Global Histone H3 Lysine Research Articles

Simvastatin-Mediated Nrf2 Activation Induces Fetal Hemoglobin and Antioxidant Enzyme Expression to Ameliorate the Phenotype of Sickle Cell Disease.

Sickle cell disease (SCD) is a pathophysiological condition of chronic hemolysis, oxidative stress, and elevated inflammation. The transcription factor Nrf2 is a master regulator of oxidative stress. Here, we report that the FDA-approved oral agent simvastatin, an inhibitor of hydroxymethyl-glutaryl coenzyme A reductase, significantly activates the expression of Nrf2 and antioxidant enzymes. Simvastatin also induces fetal hemoglobin expression in SCD patient primary erythroid progenitors and a transgenic mouse model. Simvastatin alleviates SCD symptoms by decreasing hemoglobin S sickling, oxidative stress, and inflammatory stress in erythroblasts. Particularly, simvastatin increases cellular levels of cystine, the precursor for the biosynthesis of the antioxidant reduced glutathione, and decreases the iron content in SCD mouse spleen and liver tissues. Mechanistic studies suggest that simvastatin suppresses the expression of the critical histone methyltransferase enhancer of zeste homolog 2 to reduce both global and gene-specific histone H3 lysine 27 trimethylation. These chromatin structural changes promote the assembly of transcription complexes to fetal γ-globin and antioxidant gene regulatory regions in an antioxidant response element-dependent manner. In summary, our findings suggest that simvastatin activates fetal hemoglobin and antioxidant protein expression, modulates iron and cystine/reduced glutathione levels to improve the phenotype of SCD, and represents a therapeutic strategy for further development.

3-deazaadenosine (3DA) alleviates senescence to promote cellular fitness and cell therapy efficiency in mice.

Cellular senescence is a stable type of cell cycle arrest triggered by different stresses. As such, senescence drives age-related diseases and curbs cellular replicative potential. Here, we show that 3-deazaadenosine (3DA), an S-adenosyl homocysteinase (AHCY) inhibitor, alleviates replicative and oncogene-induced senescence. 3DA-treated senescent cells showed reduced global Histone H3 Lysine 36 trimethylation (H3K36me3), an epigenetic modification that marks the bodies of actively transcribed genes. By integrating transcriptome and epigenome data, we demonstrate that 3DA treatment affects key factors of the senescence transcriptional program. Remarkably, 3DA treatment alleviated senescence and increased the proliferative and regenerative potential of muscle stem cells from very old mice in vitro and in vivo. Moreover, ex vivo 3DA treatment was sufficient to enhance the engraftment of human umbilical cord blood (UCB) cells in immunocompromised mice. Together, our results identify 3DA as a promising drug enhancing the efficiency of cellular therapies by restraining senescence.

H3K27M/I Mutations Promote Context-Dependent Transformation in Acute Myeloid Leukemia with RUNX1 Alterations

Neomorphic missense mutations in histone H3 genes (most notably K27M and K36M) are frequent in solid cancers such as diffuse intrinsic pontine gliomas (DIPG), supratentorial glioblastoma multiforme (GBM) or chondroblastomas. Despite high incidences of these mutations and their profound impact on global histone H3 lysine methylation, their relevance has not been studied in hematologic cancers.Here, we report the first identification of histone H3 mutations in AML. We find that two of 415 AML patients in the Leucegene cohort and one of 200 patients in the TCGA cohort carry either K27M (n=2) or K27I (n=1) mutations in one of twelve canonical H3 genes. No K36M or other lysine-to-methionine mutations on any of the H3 genes, and no K27M mutation on any of the replication-independent H3.3 variant genes were detected in the same cohorts. H3K27 mutations invariably occurred in the context of either truncating RUNX1 mutations or t(8;21)(q22;q22), suggesting an association with altered RUNX1 function (p < 0.003; Fisher exact test). Characterization of two of the identified H3K27 mutant patient specimens revealed that both samples contained major sub-clones with markedly reduced H3K27me2/3 levels. The H3K27 mutants but not co-occurring mutant ASXL2 or USP7 alleles were only detectable in the H3K27me2/3-low sub-clones, demonstrating that the H3K27 mutations had the most profound impact on H3K27me2/3 in these patients. Moreover, although a heterozygous TET2S1203R mutation was equally present in both H3K27me2/3-high and low sub-fractions, a homozygous RUNX1L98Sfs*22 mutation was restricted to the H3K27me2/3-low sub-clone, further strengthening the possibility that impaired RUNX1 function and reduced H3K27me2/3 associate with each other.We tested the possibility that H3K27 mutants may specifically collaborate with the product of the t(8;21) translocation (AML1-ETO-9a ; AE9a) in transduced mouse hematopoietic stem and progenitor cells (HSPCs). Selectivity of this collaboration was assessed using the combination of the H3K27 mutant gene with the MLL-AF9 fusion product. Whereas expression of H3 WT resulted in a mild competitive proliferation disadvantage in both AE9a and MLL-AF9 cells, expression of H3.3K27I/M conferred a proliferative advantage in AE9a cells in three of four H3K27I -infected and in all H3K27M- infected cultures. This was not observed in MLL-AF9 cells, where H3K27I/M expressing cells were progressively outcompeted in the same culture period. H3K27I/M -infected AE9a cells exhibited significant reductions in H3K27me2/3 and elevated levels of H3K27 acetylation (H3K27ac) compared to H3WT -infected control cells despite undiminished Ezh1, Ezh2 and Suz12 protein levels. In the context of AE9a driven AML in vivo, expression of H3K27M caused a striking disease acceleration leading to all mice reaching end stage disease with a median of 88 days, in contrast to recipients of AE9a ; empty vector or AE9a ; H3WT cells, of which none or only two of nine transplanted mice succumbed to AML within 330 days, respectively (p<0.0001, Log-rank test). Seven of nine AE9a ; H3.3K27I transplanted mice developed AML with a median latency of 208 days (p=0.0004, Log-rank test, compared to controls). Importantly, combined expression of H3K27I or H3K27M with MLL-AF9 did not change disease latencies compared to control groups, indicating selectivity.We next tested the influence of the H3K27M mutation on the differentiation and proliferation of human cord blood CD34+ HSPCs ex vivo . Relative to controls, H3.3K27M expression led to an 8-fold net expansion of a phenotypically primitive CD34+/CD45RA-/CD90+ cell population, which surprisingly lacked expression of the CD133 stem cell marker. Interestingly, granulocytic/granulo-monocytic colony-forming cells (CFU-G/GM) but not multipotent colony-forming cells (granulo/erythroid/monocytic/megakaryocytic; CFU-GEMM) were expanded in 14d cultures initiated with H3K27M transduced CD34+ CB cells when compared to controls. Xenografts of transduced CD34+ CB cells indicated a marginal loss in in vivo reconstitution activity by K27M-transduced cells, mostly at 6 and 12-week time-points.Taken together, our results represent the first description of neomorphic H3K27M/I mutations in AML. We demonstrate that these mutations have a profound impact on H3K27me2/3 and provide evidence that they selectively collaborate with impaired RUNX1 function. DisclosuresNo relevant conflicts of interest to declare.

Global Histone H3 Lysine 4 Trimethylation (H3K4me3) Landscape Changes in Response to TGFβ.

TGFβ expression acts as a biomarker of poor prognosis in prostate cancer. It plays a dual functional role in prostate cancer. In the early stages of the tumor, it acts as a tumor suppressor while at the later stages of tumor development, it promotes metastasis. The molecular mechanisms of action of TGFβ are largely understood through the canonical and non-canonical signal transduction pathways. Our understanding of the mechanisms that establish transient TGFβ stimulation into stable gene expression patterns remains incomplete. Epigenetic marks like histone H3 modifications are directly linked with gene expression and they play an important role in tumorigenesis. In this report, we performed chromatin immunoprecipitation-sequencing (ChIP-Seq) to identify the genome-wide regions that undergo changes in histone H3 Lysine 4 trimethylation (H3K4me3) occupancy in response to TGFβ stimulation. We also show that TGFβ stimulation can induce acute epigenetic changes through the modulation of H3K4me3 signals at genes belonging to special functional categories in prostate cancer. TGFβ induces the H3K4me3 on its own ligands like TGFβ, GDF1, INHBB, GDF3, GDF6, BMP5 suggesting a positive feedback loop. The majority of genes were found to be involved in the positive regulation of transcription from the RNA polymerase II promoter in response to TGFβ. Other functional categories were intracellular protein transport, brain development, EMT, angiogenesis, antigen processing, antigen presentation via MHC class II, lipid transport, embryo development, histone H4 acetylation, positive regulation of cell cycle arrest, and genes involved in mitotic G2 DNA damage checkpoints. Our results link TGFβ stimulation to acute changes in gene expression through an epigenetic mechanism. These findings have broader implications on epigenetic bases of acute gene expression changes caused by growth factor stimulation.

Global histone H3 hyperacetylation-associated epigenetic changes induced in ethephon-primed sprouts of soybean [Glycine max (L.) Merrill

The concept of plant growth regulators (PGRs) in modulating the plant physiological processes through epigenetic modification(s) is emerging. The present work aims to elucidate the histone (de)acetylation-associated epigenetic changes in ethylene (ET)-primed sprouts of soybean [Glycine max (L.) Merrill]. Our results demonstrated that ethephon (donor source of ET) priming (ETp) in soybean significantly enhanced the: (1) germination rate, (2) global histone H3 (H3ac) and H3 lysine 9 (H3K9ac) acetylation levels, (3) histone acetyltransferases activity (concomitant with reduced histone deacetylases activity), (4) cellular acetyl-CoA pool and (5) global de novo RNA synthesis. As a gene-specific case study, ETp resulted in relative reduced starch content, concomitant with an enhanced mRNA accumulation, transcriptional rate and proximal promoter H3K9ac levels of α-amylase 1 (GmαAMY1). Taken together, the proposed epigenetic role of ET as a ‘histone-code’ modulator in soybean sprouts could provide a novel insight on PGR-induced epigenetic re-programming and signifies a broader scope for future epigenetic studies in other agronomical important crops.

Abstract 2871: Global reduction of H3K4me3 improves chemotherapeutic efficacy for pediatric ependymomas

Abstract Background Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. Methods Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 occupancy was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. Results H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells, increased cell response to chemotherapy. Conclusions Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors. Key words: promoter, H3K4me3, human SETD1A, chemotherapy, pediatric ependymoma Citation Format: Guifa Xi, Nitin Wadhwani, Rintaro Hashizume, Barbara Mania-Farnell, Marcelo Bento Soares, Charles D. James, Tadanori Tomita. Global reduction of H3K4me3 improves chemotherapeutic efficacy for pediatric ependymomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2871.

Global Reduction of H3K4me3 Improves Chemotherapeutic Efficacy for Pediatric Ependymomas

BACKGROUND: Ependymomas (EPNs) are the third most common brain tumor in children. These tumors are resistant to available chemotherapeutic treatments, therefore new effective targeted therapeutics must be identified. Increasing evidence shows epigenetic alterations including histone posttranslational modifications (PTMs), are associated with malignancy, chemotherapeutic resistance and prognosis for pediatric EPNs. In this study we examined histone PTMs in EPNs and identified potential targets to improve chemotherapeutic efficacy. METHODS: Global histone H3 lysine 4 trimethylation (H3K4me3) levels were detected in pediatric EPN tumor samples with immunohistochemistry and immunoblots. Candidate genes conferring therapeutic resistance were profiled in pediatric EPN tumor samples with micro-array. Promoter H3K4me3 was examined for two candidate genes, CCND1 and ERBB2, with chromatin-immunoprecipitation coupled with real-time PCR (ChIP-PCR). These methods and MTS assay were used to verify a relationship between H3K4me3 levels and CCND1 and ERBB2, and to investigate cell viability in response to chemotherapeutic drugs in primary cultured pediatric EPN cells. RESULTS: H3K4me3 levels positively correlate with WHO grade malignancy in pediatric EPNs and are associated with progression free survival in patients with posterior fossa group A EPNs (PF-EPN-A). Reduction of H3K4me3 by silencing its methyltransferase SETD1A, in primary cultured EPN cells increased cell response to chemotherapy. CONCLUSIONS: Our results support the development of a novel treatment that targets H3K4me3 to increase chemotherapeutic efficacy in pediatric PF-EPN-A tumors.

Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos.

The ubiquitin-like, containing PHD and RING finger domains, 1 (UHRF1) protein recognizes DNA methylation and histone modification and plays a critical role in epigenetic regulation. Recently, UHRF1 was shown to have a role in DNA methylation in oocytes and early embryos. Here, we reveal that maternal UHRF1 determines the quality of mouse oocytes. We generated oocyte-specific Uhrf1-knockout mice and found that females were sterile, and few maternal UHRF1-null embryos developed into blastocysts. The UHRF1-null oocytes had an increased incidence of aneuploidy and DNA damage. In addition to defective DNA methylation, histone modification was affected during oogenesis, with UHRF1-null germinal vesicle and metaphase II-stage oocytes exhibiting reduced global histone H3 lysine 9 dimethylation levels and elevated acetylation of histone H4 lysine 12. Taken together, our results suggest that UHRF1 plays an important role in determining oocyte quality and affects epigenetic regulation of oocyte maturation as a maternal protein, which is crucial for embryo developmental potential. Further exploration of the biologic function and underlying mechanisms of maternal UHRF1 will enhance our understanding of the maternal control of the oocyte and early embryonic development.-Cao, Y., Li, M., Liu, F., Ni, X., Wang, S., Zhang, H., Sui, X., Huo, R. Deletion of maternal UHRF1 severely reduces mouse oocyte quality and causes developmental defects in preimplantation embryos.

NRF2 Mediates Epigenetic Changes in DNA and Chromatin Structure to Regulate γ-Globin Gene Expression in a Human βYAC Transgenic Mouse Model

NRF2 is the master regulator for the cellular anti-oxidative stress response and previously shown to activate γ-globin gene expression in human erythroid progenitor cells. The goal of this study was to expand on these findings by exploring the in vivo function of NRF2 using the human β-globin locus YAC transgenic (βYAC) mouse carrying the entire 248kb human β-globin locus (HBB). We observed that NRF2 activation by chronic dimethyl fumarate treatment of βYAC mice, induced human γ-globin gene expression, but had no effect on the adult β-globin gene. Subsequently in a novel βYAC/NRF2 knockout mouse model established in our laboratory, we demonstrated that NRF2 loss increased mouse erythroid CD71 levels while reducing human γ-globin gene expression during erythropoiesis in mouse embryonic E13.5 and E18.5 day fetal livers and peripheral blood. Furthermore, the ability of dimethyl fumarate to induce γ-globin gene expression was abolished after NRF2 loss. By western blot analysis of nuclear protein, we confirmed that part of the mechanism of globin gene regulation by NRF2 loss involves a decline of global histone H3 lysine 4 trimethylation levels, without changing histone acetylation. Interestingly, NRF2 loss decreased the protein levels of the DNA methylcytosine dioxygenases including TET1, TET2 and TET3. Analysis of DNA methylation/hydroxymethylation levels by DNA dot-blot assay of mouse E13.5 fetal livers isolated from βYAC/NRF2 knockout mice, showed inhibition of genome wide DNA hydroxymethylation, while DNA methylation was not affected. In addition, DNA immunoprecipitation confirmed decreased hydroxymethylation in the HBB locus control region (LCR) enhancer and γ-globin gene region. These data suggest an essential role of NRF2 in modifying chromatin structure and assembling transcription complexes to regulate γ-globin gene expression. ChIP assay to assess in vivo DNA-protein interactions showed decreased associations of histone H3 lysine 4 monomethylation and trimethylation, TATA-binding protein and RNA polymerase II to the LCR and γ-globin promoter after NRF2 loss. Final studies were conducted to evaluate long-range chromatin interactions between the LCR and individual globin genes by chromosome conformation capture assay. We observed decreased interactions between the LCR and γ-globin gene promoter region after NRF2 loss while interactions in the adult β-globin was not affected suggesting NRF2 preferentially mediates γ-globin gene regulation. In conclusion, our data suggest that NRF2 alters γ-globin expression through epigenetic DNA/histone modifications in addition to direct DNA binding. Therefore, activation of NRF2 expression using small chemical compounds is an innovative strategy to induce γ-globin gene transcription for the treatment of β-hemoglobinopathies. This work was supported by funding from the National Heart, Lung, and Blood Institute to XZ through the Hemoglobinopathy Translational Research Skills Core component of U01 grant HL117684 and R01 grant HL069234 to BSP. DisclosuresNo relevant conflicts of interest to declare.

Development of novel SUV39H2 inhibitors that exhibit growth suppressive effects in mouse xenograft models and regulate the phosphorylation of H2AX.

Protein methyltransferase SUV39H2 was reported to methylate histone H2AX at lysine 134 and enhance the formation of phosphorylated H2AX (γ-H2AX), which causes chemoresistance of cancer cells. We found that a series of imidazo[1,2-a]pyridine compounds that we synthesized could inhibit SUV39H2 methyltransferase activity. One of the potent compounds, OTS193320, was further analyzed in in vitro studies. The compound decreased global histone H3 lysine 9 tri-methylation levels in breast cancer cells and triggered apoptotic cell death. Combination of OTS193320 with doxorubicin (DOX) resulted in reduction of γ-H2AX levels as well as cancer cell viability compared to a single agent OTS193320 or DOX. Further optimization of inhibitors and their in vivo analysis identified a compound, OTS186935, which revealed significant inhibition of tumor growth in mouse xenograft models using MDA-MB-231 breast cancer cells and A549 lung cancer cells without any detectable toxicity. Our results suggest that the SUV39H2 inhibitors sensitize cancer cells to DOX by reduction of γ-H2AX levels in cancer cells, and collectively demonstrate that SUV39H2 inhibition warrants further investigation as a novel anti-cancer therapy.

H3K27M/I mutations promote context-dependent transformation in acute myeloid leukemia with RUNX1 alterations

AbstractNeomorphic missense mutations affecting crucial lysine residues in histone H3 genes significantly contribute to a variety of solid cancers. Despite the high prevalence of H3K27M mutations in pediatric glioblastoma and their well-established impact on global histone H3 lysine 27 di- and trimethylation (H3K27me2/3), the relevance of these mutations has not been studied in acute myeloid leukemia (AML). Here, we report the first identification of H3K27M and H3K27I mutations in patients with AML. We find that these lesions are major determinants of reduced H3K27me2/3 in these patients and that they are associated with common aberrations in the RUNX1 gene. We demonstrate that H3K27I/M mutations are strong disease accelerators in a RUNX1-RUNX1T1 AML mouse model, suggesting that H3K27me2/3 has an important and selective leukemia-suppressive activity in this genetic context.

Abstract 4041: SUV39H2 inhibition enhances sensitivity of breast cancer cells to doxorubicin through downregulation of γ-H2AX production

Abstract Accumulating evidence has demonstrated the biological importance of protein methyltransferases in human tumorigenesis, and several small molecular inhibitors targeting these enzymes have been developed. Moreover, inhibiting pathways involved in DNA repair has been shown to enhance the cytotoxicity of DNA-damaging agents. Herein, we report the development of a potent SUV39H2 inhibitor (OTS193320) that decreases global histone H3 lysine 9 tri-methylation (H3K9me3) levels in cancer cells and attenuates cancer cell proliferation, and suppresses the tumor growth in mouse xenograft models. SUV39H2 (Suppressor of variegation 3-9 homolog 2), is a protein methyltransferase known to methylate histone H3 at lysine 9 (H3K9), and recently reported to methylate histone H2AX at lysine 134, which enhances the accumulation of phosphorylated H2AX (γ-H2AX) and regulates the DNA repair pathway in human cancer. SUV39H2 is highly expressed in many cancer types, including lung and breast cancers, while its expression levels are restricted to testis in normal adult tissues. Exposure of OTS193320 to two triple negative breast cancer cell lines attenuated H3K9me3 levels in a dose-dependent manner and triggered apoptotic cell death. Combination of OTS193320 and doxorubicin (DOX) resulted in reduction of γ-H2AX levels compared to single agent DOX, as visualized on western blot and immunocytochemical analysis. Furthermore, combination therapy attenuated the levels of p53-binding protein 1, which is reported to co-localize with γ-H2AX foci, compared to single agent DOX. Cell viability assays demonstrated a significant growth suppressive effect when OTS193320 was combined with DOX, compared to single agent treatment of either drug, suggesting chemosensitization to DOX. Importantly, in a mouse xenograft model of A549 lung cancer cells, we observed a tumor growth inhibition of 60.8% at day 14 with 25mg/kg intravenous administration of the inhibitor, without significant body weight loss or toxicity. Immunohistochemical staining of tumors treated with the inhibitor demonstrated a significantly lower number of Ki-67 positive cells and attenuated distribution patterns of H3K9me3 compared to the control tumors. Collectively, our results demonstrate that SUV39H2 inhibition may be a promising approach to develop a novel class of anti-cancer treatment. Citation Format: Theodore Vougiouklakis, Vassiliki Saloura, Yo Matsuo, Yusuke Nakamura. SUV39H2 inhibition enhances sensitivity of breast cancer cells to doxorubicin through downregulation of γ-H2AX production [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4041. doi:10.1158/1538-7445.AM2017-4041

Global Regulation of Plant Immunity by Histone Lysine Methyl Transferases.

Posttranslational modification of histones modulates gene expression affecting diverse biological functions. We showed that the Arabidopsis thaliana histone methyl transferases SET DOMAIN GROUP8 (SDG8) and SDG25 regulate pep1-, flg22-, and effector-triggered immunity as well as systemic acquired resistance. Genome-wide basal and induced transcriptome changes regulated by SDG8 and/or SDG25 showed that two genes of the SDG-dependent transcriptome, CAROTENOID ISOMERASE2 (CCR2) and ECERIFERUM3 (CER3), were also required for plant immunity, establishing mechanisms in defense functions for SDG8 and SDG25. CCR2 catalyzes the biosynthesis of carotenoids, whereas CER3 is involved in the biosynthesis of cuticular wax. SDG8 and SDG25 affected distinct and overlapping global and locus-specific histone H3 lysine 4 (H3K4) and histone H3 lysine 36 (H3K36) methylations. Loss of immunity in sdg mutants was attributed to altered global and CCR2- and CER3-specific histone lysine methylation (HLM). Loss of immunity in sdg, ccr2, and cer3 mutants was also associated with diminished accumulation of lipids and loss of cuticle integrity. In addition, sdg8 and sdg25 mutants were impaired in H2B ubiquitination (H2Bubn) at CCR2, CER3, and H2Bubn regulated R gene, SNC1, revealing crosstalk between the two types of histone modifications. In summary, SDG8 and SDG25 contribute to plant immunity directly through HLM or indirectly through H2Bubn and by regulating expression of plant immunity genes, accumulation of lipids, biosynthesis of carotenoids, and maintenance of cuticle integrity.

Cellular functions of MLL/SET-family histone H3 lysine 4 methyltransferase components

The MLL/SET family of histone H3 lysine 4 methyltransferases form enzyme complexes with core subunits ASH2L, WDR5, RbBP5, and DPY-30 (often abbreviated WRAD), and are responsible for global histone H3 lysine 4 methylation, a hallmark of actively transcribed chromatin in mammalian cells. Accordingly, the function of these proteins is required for a wide variety of processes including stem cell differentiation, cell growth and division, body segmentation, and hematopoiesis. While most work on MLL-WRAD has focused on the function this core complex in histone methylation, recent studies indicate that MLL-WRAD proteins interact with a variety of other proteins and lncRNAs and can localize to cellular organelles beyond the nucleus. In this review, we focus on the recently described activities and interacting partners of MLL-WRAD both inside and outside the nucleus.

Quantification of Histone H3 Lys27 Trimethylation (H3K27me3) by High-Throughput Microscopy Enables Cellular Large-Scale Screening for Small-Molecule EZH2 Inhibitors

EZH2 inhibition can decrease global histone H3 lysine 27 trimethylation (H3K27me3) and thereby reactivates silenced tumor suppressor genes. Inhibition of EZH2 is regarded as an option for therapeutic cancer intervention. To identify novel small-molecule (SMOL) inhibitors of EZH2 in drug discovery, trustworthy cellular assays amenable for phenotypic high-throughput screening (HTS) are crucial. We describe a reliable approach that quantifies changes in global levels of histone modification marks using high-content analysis (HCA). The approach was validated in different cell lines by using small interfering RNA and SMOL inhibitors. By automation and miniaturization from a 384-well to 1536-well plate, we demonstrated its utility in conducting phenotypic HTS campaigns and assessing structure-activity relationships (SAR). This assay enables screening of SMOL EZH2 inhibitors and can advance the mechanistic understanding of H3K27me3 suppression, which is crucial with regard to epigenetic therapy. We observed that a decrease in global H3K27me3, induced by EZH2 inhibition, comprises two distinct mechanisms: (1) inhibition of de novo DNA methylation and (II) inhibition of dynamic, replication-independent H3K27me3 turnover. This report describes an HCA assay for primary HTS to identify, profile, and optimize cellular active SMOL inhibitors targeting histone methyltransferases, which could benefit epigenetic drug discovery.

Abstract LB-82: MLL2 maintains neoplastic cell growth and global histone H3 lysine 4 monomethylation

Abstract One striking theme emerging from recent findings in cancer genetics is that chromatin remodeling and histone methylation modifiers are frequently altered in human cancers. Among these newly identified cancer genes, one of the best examples is the gene encoding MLL2 (KMT2D, a.k.a. ALR/MLL4), a histone lysine methyltransferase that plays an important role in regulating gene transcription. Genetic alterations suggestive of a functional deficiency in MLL2 and other genes in the same pathways are common. The identification of alterations in MLL2 suggests potential new opportunities for therapeutics, and highlights an urgent need to understand the underlying tumorigenic mechanism. Filling such a knowledge gap has been challenging, due to the lack of appropriate assays for the gigantic (∼600 kDa), understudied MLL2 protein. To overcome the difficulty, we have used innovative somatic gene editing-based assays to determine the effect of an MLL2 deficiency on neoplastic cells. In particular, we have used homologous recombination- and nuclease-mediated gene editing approaches to generate a panel of isogenic human cancer cell lines that differ with respect to their endogenous MLL2 status. Our studies found that an MLL2 deficiency results in attenuated cancer cell proliferation and defective cell migration. We identified direct transcriptional target genes and revealed the connection of MLL2 to multiple cellular signaling pathways. Analysis of histone H3 modifications revealed that MLL2 is essential for maintaining the level of global histone H3 lysine 4 (H3K4) monomethylation and that its enzymatic SET domain is directly responsible for this function. Furthermore, we found that a majority of MLL2 binding sites are located in regions of potential enhancer elements. The finding concerning enhancer elements is significant, as enhancer elements have increasingly been recognized as critically involved in tumorigenesis. Together, these findings revealed the role of MLL2 in mediating diverse signaling pathways and regulating enhancer elements in human cells, and shed light on the tumorigenic role of its deficiency. Our study supports that MLL2 has distinct roles in neoplastic cells, as opposed to pre-neoplastic cells, and that inhibiting MLL2 may be a viable strategy for cancer therapeutics. Citation Format: Changcun Guo, Lee H. Chen, Yafen Huang, Chun-chi Chang, Ping Wang, Christopher J. Pirozzi, Xiaoxia Qin, Xuhui Bao, Paula K. Greer, Roger E. McLendon, Hai Yan, Stephen T. Keir, Darell D. Bigner, Yiping He. MLL2 maintains neoplastic cell growth and global histone H3 lysine 4 monomethylation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr LB-82. doi:10.1158/1538-7445.AM2014-LB-82

Chromatin Remodeling of Embryonic Stem Cells by E1A Alters Global Histone H3 Lysine 18 Acetylation and Loss of Pluripotency

Chromatin Remodeling of Embryonic Stem Cells by E1A Alters Global Histone H3 Lysine 18 Acetylation and Loss of Pluripotency

KMT2D maintains neoplastic cell proliferation and global histone H3 lysine 4 monomethylation.

KMT2D (lysine (K)-specific methyltransferase 2D), formerly named MLL2 (myeloid/lymphoid or mixed-lineage leukemia 2, also known as ALR/MLL4), is a histone methyltransferase that plays an important role in regulating gene transcription. In particular, it targets histone H3 lysine 4 (H3K4), whose methylations serve as a gene activation mark. Recently, KMT2D has emerged as one of the most frequently mutated genes in a variety of cancers and in other human diseases, including lymphoma, medulloblastoma, gastric cancer, and Kabuki syndrome. Mutations in KMT2D identified thus far point to its loss-of-function in pathogenesis and suggest its role as a tumor suppressor in various tissues. To determine the effect of a KMT2D deficiency on neoplastic cells, we used homologous recombination- and nuclease-mediated gene editing approaches to generate a panel of isogenic colorectal and medulloblastoma cancer cell lines that differ with respect to their endogenous KMT2D status. We found that a KMT2D deficiency resulted in attenuated cancer cell proliferation and defective cell migration. Analysis of histone H3 modifications revealed that KMT2D was essential for maintaining the level of global H3K4 monomethylation and that its enzymatic SET domain was directly responsible for this function. Furthermore, we found that a majority of KMT2D binding sites are located in regions of potential enhancer elements. Together, these findings revealed the role of KMT2D in regulating enhancer elements in human cells and shed light on the tumorigenic role of its deficiency. Our study supports that KMT2D has distinct roles in neoplastic cells, as opposed to normal cells, and that inhibiting KMT2D may be a viable strategy for cancer therapeutics.

Identification of EZH2 and EZH1 Small Molecule Inhibitors with Selective Impact on Diffuse Large B Cell Lymphoma Cell Growth

The histone methyltransferase enhancer of Zeste homolog 2 (EZH2) is a candidate oncogene due to its prevalent overexpression in malignant diseases, including late stage prostate and breast cancers. The dependency of cancer cells on EZH2 activity is also predicated by recurrent missense mutations residing in the catalytic domain of EZH2 that have been identified in subtypes of diffuse large B cell lymphoma, follicular lymphoma and melanoma. Herein, we report the identification of a highly selective small molecule inhibitor series of EZH2 and EZH1. These compounds inhibit wild-type and mutant versions of EZH2 with nanomolar potency, suppress global histone H3-lysine 27 methylation, affect gene expression, and cause selective proliferation defects. These compounds represent a structurally distinct EZH2 inhibitor chemotype for the exploration of the role of Polycomb Repressive Complex 2-mediated H3K27 methylation in various biological contexts.

The Histone Methyltransferase SDG724 Mediates H3K36me2/3 Deposition at MADS50 and RFT1 and Promotes Flowering in Rice

Chromatin modifications affect flowering time in the long-day plant Arabidopsis thaliana, but the role of histone methylation in flowering time regulation of rice (Oryza sativa), a short-day plant, remains to be elucidated. We identified a late-flowering long vegetative phase1 (lvp1) mutant in rice and used map-based cloning to reveal that lvp1 affects the SET domain group protein 724 (SDG724). SDG724 functions as a histone methyltransferase in vitro and contributes to a major fraction of global histone H3 lysine 36 (H3K36) methylation in vivo. Expression analyses of flowering time genes in wild-type and lvp1 mutants revealed that Early heading date1, but not Heading date1, are misregulated in lvp1 mutants. In addition, the double mutant of lvp1 with photoperiod sensitivity5 (se5) flowered later than the se5 single mutant, indicating that lvp1 delays flowering time irrespective of photoperiod. Chromatin immunoprecipitation assays showed that lvp1 had reduced levels of H3K36me2/3 at MADS50 and RFT1. This suggests that the divergent functions of paralogs RFT1 and Hd3a, and of MADS50 and MADS51, are in part due to differential H3K36me2/3 deposition, which also correlates with higher expression levels of MADS50 and RFT1 in flowering promotion in rice.