53 genes encoding epigenetic enzymes regulating histone methylation by using AML cell lines with monosomy 7: KG-1 and F-36P cells. We found that the siRNA-mediated knockdown of EED, BRD4 or BRDT significantly decreased cell viability in AML cells with monosomy 7 compared to K562 cells as the control without monosomy 7. Stable knockdown of EED expression by retroviral transduction of shRNA markedly attenuated proliferation as well as increased apoptosis specifically in leukemia cells with monosomy 7. Surprisingly, pharmacologic inhibition of EED, which blocks the binding of EED to the polycomb repressive complex 2 (PRC2), was only marginally effective for the monosomy 7 leukemia cells. These results suggest that EED supports survival of the monosomy 7 leukemia cells in a PRC2-independent manner. Recent studies showed that EED is not only a component of PRC2 but also interacts with the polycomb repressive complex 1 (PRC1). In contrast to modest effects of PRC2 inhibition, inhibition of PRC1 activity showed marked efficacy specifically in leukemia cells with monosomy 7. Co-inhibition of PRC1 and PRC2 synergistically suppressed proliferation of the monosomy 7-AML cells. These results collectively suggest that while both PRC1 and PRC2 cooperatively function, the PRC1 rather than PRC2 has a predominant role in the survival of monosomy 7 leukemia cells. In addition to EED, we identified BRD4 as an essential survival factors in AML with monosomy 7. Although inhibition of BET bromodomain proteins have already shown to be effective for a variety of AML, we showed that blockade of bromodomain proteins preferentially affected cellular proliferation of AML cells with monosomy 7. We then explored whether the deletion of specific genes within the chromosome 7 was responsible for increased sensitivity to BET bromodomain protein inhibition. K562 cells were individually treated with the siRNAs against 22 genes in the presence of a BET bromodomain inhibitor JQ1. We found that knockdown of MLL3 or MLL5 resulted in increased apoptosis of the JQ1-treated K562 cells. We also confirmed that pharmacological inhibition of histone methyltransferase activity of MLL3 and MLL5 showed synergistic effects with JQ1 treatment in the leukemia cells. Conclusion: We identified EED and BRD4 as novel promising therapeutic targets in AML with monosomy 7. Disclosures Kagoya: Kyowa Kirin: Speakers Bureau; Nihon Shinyaku: Research Funding. Miyauchi:kyowa Kirin: Research Funding. Kurokawa:Novartis Pharma K.K.: Research Funding; Celgene K.K.: Consultancy, Speakers Bureau; Astellas Pharma Inc.: Research Funding, Speakers Bureau; Kyowa Hakko Kirin Co., Ltd.: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Eisai Co., Ltd.: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma Co.,Ltd.: Research Funding, Speakers Bureau; Teijin Limited: Research Funding; Boehringer Ingelheim: Speakers Bureau; Yakult Honsha Company: Speakers Bureau; Nippon Shinyaku Co., Ltd.: Research Funding; Shionogi & Co., Ltd: Consultancy, Honoraria; Bioverativ Japan ltd.: Consultancy; Otsuka Pharmaceutical Co., Ltd.: Research Funding, Speakers Bureau; Daiichi Sankyo Conpany: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Chugai Pharmaceutical Company: Consultancy, Research Funding, Speakers Bureau; Pfizer Japan Inc.: Research Funding; MSD K.K.: Consultancy, Research Funding, Speakers Bureau; Takeda Pharmaceutical Company Limited.: Research Funding, Speakers Bureau; ONO PHARMACEUTICAL CO., LTD.: Speakers Bureau; Shire Japan K.K.: Speakers Bureau; Janssen Pharmaceutical K.K.: Speakers Bureau.

Meiotic recombination is required for correct segregation of chromosomes to gametes and to generate genetic diversity. In mice and humans, DNA double-strand breaks (DSBs) are initiated by SPO11 at recombination hotspots activated by PRDM9-catalyzed histone modifications on open chromatin. However, the DSB-initiating and repair proteins are associated with a linear proteinaceous scaffold called the chromosome axis, the core of which is composed of cohesin proteins. STAG3 is a stromalin subunit common to all meiosis-specific cohesin complexes. Mutations of meiotic cohesin proteins, especially STAG3, perturb both axis formation and recombination in the mouse, prompting determination of how the processes are mechanistically related. Protein interaction and genetic analyses revealed that PRDM9 interacts with STAG3 and REC8 in cooperative relationships that promote normal levels of meiotic DSBs at recombination hotspots in spermatocytes. The efficacy of the Prdm9-Stag3 genetic interaction in promoting DSB formation depends on PRDM9-mediated histone methyltransferase activity. Moreover, STAG3 deficiency has a major effect on DSB number even in the absence of PRDM9, showing that its role is not restricted to canonical PRDM9-activated hotspots. STAG3 and REC8 promote axis localization of the DSB-promoting proteins HORMAD1, IHO1, and MEI4, as well as SPO11 activity. These results establish that PRDM9 and axis-associated cohesin complexes together coordinate and facilitate meiotic recombination by recruiting key proteins for initiation of DSBs, thereby associating activated hotspots with DSB-initiating complexes on the axis.

Background Loss of function alleles in SETD1A are a genetic risk factor for developing schizophrenia. SETD1A encodes a protein possessing histone methyltransferase activity. Specifically, it catalyses the methylation of histone H3 at Lys4, a modification indicative of active regulatory DNA function. Reduced SETD1A expression is therefore likely to have a major impact on global gene expression via alterations to the histone code. To understand the downstream genes and pathways altered by SETD1A expression we performed gene expression profiling of a neural cell line in which SETD1A expression had been reduced. Methods SETD1A expression was reduced in the SH-SY5Y neuroblastoma cell line using two non-overlapping siRNAs. A non-targeting siRNA was used as a control. Four days after transfection cells were harvested for RNA extraction. Transcriptional profiling was performed using Illumina microarrays. Differential expressed probes were identified using t tests and gene ontology enrichment analysis performed using DAVID. Quantitative PCR was used to verify the microarray findings for a subset of gene transcripts. We tested for enrichment of association with schizophrenia amongst the differential expressed probes using MAGMA. Results Both targeting siRNAs reduced SETD1A RNA expression ~50% compared to control. Gene expression profiling identified 1131 differentially expressed probes (p Discussion Reduced SETD1A levels lead to widespread changes in the expression of genes involved in diverse process, consistent with its role in general transcription. However, we observed altered expression of known regulators of neuronal development and function. Our results also highlight mitochondrial function as important downstream consequences of reduced SETD1A expression. Importantly, we show that gene expression changes after knock-down of a rare variant schizophrenia risk gene are enriched for a common variant association signal, indicating a point of molecular convergence in risk mechanisms.

Summary Dpy-30 is a regulatory subunit controlling the histone methyltransferase activity of the KMT2 enzymes in vivo. Paradoxically, in vitro methyltransferase assays revealed that Dpy-30 only modestly participates in the positive heterotypic allosteric regulation of these methyltransferases. Detailed genome-wide, molecular and structural studies reveal that an extensive network of interactions taking place at the interface between Dpy-30 and Ash2L are critical for the correct placement, genome-wide, of H3K4me2 and H3K4me3 but marginally contribute to the methyltransferase activity of KMT2 enzymes in vitro. Moreover, we show that H3K4me2 peaks persisting following the loss of Dpy-30 are found in regions of highly transcribed genes, highlighting an interplay between Complex of Proteins Associated with SET1 (COMPASS) kinetics and the cycling of RNA polymerase to control H3K4 methylation. Overall, our data suggest that Dpy-30 couples its modest positive heterotypic allosteric regulation of KMT2 methyltransferase activity with its ability to help the positioning of SET1/COMPASS to control epigenetic signaling.

PRDM proteins belong to the SET domain protein family, which is involved in the regulation of gene expression. Although few PRDM members possess histone methyltransferase activity, the molecular mechanisms by which the other members exert transcriptional regulation remain to be delineated. In this study, we find that Prdm5 is highly expressed in mouse embryonic stem (mES) cells and exploit this cellular system to characterize molecular functions of Prdm5. By combining proteomics and next-generation sequencing technologies, we identify Prdm5 interaction partners and genomic occupancy. We demonstrate that although Prdm5 is dispensable for mES cell maintenance, it directly targets genomic regions involved in early embryonic development and affects the expression of a subset of developmental regulators during cell differentiation. Importantly, Prdm5 interacts with Ctcf, cohesin, and TFIIIC and cooccupies genomic loci. In summary, our data indicate how Prdm5 modulates transcription by interacting with factors involved in genome organization in mouse embryonic stem cells.

The symptoms of ataxia-telangiectasia (A-T) include a progressive neurodegeneration caused by ATM protein deficiency. We previously found that nuclear accumulation of histone deacetylase-4, HDAC4, contributes to this degeneration; we now report that increased trimethylation of histone H3 on Lys27 (H3K27me3) mediated by polycomb repressive complex 2 (PRC2) is also important in the A-T phenotype. Enhancer of zeste homolog 2 (EZH2), a core catalytic component of PRC2, is a new ATM kinase target, and ATM-mediated phosphorylation of EZH2 on Ser734 reduces protein stability. Thus, PRC2 formation is elevated along with H3K27me3 in ATM deficiency. Chromatin immunoprecipitation and sequencing showed an increase in H3K27me3 'marks' and a dramatic shift in their location. The change of H3K27me3 chromatin-binding pattern is directly related to cell cycle reentry and cell death of ATM-deficient neurons. Lentiviral knockdown of EZH2 rescued Purkinje cell degeneration and behavioral abnormalities in Atm(-/-) mice, demonstrating that EZH2 hyperactivity is another key factor in A-T neurodegeneration.

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.

Appropriate gene expression relies on the sophisticated interplay between genetic and epigenetic factors. Histone acetylation and an open chromatin configuration are key features of transcribed regions and are mainly present around active promoters. Our recent identification of the SET-domain containing protein UpSET established a new functional link between the modulation of open chromatin features and active recruitment of well-known co-repressors in metazoans. Structurally, the SET domain of UpSET resembles H3K4 and H3K36 methyltransferases; however, it is does not confer histone methyltransferase activity. Rather than methylating histones to regulate gene expression like other SET domain-containing proteins, UpSET fine-tunes transcription by modulating the chromatin structure around active promoters resulting in suppression of expression of off-target genes or nearby repetitive elements. Chromatin modulation by UpSET occurs in part through its interaction with histone deacetylases. Here, we discuss th...

MicroRNAs are short noncoding RNAs, known regulators of several signaling pathways cell differentiation and proliferation, development, and apoptosis, which are deregulated in acute leukemia. Mixed lineage leukemia (MLL) gene encodes a protein with histone methyltransferase activity, which is essential for the fine tuning of hematopoietic stem cell development and differentiation through the regulation of HOXA and MEIS1. MLL gene rearrangements characterize both acute myeloid and acute lymphoblastic leukemia associated with poor outcomes. MicroRNAs and MLL rearrangements are in tight association regulating each other expression, affecting cell cycle regulators, and composing complex networks with factors involved in leukemogenesis such as MYC and FLT3. MLL fusion genes are also capable of recruiting DNA methyltransferases at microRNAs promoters controlling their expression through epigenetic changes. Direct drug targeting of MLL has been difficult to achieve, and in this context, microRNA expression modulation represents an attractive approach.

The equilibrium between self-renewal and differentiation of hematopoietic stem cells is regulated by epigenetic mechanisms. In particular, Polycomb-group (PcG) proteins have been shown to be involved in this process by repressing genes involved in cell-cycle regulation and differentiation. PcGs are histone modifiers that reside in two multi-protein complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). The existence of multiple orthologs for each Polycomb gene allows the formation of a multitude of distinct PRC1 and PRC2 sub-complexes. Changes in the expression of individual PcG genes are likely to cause perturbations in the composition of the PRC, which affect PRC enzymatic activity and target selectivity. An interesting recent development is that aberrant expression of, and mutations in, PcG genes have been shown to occur in hematopoietic neoplasms, where they display both tumor-suppressor and oncogenic activities. We therefore comprehensively reviewed the latest research on the role of PcG genes in normal and malignant blood cell development. We conclude that future research to elucidate the compositional changes of the PRCs and methods to intervene in PRC assembly will be of great therapeutic relevance to combat hematological malignancies. Leukemia (2013) 27, 523-533; doi:10.1038/leu.2012.368

The equilibrium between self-renewal and differentiation of hematopoietic stem cells is regulated by epigenetic mechanisms. In particular, Polycomb-group (PcG) proteins have been shown to be involved in this process by repressing genes involved in cell-cycle regulation and differentiation. PcGs are histone modifiers that reside in two multi-protein complexes: Polycomb Repressive Complex 1 and 2 (PRC1 and PRC2). The existence of multiple orthologs for each Polycomb gene allows the formation of a multitude of distinct PRC1 and PRC2 sub-complexes. Changes in the expression of individual PcG genes are likely to cause perturbations in the composition of the PRC, which affect PRC enzymatic activity and target selectivity. An interesting recent development is that aberrant expression of, and mutations in, PcG genes have been shown to occur in hematopoietic neoplasms, where they display both tumor-suppressor and oncogenic activities. We therefore comprehensively reviewed the latest research on the role of PcG genes in normal and malignant blood cell development. We conclude that future research to elucidate the compositional changes of the PRCs and methods to intervene in PRC assembly will be of great therapeutic relevance to combat hematological malignancies. Leukemia (2013) 27, 523-533; doi:10.1038/leu.2012.368

Daily cyclical expression of thousands of genes in tissues such as the liver is orchestrated by the molecular circadian clock, the disruption of which is implicated in metabolic disorders and cancer. Although we understand much about the circadian transcription factors that can switch gene expression on and off, it is still unclear how global changes in rhythmic transcription are controlled at the genomic level. Here, we demonstrate circadian modification of an activating histone mark at a significant proportion of gene loci that undergo daily transcription, implicating widespread epigenetic modification as a key node regulated by the clockwork. Furthermore, we identify the histone-remodelling enzyme mixed lineage leukemia (MLL)3 as a clock-controlled factor that is able to directly and indirectly modulate over a hundred epigenetically targeted circadian “output” genes in the liver. Importantly, catalytic inactivation of the histone methyltransferase activity of MLL3 also severely compromises the oscillation of “core” clock gene promoters, including Bmal1 , mCry1 , mPer2 , and Rev-erbα , suggesting that rhythmic histone methylation is vital for robust transcriptional oscillator function. This highlights a pathway by which the clockwork exerts genome-wide control over transcription, which is critical for sustaining temporal programming of tissue physiology.

Polycomb group (PcG) protein complexes repress developmental regulator genes by modifying their chromatin. How different PcG proteins assemble into complexes and are recruited to their target genes is poorly understood. Here, we report the crystal structure of the core of the Drosophila PcG protein complex Pleiohomeotic (Pho)-repressive complex (PhoRC), which contains the Polycomb response element (PRE)-binding protein Pho and Sfmbt. The spacer region of Pho, separated from the DNA-binding domain by a long flexible linker, forms a tight complex with the four malignant brain tumor (4MBT) domain of Sfmbt. The highly conserved spacer region of the human Pho ortholog YY1 binds three of the four human 4MBT domain proteins in an analogous manner but with lower affinity. Comparison of the Drosophila Pho:Sfmbt and human YY1:MBTD1 complex structures provides a molecular explanation for the lower affinity of YY1 for human 4MBT domain proteins. Structure-guided mutations that disrupt the interaction between Pho and Sfmbt abolish formation of a ternary Sfmbt:Pho:DNA complex in vitro and repression of developmental regulator genes in Drosophila. PRE tethering of Sfmbt by Pho is therefore essential for Polycomb repression in Drosophila. Our results support a model where DNA tethering of Sfmbt by Pho and multivalent interactions of Sfmbt with histone modifications and other PcG proteins create a hub for PcG protein complex assembly at PREs.

In vertebrate retinal development, various transcription factors are known to execute essential activities in gene regulation. Although epigenetic modification is considered to play a pivotal role in retinal development, the exactin vivorole of epigenetic regulation is still poorly understood. We observed that G9a histone methyltransferase, which methylates histone H3 at lysine 9 (H3K9), is substantially expressed in the mouse retina throughout development. To addressin vivoG9a function in the mouse retina, we ablatedG9ain retinal progenitor cells by conditional gene knock-out (G9a Dkk3CKO). TheG9a Dkk3CKO retina exhibited severe morphological defects, including photoreceptor rosette formation, a partial loss of the outer nuclear layer, elevated cell death, and persistent cell proliferation. Progenitor cell-related genes, including several cyclins, Hes1, Chx10, and Lhx2, are methylated on histone H3K9 in the wild-type retina, but they were defective in H3K9 methylation and improperly upregulated at late developmental stages in theG9a Dkk3CKO retina. Notably, conditional depletion ofG9ain postmitotic photoreceptor precursors (G9a CrxCKO) led to the development of an almost normal retina, indicating that G9a activity mainly in retinal progenitor cells, but not in photoreceptor precursors, is essential for normal terminal differentiation of and survival of the retina. Our results suggest that proper epigenetic marks in progenitor cells are important for subsequent appropriate terminal differentiation and survival of retinal cells by repressing progenitor cell-related genes in differentiating retinal cells.

Polycomb repressive complex 2 (PRC2) catalyzes the monomethylation, dimethylation, and trimethylation of histone H3 Lys27 (H3K27) and acts as a central epigenetic regulator that marks the repressive chromatin domain. Embryonic ectoderm development (EED), an essential component of PRC2, interacts with trimethylated H3K27 (H3K27me3) through the aromatic cage structure composed of its three aromatic amino acids, Phe97, Trp364, and Tyr365. This interaction allosterically activates the histone methyltransferase activity of PRC2 and thereby propagates repressive histone marks. In this study, we report the analysis of knock-in mice harboring the myeloid disorder-associated EED Ile363Met (I363M) mutation, analogous to the EED aromatic cage mutants. The I363M homozygotes displayed a remarkable and preferential reduction of H3K27me3 and died at midgestation. The heterozygotes increased the clonogenic capacity and bone marrow repopulating activity of hematopoietic stem/progenitor cells (HSPCs) and were susceptible to leukemia. Lgals3, a PRC2 target gene encoding a multifunctional galactose-binding lectin, was derepressed in I363M heterozygotes, which enhanced the stemness of HSPCs. Thus, our work provides in vivo evidence that the structural integrity of EED to H3K27me3 propagation is critical, especially for embryonic development and hematopoietic homeostasis, and that its perturbation increases the predisposition to hematologic malignancies.

KMT2A mutations cause Wiedemann-Steiner syndrome (WDSTS), which is characterized by hypertrichosis cubiti, short stature, and distinct facial features in general. Here, we report two Chinese boys with novel nonsense KMT2A mutations. Most of their phenotypes are concordant with WDSTS. They, however, lack the key WDSTS feature-hypertrichosis cubiti. Additionally, their transverse palmar creases are absent. We further summarized the genotypes and phenotypes of the KMT2A mutation carriers. The consensus phenotypes include postnatal growth retardation, developmental delay, short stature, and intellectual disability. The common facial features include thick eyebrows, long eyelashes, downslanting, and narrow palpebral fissures, wide nasal bridge, and broad nasal tip. They have generalized hypertrichosis. A hairy back can be observed as frequently as hairy elbows in patients with KMT2A mutations. Absent palmar proximal transverse creases are only observed in these two Chinese boys. This might be due to the difference in ethnic background. Thus far, all mutations in KMT2A are located before the FYRC domain. They would truncate KMT2A mRNA transcripts. Haploinsufficiency of the histone methyltransferase activity would therefore influence transcriptional regulation. © 2016 Wiley Periodicals, Inc.

Some studies have linked age-related beneficial effects of exercise and epigenetic mechanisms. Although, the impact of treadmill exercise on histone acetylation, histone and DNA methylation marks in aged cortices yet remains poorly understood. Considering the role of frontal cortex on brain functions, we investigated the potential of different exercise protocols, single session and daily exercise, to modulate epigenetic marks, namely global H4 acetylation, histone methyltransferase activity (HMT H3K27) and levels of DNA methytransferase (DNMT1 and DNMT3b) in prefrontal cortices from 3 and 21-months aged Wistar rats. The animals were submitted to two treadmill exercise protocols, single session (20min) or daily moderate (20min/day during 14days). The daily exercise protocol induced an increased in histone H4 acetylation levels in prefrontal cortices of 21-months-old rats, without any effects in young adult group. DNMT3b levels were increased in aged cortices of animals submitted to single session of exercise. These results indicate that prefrontal cortex is susceptible to epigenetic changes in a protocol dependent-manner and that H4 acetylation levels and DNMT3b content changes might be linked at least in part to exercise-induced effects on brain functions.

Hematopoietic stem cells were once considered identical. However, in the mid-1990s, it became apparent that stem cells from a person's early developmental phases are superior to those from adults, and aged stem cells are defective compared with young stem cells. It has since become clear that polycomb group proteins are important regulators of stem cell functioning. Polycomb group proteins are chromatin-associated proteins involved in writing or reading epigenetic histone modifications. Polycomb group proteins are involved in normal blood cell formation, in cancer, and possibly in aging. In this review, we describe how the different phases of hematopoietic stem cells-birth, maintenance, functional decline, derailment, and death-are continuous processes that may be controlled by polycomb group proteins.

International audience; Weaver syndrome (WS) is a rare congenital disorder characterized by generalized overgrowth, macrocephaly, specific facial features, accelerated bone age, intellectual disability, and susceptibility to cancers. De novo mutations in the enhancer of zeste homolog 2 (EZH2) have been shown to cause WS. EZH2 is a histone methyltransferase that acts as the catalytic agent of the polycomb-repressive complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating histone methyltransferase activity of mutant EZH2 from various cancers have been reported, while WS-associated mutations remain poorly characterized. To investigate the role of EZH2 in WS, we performed functional studies using artificially-assembled PRC2 complexes containing mutagenized human EZH2 that reflected the codon changes predicted from patients with WS. We found that WS-associated amino acid alterations reduce the histone methyltransferase function of EZH2 in this in vitro assay. Our results support the hypothesis that WS is caused by constitutional mutations in EZH2 that alter the histone methyltransferase function of PRC2. However, histone methyltransferase activities of different EZH2 variants do not appear to correlate directly with the phenotypic variability between WS patients and individuals with a common c.553G\textgreaterC (p.Asp185His) polymorphism in EZH2. This article is protected by copyright. All rights reserved

EZH2 is a histone methyltransferase whose functions in stem cells and tumor cells are well established. Accumulating evidence shows that EZH2 has critical roles in T cells and could be a promising therapeutic target for several immune diseases. To further reveal the novel functions of EZH2 in human T cells, protein co-immunoprecipitation combined mass spectrometry was conducted and several previous unknown EZH2-interacting proteins were identified. Of them, we focused on a DNA damage responsive protein, Ku80, because of the limited knowledge regarding EZH2 in the DNA damage response. Then, we demonstrated that instead of being methylated by EZH2, Ku80 bridges the interaction between the DNA-dependent protein kinase (DNA-PK) complex and EZH2, thus facilitating EZH2 phosphorylation. Moreover, EZH2 histone methyltransferase activity was enhanced when Ku80 was knocked down or DNA-PK activity was inhibited, suggesting DNA-PK-mediated EZH2 phosphorylation impairs EZH2 histone methyltransferase activity. On the other hand, EZH2 inhibition increased the DNA damage level at the late phase of T-cell activation, suggesting EZH2 involved in genomic integrity maintenance. In conclusion, our study is the first to demonstrate that EZH2 is phosphorylated by the DNA damage responsive complex DNA-PK and regulates DNA damage-mediated T-cell apoptosis, which reveals a novel functional crosstalk between epigenetic regulation and genomic integrity.

The best-understood mechanism by which EZH2 exerts its oncogenic function is through polycomb repressive complex 2 (PRC2)-mediated gene repression, which requires its histone methyltransferase activity. However, small-molecule inhibitors of EZH2 that selectively target its enzymatic activity turn out to be potent only for lymphoma cells with EZH2-activating mutation. Intriguingly, recent discoveries, including ours, have placed EZH2 into the category of transcriptional coactivators and thus raised the possibility of noncanonical signaling pathways. However, it remains unclear how EZH2 switches to this catalytic independent function. In the current study, using natural killer/T-cell lymphoma (NKTL) as a disease model, we found that phosphorylation of EZH2 by JAK3 promotes the dissociation of the PRC2 complex leading to decreased global H3K27me3 levels, while it switches EZH2 to a transcriptional activator, conferring higher proliferative capacity of the affected cells. Gene expression data analysis also suggests that the noncanonical function of EZH2 as a transcriptional activator upregulates a set of genes involved in DNA replication, cell cycle, biosynthesis, stemness, and invasiveness. Consistently, JAK3 inhibitor was able to significantly reduce the growth of NKTL cells, in an EZH2 phosphorylation-dependent manner, whereas various compounds recently developed to inhibit EZH2 methyltransferase activity have no such effect. Thus, pharmacological inhibition of JAK3 activity may provide a promising treatment option for NKTL through the novel mechanism of suppressing noncanonical EZH2 activity.

Purpose: A deregulated epigenome contributes to the transformed phenotype of mantle cell lymphoma (MCL). This involves activity of the polycomb repressive complex (PRC) 2, containing three core proteins, EZH2, SUZ12, and EED, in which the SET domain of EZH2 mediates the histone methyltransferase activity. We determined the effects of 3-deazaneplanocin A (DZNep), an S-adenosylhomocysteine hydrolase inhibitor, and/or pan-histone deacetylase inhibitor panobinostat (PS) on cultured and primary MCL cells. Experimental Design: Following treatment with DZNep and/or PS, apoptosis and the levels and activity of EZH2 and PRC2 proteins in cultured and primary MCL cells were determined. Results: Treatment with DZNep depleted EZH2, SUZ12, and 3MeK27H3 in the cultured human MCL cells. DZNep also increased expression of p21, p27, and FBXO32, whereas it depleted Cyclin D1 and Cyclin E1 levels in MCL cells. In addition, DZNep treatment induced cell-cycle arrest and apoptosis in cultured and primary MCL cells. Furthermore, as compared with treatment with each agent alone, cotreatment with DZNep and PS caused greater depletion of EZH2, SUZ12, 3MeK27H3, and Cyclin D1 levels, whereas it induced greater expression of FBXO32, p16, p21, and p27. Combined treatment with DZNep and PS synergistically induced apoptosis of cultured and primary MCL cells while relatively sparing normal CD34 + cells. Cotreatment with DZNep and PS also caused significantly greater inhibition of tumor growth of JeKo-1 xenografts in NOD/SCID mice. Conclusions: These preclinical in vitro and in vivo findings show that cotreatment with DZNep and PS is an active combined epigenetic therapy worthy of further in vivo testing against MCL. Clin Cancer Res; 18(22); 6227–38. ©2012 AACR.

The polycomb repressive complex 2 (PRC2) is a highly conserved histone H3 lysine 27 methyltransferase that regulates the expression of developmental genes. Inactivating mutations of the catalytic component of PRC2, EZH2, are seen in myeloid disorders. We reasoned that the other 2 core PRC2 components, SUZ12 and EED, may also be mutational targets in these diseases, as well as associated factors such as JARID2. SUZ12 mutations were identified in 1 of 2 patients with myelodysplastic syndrome/myeloproliferative neoplasms with 17q acquired uniparental disomy and in 2 of 2 myelofibrosis cases with focal 17q11 deletions. All 3 were missense mutations affecting the highly conserved VEFS domain. Analysis of a further 146 myelodysplastic syndrome/myeloproliferative neoplasm patients revealed an additional VEFS domain mutant, yielding a total mutation frequency of 1.4% (2 of 148). We did not find mutations of JARID2 or EED in association with acquired uniparental disomy for chromosome 6p or 11q, respectively; however, screening unselected cases identified missense mutations in EED (1 of 148; 1%) and JARID2 (3 of 148; 2%). All 3 SUZ12 mutations tested and the EED mutation reduced PRC2 histone methyltransferase activity in vitro, demonstrating that PRC2 function may be compromised in myeloid disorders by mutation of distinct genes.

Changes of histone modification status at critical lineage-specifying gene loci in multipotent precursors can influence cell fate commitment. The contribution of these epigenetic mechanisms to natural killer (NK) cell lineage determination from common lymphoid precursors is not understood. Here we investigate the impact of histone methylation repressive marks (H3 Lys27 trimethylation; H3K27(me3)) on early NK cell differentiation. We demonstrate that selective loss of the histone-lysine N-methyltransferase Ezh2 (enhancer of zeste homolog 2) or inhibition of its enzymatic activity with small molecules unexpectedly increased generation of the IL-15 receptor (IL-15R) CD122(+) NK precursors and mature NK progeny from both mouse and human hematopoietic stem and progenitor cells. Mechanistic studies revealed that enhanced NK cell expansion and cytotoxicity against tumor cells were associated with up-regulation of CD122 and the C-type lectin receptor NKG2D. Moreover, NKG2D deficiency diminished the positive effects of Ezh2 inhibitors on NK cell commitment. Identification of the contribution of Ezh2 to NK lineage specification and function reveals an epigenetic-based mechanism that regulates NK cell development and provides insight into the clinical application of Ezh2 inhibitors in NK-based cancer immunotherapies.

Ubiquitination typically occurs through the sequential action of three enzymes catalyzing ubiquitin activation (E1), conjugation (E2), and ligation (E3) and regulates diverse eukaryotic cellular processes. Although monoubiquitination commonly confers nondegradative activities, mechanisms underlying its temporal and spatial regulation and functional plasticity still remain largely unknown. Here we demonstrate that SETDB1, a major histone H3K9 methyltransferase is monoubiquitinated at the evolutionarily conserved lysine-867 in its SET-Insertion domain. This ubiquitination is directly catalyzed by UBE2E family of E2 enzymes in an E3-independent manner while the conjugated-ubiquitin (Ub) is protected from active deubiquitination. The resulting constitutive lysine-867 monoubiquitination is essential for SETDB1's enzymatic activity and endogenous retrovirus silencing in murine embryonic stem cells. Furthermore, the canonical hydrophobic patch on the conjugated-Ub is critical for Ub protection and function. Together, our findings highlight an E3-independent mechanism for monoubiquitination and reveal mechanistic details of SETDB1's enzymatic activity and the functional significance of its SET-Insertion.

Both EZH2 and NF-κB contribute to aggressive breast cancer, yet whether the two oncogenic factors have functional crosstalk in breast cancer is unknown. Here, we uncover an unexpected role of EZH2 in conferring the constitutive activation of NF-κB target gene expression in ER-negative basal-like breast cancer cells. This function of EZH2 is independent of its histone methyltransferase activity but requires the physical interaction with RelA/RelB to promote the expression of NF-κB targets. Intriguingly, EZH2 acts oppositely in ER-positive luminal-like breast cancer cells and represses NF-κB target gene expression by interacting with ER and directing repressive histone methylation on their promoters. Thus, EZH2 functions as a double-facet molecule in breast cancers, either as a transcriptional activator or repressor of NF-κB targets, depending on the cellular context. These findings reveal an additional mechanism by which EZH2 promotes breast cancer progression and underscore the need for developing context-specific strategy for therapeutic targeting of EZH2 in breast cancers.