Dimethylation Of Histone H3 Lysine Research Articles

Targeting the histone methyltransferase G9a activates imprinted genes and improves survival of a mouse model of Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is an imprinting disorder caused by a deficiency of paternally expressed gene(s) in the 15q11-q13 chromosomal region. The regulation of imprinted gene expression in this region is coordinated by an imprinting center (PWS-IC). In individuals with PWS, genes responsible for PWS on the maternal chromosome are present, but repressed epigenetically, which provides an opportunity for the use of epigenetic therapy to restore expression from the maternal copies of PWS-associated genes. Through a high-content screen (HCS) of >9,000 small molecules, we discovered that UNC0638 and UNC0642-two selective inhibitors of euchromatic histone lysine N-methyltransferase-2 (EHMT2, also known as G9a)-activated the maternal (m) copy of candidate genes underlying PWS, including the SnoRNA cluster SNORD116, in cells from humans with PWS and also from a mouse model of PWS carrying a paternal (p) deletion from small nuclear ribonucleoprotein N (Snrpn (S)) to ubiquitin protein ligase E3A (Ube3a (U)) (mouse model referred to hereafter as m+/pΔS-U). Both UNC0642 and UNC0638 caused a selective reduction of the dimethylation of histone H3 lysine 9 (H3K9me2) at PWS-IC, without changing DNA methylation, when analyzed by bisulfite genomic sequencing. This indicates that histone modification is essential for the imprinting of candidate genes underlying PWS. UNC0642 displayed therapeutic effects in the PWS mouse model by improving the survival and the growth of m+/pΔS-U newborn pups. This study provides the first proof of principle for an epigenetics-based therapy for PWS.

Effects of lipopolysaccharide on maturation of bovine oocyte in vitro and its possible mechanisms.

Lipopolysaccharide disturbs the secretion of gonadotropin, endometrial function and implantation efficiency. However, there is little information regarding the effects of lipopolysaccharide on cyclic ovary activity, especially oocyte maturation. Therefore, we aimed to investigate the effects of lipopolysaccharide on the maturation potential of bovine oocytes. We found that lipopolysaccharide exposure significantly decreased the first polar body extrusion rate and delayed the cell cycle progression. The abnormal spindle rate was significantly increased in lipopolysaccharide treatment group, accompanied by disrupted localization and level of phosphorylated mitogen-activated protein kinase (p-MAPK). Moreover, lipopolysaccharide treatment significantly increased intracellular reactive oxygen species (ROS) levels and the early apoptotic rate in oocytes. The pro-apoptotic caspase-3 and Bax mRNA levels and caspase-3 protein level were significantly increased, whereas the anti-apoptotic Bcl-2 and XIAP transcript abundance were significantly decreased in lipopolysaccharide exposure group. Furthermore, the dimethyl-histone H3 lysine 4 (H3K4me2) level was significantly increased, while the DNA methylation (5-mC) and dimethyl-histone H3 lysine 9 (H3K9me2) levels were markedly decreased in oocytes treated with lipopolysaccharide. In conclusion, lipopolysaccharide exposure inhibits the maturation potential of bovine oocytes by affecting cell cycle, cytoskeletal dynamics, oxidative stress, and epigenetic modifications.

Cooperative Gene Repression By DNA Methylation and LSD1-Mediated Enhancer Inactivation in Acute Myeloid Leukemia

Epigenetic silencing of tumor suppressor genes, mediated by aberrant DNA hypermethylation and repressive histone modifications, is a hallmark of acute myeloid leukemia (AML). Novel epigenetic therapies are emerging, but single-agent approaches targeting only one category of epigenetic marks have limited therapeutic activity. We show that combinatorial treatment strategy targeting epigenetic modifications of both promoter and enhancer elements can improve therapeutic intervention.As current cell lines do not accurately represent the heterogeneous nature of AML, we established an ex vivo culturing system enabling us to propagate primary AML specimens long enough to test epigenetic therapeutics (48 specimens expanded 4-6 weeks; 6 specimens expanded >12 months; 39 specimen fail to expand). We tested 52 expandable primary AMLs with a combination therapy utilizing the DNA demethylating agent 5-Azacytidine (5-Aza) and a novel small molecule inhibitor against LSD1, a histone demethylase that removes enhancer-associated marks (mono-/di-methylated histone H3 lysine 4; H3K4me1/2). In order to avoid DNA damage-induced cytotoxicity, we applied a non-DNA damaging dose of 5-Aza (≤ 200 nM). 5-Aza treatment had modest inhibition of cell growth for the majority of cases, but did not appreciably alter cell viability. Treatment with the LSD1 inhibitor (LSD1i) substantially impaired cell growth and survival of ~80% (p<0.001) of cultured primary AMLs. Notably, we found that dual combination therapy using LSD1i and 5-Aza demonstrated significantly improved inhibition of cell viability and growth in most LSD1i-resistant AML cases. Targeted resequencing analysis of 200 genes recurrently mutated in myeloid malignancies revealed an association of mutations with therapeutic activity; TET2mut AML cases without concurrent DNMT3A mutations were most substantially impaired by combinatorial treatment (p<0.001). Also, leukemia burden was significantly reduced after combination therapy in NSG mice transplanted with luciferase-labelled TET2mut patient-derived AML cells (p<0.0002).To gain insight into mechanism, we performed a genome-wide integrative analysis of the DNA methylome (Enhanced Reduced Representation Bisulfite Sequencing), LSD1 occupancy sites (ChIP-sequencing) and gene expression profiling (RNA-sequencing) in a TET2mut patient-derived AML sample. Single-agent LSD1i treatment led to induction of genes associated with myeloid differentiation. Even though 5-Aza induced substantial hypomethylation, there was only a minor effect on gene expression. However, combination therapy did induce greater de-repression of genes, in particular those where 5-Aza induced hypomethylation at promoters and at LSD1-occupied enhancers. Combination treatment especially potentiated induction of differentiation genes, suggesting potential anti-leukemia stem cell effects. Accordingly, combination therapy resulted in impaired colony formation potential (p=0.007). Moreover, limiting dilution analysis in NSG mice demonstrated significant impairment of leukemia-initiating cells (p=0.0025) after 5-Aza+LSD1i combination therapy when compared to either drug alone.To determine what genes mediate the effect of combination therapy, we studied genes preferentially induced by 5-Aza+LSD1i. GATA2, a transcription factor involved in hematopoietic differentiation, was more potently induced by combination therapy in primary AML samples vs. either drug alone. We found LSD1 occupied at the GATA2 distal hematopoietic enhancer and verified by using quantitative ChIP analysis that inhibition of LSD1 induced an increase of H3K4me2 and H3K27ac, a mark that reflects enhancer activation. Overexpression of GATA2 using viral transduction in two patient-derived AML cases caused partial differentiation and attenuated leukemia growth. These results indicated that cellular responses to combination therapy are in part mediated by induction of GATA2.In summary, our results provide a novel link for therapeutic approaches targeting redundant epigenetic silencing of tumor suppressors via cooperative enhancer and enhancer-promoter activation. DisclosuresDuy:GlaxoSmithKline: Research Funding. Mohammad:GlaxoSmithKline: Employment. Smitheman:GlaxoSmithKline: Employment. Guzman:Cellectis: Research Funding. Kruger:GlaxoSmithKline: Employment. Creasy:GlaxoSmithKline: Employment. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Melnick:Janssen: Research Funding.

α-Synuclein enhances histone H3 lysine-9 dimethylation and H3K9me2-dependent transcriptional responses.

α-Synuclein (αS) is a protein linked to Parkinson’s disease (PD) and related neurodegenerative disorders. It is mostly localized within synapses, but αS has also been suggested to play a role in the nucleus. We used transgenic Drosophila and inducible SH-SY5Y neuroblastoma cells to investigate the effects of αS on chromatin with a particular focus on histone modifications. Overexpression of αS in male flies as well as in retinoic acid pre-treated neuroblastoma cells led to an elevation of histone H3K9 methylations, mostly mono- (H3K9me1) and di- (H3K9me2). The transient increase of H3K9 methylation in αS-induced SH-SY5Y cells was preceded by mRNA induction of the euchromatic histone lysine N-methyltransferase 2 (EHMT2). EHMT2 and H3K9me2 can function within the REST complex. Chromatin immunoprecipitation (ChIP) analyses of selected candidate, REST regulated genes showed significantly increased H3K9me2 promoter occupancy of genes encoding the L1CAM cell adhesion molecule and the synaptosomal-associated protein SNAP25, whose reduced expression levels were confirmed by RT-qPCR in αS induced cells. Treatment with EHMT inhibitor UNC0638 restored the mRNA levels of L1CAM and SNAP25. Thus, αS overexpression enhances H3K9 methylations via ΕΗΜΤ2 resulting in elevated H3K9me2 at the SNAP25 promoter, possibly affecting SNARE complex assembly and hence synaptic vesicle fusion events regulated by αS.

Discovery of novel small molecule inhibitors of lysine methyltransferase G9a and their mechanism in leukemia cell lines

Lysine methyltransferase G9a regulates the transcription of multiple genes by primarily catalyzing mono- and di-methylation of histone H3 lysine 9, as well as several non-histone lysine sites. An attractive therapeutic target in treating leukemia, knockout studies of G9a in mice have found dramatically slowed proliferation and self-renewal of acute myeloid leukemia (AML) cells due to the attenuation of HoxA9-dependent transcription. In this study, a series of compounds were identified as potential inhibitors through structure-based virtual screening. Among these compounds, a new G9a inhibitor, DCG066, was confirmed by in vitro biochemical, and cell based enzyme assays. DCG066 has a novel molecular scaffold unlike other G9a inhibitors presently available. Similar to G9a’s histone substrate, DCG066 can bind directly to G9a and inhibit methyltransferase activity in vitro. In addition to suppressing G9a methyltransferase activity and reducing histone H3 methylation levels, DCG066 displays low cytotoxicity in leukemia cell lines with high levels of G9a expression, including K562. This work presents DCG066 as an inhibitor of G9a with a novel structure, providing both a lead in G9a inhibitor design and a means for probing the functionality of G9a.

Inhibition of G9a/GLP Complex Promotes Long-Term Potentiation and Synaptic Tagging/Capture in Hippocampal CA1 Pyramidal Neurons.

Epigenetic regulations play an important role in regulating the learning and memory processes. G9a/G9a-like protein (GLP) lysine dimethyltransferase complex controls a prominent histone H3 lysine9 dimethylation (H3K9me2) that results in transcriptional silencing of the chromatin. Here, we report that the inhibition of G9a/GLP complex by either of the substrate competitive inhibitors UNC 0638 or BIX 01294 reinforces protein synthesis-independent long-term potentiation (early-LTP) to protein synthesis-dependent long-term potentiation (late-LTP). The reinforcement effect was observed if the inhibitors were present during the induction of early-LTP and in addition when G9a/GLP complex inhibition was carried out by priming of synapses within an interval of 30 min before or after the induction of early-LTP. Surprisingly, the reinforced LTP by G9a/GLP complex inhibition was able to associate with a weak plasticity event from nearby independent synaptic populations, resulting in synaptic tagging/capture (STC). We have identified brain-derived neurotrophic factor (BDNF) as a critical plasticity protein that maintains G9a/GLP complex inhibition-mediated LTP facilitation and its STC. Our study reveals an epigenetic mechanism for promoting plasticity and associativity by G9a/GLP complex inhibition, and it may engender a promising epigenetic target for enhancing memory in neural networks.

Interaction of DNA demethylase and histone methyltransferase upregulates Nrf2 in 5-fluorouracil-resistant colon cancer cells.

We recently reported that DNA demethylase ten-eleven translocation 1 (TET1) upregulates nuclear factor erythroid 2-related factor 2 (Nrf2) in 5-fluorouracil-resistant colon cancer cells (SNUC5/5-FUR). In the present study, we examined the effect of histone modifications on Nrf2 transcriptional activation. Histone deacetylase (HDAC) and histone acetyltransferase (HAT) were respectively decreased and increased in SNUC5/5-FUR cells as compared to non-resistant parent cells. Mixed-lineage leukemia (MLL), a histone methyltransferase, was upregulated, leading to increased trimethylation of histone H3 lysine 4, while G9a was downregulated, leading to decreased dimethylation of histone H3 lysine 9. siRNA-mediated MLL knockdown decreased levels of Nrf2 and HO-1 to a greater extent than did silencing HAT1. Host cell factor 1 (HCF1) was upregulated in SNUC5/5-FUR cells, and we observed interaction between HCF1 and MLL. Upregulation of O-GlcNAc transferase (OGT), an activator of HCF1, was also associated with HCF1-MLL interaction. In SNUC5/5-FUR cells, a larger fraction of OGT was bound to TET1, which recruits OGT to the Nrf2 promoter region, than in SNUC5 cells. These findings indicate that SNUC5/5-FUR cells are under oxidative stress, which induces expression of histone methylation-related proteins as well as DNA demethylase, leading to upregulation of Nrf2 and 5-FU resistance.

The regulation of TIM-3 transcription in T cells involves c-Jun binding but not CpG methylation at the TIM-3 promoter.

Tim-3 is an immunomodulatory protein that is expressed constitutively on monocytes but is induced in activated T cells. The mechanisms involved in the regulation of TIM-3 transcription are poorly understood. In the present study, we investigated whether methylation of the TIM-3 promoter is involved in regulatingTIM-3 transcription in T cells, and identified a transcription factor that regulates TIM-3 transcription by associating with the TIM-3 minimal promoter region. Pyrosequencing of the TIM-3 promoter up to -1440bp revealed 11 hypermethylated CpG sites and 4 hypomethylated CpG sites in human CD4(+) T cells as well as in CD11b(+) cells. Dimethylation of histone H3 lysine 4 (H3K4), a mark of transcriptional activation, was predominantly found in the proximal TIM-3 promoter -954 to -34bp region, whereas trimethylation of H3K9 and H3K27, which are markers of transcriptional suppression, were mostly observed in the distal promoter -1549 to -1048bp region in human CD4(+) T cells and CD11b(+) cells. However, no change in the methylation status of CpG sites and the histone H3 in the TIM-3 promoter was found during induction of TIM-3 transcription in T cells. Finally, AP-1 involvement in TIM-3 transcription was shown in relation with the TIM-3 minimal promoter -146 to +144bp region. The present study defines the minimal TIM-3 promoter region and demonstrates its interaction with c-Jun during TIM-3 transcription in CD4(+) T cells.

Analysis of gene expression and histone modification between C4 and non-C4 homologous genes of PPDK and PCK in maize.

More efficient photosynthesis has allowed C4 plants to adapt to more diverse ecosystems (such as hot and arid conditions) than C3 plants. To better understand C4 photosynthesis, we investigated the expression patterns of C4 genes (C4PPDK and PCK1) and their non-C4 homologous genes (CyPPDK1, CyPPDK2, and PCK2) in the different organs of maize (Zea mays). Both C4 genes and non-C4 genes showed organ-dependent expression patterns. The mRNA levels of C4 genes were more abundant in leaf organ than in seeds at 25days after pollination (DAP), while non-C4 genes were mainly expressed in developing seeds. Further, acetylation of histone H3 lysine 9 (H3K9ac) positively correlates with mRNA levels of C4 genes (C4PPDK and PCK1) in roots, stems, leaves, and seeds at 25 DAP, acetylation of histone H4 lysine 5 (H4K5ac) in the promoter regions of both C4 (C4PPDK and PCK1) and non-C4 genes (CyPPDK1, CyPPDK2, and PCK2) correlated well with their transcripts abundance in stems. In photosynthetic organs (stems and leaves), dimethylation of histone H3 lysine 9 (H3K9me2) negatively correlated with mRNA levels of both C4 and non-C4 genes. Taken together, our data suggest that histone modification was involved in the transcription regulation of both C4 genes and non-C4 genes, which might provide a clue of the functional evolution of C4 genes.

Inhibition of Lysine-Specific Demethylase-1 (LSD1/KDM1A) Promotes the Adipogenic Differentiation of hESCs Through H3K4 Methylation.

Given their totipotency, human embryonic stem cells (hESCs) can differentiate into all types of cells, including adipocytes, and provide an excellent research model for studying diseases associated with the metabolism of adipocytes, such as obesity and diabetes mellitus. Epigenetic regulation, including DNA methylation and histone modification, plays an essential role in the development and differentiation of hESCs. Lysine-specific demethylase 1 (LSD1), a well-characterized histone-modifying enzyme, demethylates dimethylated histone H3 lysine 4 (H3K4) through a flavin adenine dinucleotide (FAD)-dependent oxidative reaction. LSD1 affects the growth and differentiation of human and mouse ES cells, and the deletion of this gene in mice leads to embryonic lethality. Here, we investigated the functional role of LSD1 during the adipogenic differentiation of hESCs involving the demethylation of H3K4. We also found that treating hESCs with the LSD1 inhibitor CBB1007 promotes the adipogenic differentiation of hESCs.

Depletion of G9a gene induces cell apoptosis in human gastric carcinoma.

G9a is a mammalian histone methyltransferase that contributes to the epigenetic silencing of tumor suppressor genes. Evidence suggests that G9a is required to maintain the malignant phenotype, but little documentation show the role of G9a function in mediating tumor growth. We retrospectively analyzed the protein of G9a and monomethylated histone H3 lysine 9 (H3K9 me1), and dimethylated histone H3 lysine 9 (H3K9 me2) in 175 cases of gastric carcinoma by immunohistochemistry. RNAi-based inhibition of G9a in MGC803 cancer cell line was studied. G9a depletion was done by transient transfection using Lipofectamine 2000. Depletion efficiency of G9a was tested using real-time PCR and western blot analysis. Cell apoptosis and proliferation were detected by TUNEL assay and MTT, respectively. The proteins of H3K9 me1, me2, trimethylation of H3K9 (H3K9 me3), monomethylated histone H3 lysine 27 (H3K27 me1), dimethylated histone H3 lysine 27 (H3K27 me2) and histone acetylated H3, apoptotic proteins were studied by western blot analysis. G9a and H3K9 me2 expression was higher in gastric cancer cells compared to the control (p<0.05). Both G9a and H3K9 me2 were positively correlated with the degree of differentiation, depth of infiltration, lymphatic invasions and tumor-node-metastasis stage in gastric carcinoma, (p<0.05). RNAi-mediated knockdown of G9a induced cell apoptosis and inhibited cell proliferation. Depletion of G9a reduced the levels of H3K9 me1 and me2, H3K27 me1 and me2. Nonetheless, it did not activate acetylation of H3 and H3K9 me3. These data suggest that G9a is required in tumorigenesis, and correlated with prognosis. Furthermore, G9a plays a critical role in regulating epigenetics. Depletion of G9a inhibits cell growth and induces cells apoptosis in gastric cancer. It might be of therapeutic benefit in gastric cancers.

Sulforaphane modulates telomerase activity via epigenetic regulation in prostate cancer cell lines.

Epidemiologic studies have revealed that diets rich in sulforaphane (SFN), an isothiocyanate present in cruciferous vegetables, are associated with a marked decrease in prostate cancer incidence. The chemo-preventive role of SFN is associated with its histone de-acetylase inhibitor activity. However, the effect of SFN on chromatin composition and dynamic folding, especially in relation to HDAC inhibitor activity, remains poorly understood. In this study, we found that SFN can inhibit the expression and activity of human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomerase, in 2 prostate cancer cell lines. This decrease in gene expression is correlated with SFN-induced changes in chromatin structure and composition. The SFN-mediated changes in levels of histone post-translational modifications, more specifically acetylation of histone H3 lysine 18 and di-methylation of histone H3 lysine 4, 2 modifications linked with high risk of prostate cancer recurrence, were associated with regulatory elements within the hTERT promoter region. Chromatin condensation may also play a role in SFN-mediated hTERT repression, since expression and recruitment of MeCP2, a known chromatin compactor, were altered in SFN treated prostate cancer cells. Chromatin immuno-precipitation (ChIP) of MeCP2 showed enrichment over regions of the hTERT promoter with increased nucleosome density. These combined results strongly support a role for SFN in the mediation of epigenetic events leading to the repression of hTERT in prostate cancer cells. This ability of SFN to modify chromatin composition and structure associated with target gene expression provides a new model by which dietary phytochemicals may exert their chemoprevention activity.

Abstract 3509: Novel dual inhibitors of LSD1-HDAC for treatment of cancer

Abstract Introduction: LSD1 is a flavin adenine dinucleotide (FAD)-dependent amine oxidase that removes methyl groups from mono- or dimethylated histone H3 lysine 4. LSD1 has been reported to be overexpressed in many malignant tumors, including breast, colon, prostate, lung, gastric cancers and others. Studies have shown that LSD1 may contribute to acute myelogenous leukaemia pathogenesis and patients that are non responders to ATRA have responded to LSD1-ATRA combination treatment in clinic. Recent studies have shown that there is cross-talk between two components of CoREST complex, LSD1 and HDAC which provides an advantage to cancer cell proliferation and survival by regulating key signalling pathways. Accordingly, combined inhibition of LSD1 and HDAC has been shown to be more efficacious in inhibiting the growth of glioblastoma, AML and breast cancer. Therefore, dual inhibitors targeting both LSD1 and HDAC could be useful in treating several cancers effectively without enhancing systemic toxicity mediated by administration of multiple drugs. To test the hypothesis, we have developed a set of molecules that either have LSD1 activity alone or dual activity on LSD1 and HDAC. Methods: Computational chemistry approaches were used to design LSD1 specific and LSD1-HDAC dual inhibitors. To assess in vitro LSD1 potency, TR-FRET assay was used. For assessing in vitro HDAC activity fluorescence based pan-HDAC activity assay was performed. Western blotting was used to assess biomarkers of LSD1 and HDAC inhibition. Alamar blue cytotoxicity assay was used to assess cell proliferation. Results: In vitro potency of these compounds on LSD1 ranged between,0.007 to 0.2 μM, which was comparable to literature molecules. Pan-HDAC potency of these molecules in the in vitro assay was comparable to the approved drug, Vorinostat. H3K4 di- and mono-methyl markers and H3K9 acetylation levels respectively, were used as target engagement biomarkers for LSD1and HDAC inhibition. Treatment of leukaemia cells with LSD1or LSD1-HDAC inhibitors resulted in a dose-dependent increase in the levels of H3K4 di-and mono methylation. In addition, compounds with LSD1-HDAC dual activity showed a clear increase in H3K9 acetylation levels as well. In the cell proliferation assay, compounds with LSD1 activity alone did not show any effect up to 72 h of treatment. However, compounds with LSD1-HDAC dual activity showed a strong effect in inhibiting cell proliferation in multiple cell lines, with IC50s ranging from 0.02 to 2 μM, with leukaemia cell lines being generally more sensitive than solid tumors. Conclusion: The data obtained demonstrate that it is feasible to design dual LSD1-HDAC inhibitors that retain individual activity and potently inhibit cell proliferation, and such inhibitors could serve as powerful therapeutic agents for cancer. Further understanding of mechanisms to identify the right tool compound is in progress. Citation Format: Dhanalakshmi Sivanandhan, Sridharan Rajagopalan, Sreekala Nair, Purushottam Dewang, Durga Prasanna Kumar, Chandrika Mulakala, Lavanya Mahadevan, Neema Skariah, Venkata Giri Kavuru, Damodara Kuntrapaku, Suchitra Sajja, Mohammad Zainuddin, Krishnakumar V, Ritu Singh, Swarnakumari V, Ramachandraiah Gosu, Jayashree Aiyar, Pravin Iyer, Sriram Rajagopal. Novel dual inhibitors of LSD1-HDAC for treatment of cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3509. doi:10.1158/1538-7445.AM2015-3509

A Systematic Review of Histone Lysine‐Specific Demethylase 1 and Its Inhibitors

Histone lysine-specific demethylase 1 (LSD1) is the first discovered and reported histone demethylase by Dr. Shi Yang's group in 2004. It is classified as a member of amine oxidase superfamily, the common feature of which is using the flavin adenine dinucleotide (FAD) as its cofactor. Since it is located in cell nucleus and acts as a histone methylation eraser, LSD1 specifically removes mono- or dimethylated histone H3 lysine 4 (H3K4) and H3 lysine 9 (H3K9) through formaldehyde-generating oxidation. It has been indicated that LSD1 and its downstream targets are involved in a wide range of biological courses, including embryonic development and tumor-cell growth and metastasis. LSD1 has been reported to be overexpressed in variety of tumors. Inactivating LSD1 or downregulating its expression inhibits cancer-cell development. LSD1 targeting inhibitors may represent a new insight in anticancer drug discovery. This review summarizes recent studies about LSD1 and mainly focuses on the basic physiological function of LSD1 and its involved mechanisms in pathophysiologic conditions, as well as the development of LSD1 inhibitors as potential anticancer therapeutic agents.

A Proteomic Strategy Identifies Lysine Methylation of Splicing Factor snRNP70 by the SETMAR Enzyme

The lysine methyltransferase (KMT) SETMAR is implicated in the response to and repair of DNA damage, but its molecular function is not clear. SETMAR has been associated with dimethylation of histone H3 lysine 36 (H3K36) at sites of DNA damage. However, SETMAR does not methylate H3K36 in vitro. This and the observation that SETMAR is not active on nucleosomes suggest that H3K36 methylation is not a physiologically relevant activity. To identify potential non-histone substrates, we utilized a strategy on the basis of quantitative proteomic analysis of methylated lysine. Our approach identified lysine 130 of the mRNA splicing factor snRNP70 as a SETMAR substrate in vitro, and we show that the enzyme primarily generates monomethylation at this position. Furthermore, we show that SETMAR methylates snRNP70 Lys-130 in cells. Because snRNP70 is a key early regulator of 5' splice site selection, our results suggest a model in which methylation of snRNP70 by SETMAR regulates constitutive and/or alternative splicing. In addition, the proteomic strategy described here is broadly applicable and is a promising route for large-scale mapping of KMT substrates.

WHSC1 promotes oncogenesis through regulation of NIMA-related kinase-7 in squamous cell carcinoma of the head and neck.

Squamous cell carcinoma of the head and neck (SCCHN) is a relatively common malignancy with suboptimal long-term prognosis, thus new treatment strategies are urgently needed. Over the last decade, histone methyltransferases (HMT) have been recognized as promising targets for cancer therapy, but their mechanism of action in most solid tumors, including SCCHN, remains to be elucidated. This study investigated the role of Wolf-Hirschhorn syndrome candidate 1 (WHSC1), an NSD family HMT, in SCCHN. Immunohistochemical analysis of locoregionally advanced SCCHN, dysplastic, and normal epithelial tissue specimens revealed that WHSC1 expression and dimethylation of histone H3 lysine 36 (H3K36me2) were significantly higher in SCCHN tissues than in normal epithelium. Both WHSC1 expression and H3K36me2 levels were significantly correlated with histologic grade. WHSC1 knockdown in multiple SCCHN cell lines resulted in significant growth suppression, induction of apoptosis, and delay of the cell-cycle progression. Immunoblot and immunocytochemical analyses in SCCHN cells demonstrated that WHSC1 induced H3K36me2 and H3K36me3. Microarray expression profile analysis revealed NIMA-related kinase-7 (NEK7) to be a downstream target gene of WHSC1, and chromatin immunoprecipitation (ChIP) assays showed that NEK7 was directly regulated by WHSC1 through H3K36me2. Furthermore, similar to WHSC1, NEK7 knockdown significantly reduced cell-cycle progression, indicating that NEK7 is a key player in the molecular pathway regulated by WHSC1. WHSC1 possesses oncogenic functions in SCCHN and represents a potential molecular target for the treatment of SCCHN.

Early-life exposure to high-fat diet may predispose rats to gender-specific hepatic fat accumulation by programming Pepck expression

Phosphoenolpyruvate carboxykinase (PEPCK) produces phosphoenolpyruvate during glyceroneogenesis. We previously demonstrated that a high-fat diet during pregnancy induced Pepck mRNA expression in neonatal rat pups, which is characterized by histone modifications in specific regions of the gene (Strakovsky RS, Zhang X, Zhou D, Pan YX. Gestational high fat diet programs hepatic phosphoenolpyruvate carboxykinase gene expression and histone modification in neonatal offspring rats. The Journal of Physiology 2011;589:2707–17). In the present study, we investigated whether these alterations persistent in adult offspring. Dams were fed either control or high-fat diet throughout gestation and lactation. Offspring were placed on control diet after weaning, generating C/C and HF/C groups. Liver was collected at 12 weeks of age. Hepatic nicotinamide adenine dinucleotide (reduced) (NADH) level was increased in both genders, but fat accumulation occurred only in liver of female offspring in HF/C group. This was accompanied by a significant increase of Pepck and fatty acid synthase (Fasn) mRNA expression in only female liver. The induction of Pepck gene expression in females was associated with increased dimethylated histone H3 lysine 4 level in multiple regions of the gene. Meanwhile, acetylated histone H3 and trimethylated histone H3 lysine 4 were induced at a specific coding region in HF/C, accompanied by decreased trimethylated histone H3 lysine 9 level at the promoter of female offspring. In conclusion, maternal high-fat diet programs Pepck expression through histone modifications in adult female offspring. Persistent Pepck induction in females may contribute to increased triglyceride synthesis, together with induced Fasn expression and NADH levels, which may lead to increased fat deposition in a gender-specific manner.

Abstract 96: Epigenetic And Transcriptional Regulation Of Induced Pluripotent Stem Cells In Patients With Hypoplastic Left Heart Syndrome

Background: Although a number of studies have uncovered heterozygous mutations in cardiac regulatory genes caused hypoplastic left heart syndrome (HLHS), the identified genetic variants may not be directly correlated with the disease development. The aim of this study is to determine the epigenetic and transcriptional network responsible for myocardial patterning and morphogenesis during cardiac development in HLHS by using patient-derived induced pluripotent stem (iPS) cells. Methods: Five-independent iPS cell lines were generated from HLHS and biventricle (BV) heart-derived cardiac progenitor cells (CPCs). Global gene expression analysis, real-time RT-PCR, mutation analysis, ChIP assay, and cardiac-specific gene promoter activities were examined during differentiation. Results: We found one synonymous single nucleotide polymorphism in NKX2-5 and five in NOTCH1, respectively. Cardiac transcriptional factors such as NKX2-5, HAND1, and NOTCH/HEY, those are known to drive primary heart field and outflow tract development, were significantly reduced in HLHS-derived iPS cells after differentiation compared with BV- and control 201B7 iPS-derived cardiomyocytes. ChIP assay showed that a marked decrease in dimethylated histone H3-lysine 4 and acetylated histone H3 was found within the NKX2-5 promoter regions, whereas a significantly increased trimethylated H3-lysine 27 was identified in differentiated HLHS-derived iPS cells. To specify the target transcripts responsible for cardiac development of HLHS, cardiac troponin-T and natriuretic peptide A promoter analyses were performed. We found that both promoter activities were significantly suppressed in HLHS-derived CPCs and iPS cells compared with those from BV hearts. These repressed promoter activities could be fully restored by transient transfection of NKX2-5, HAND1, and NOTCH1 genes into these stem/progenitor cells by synergistic manner. Conclusions: These findings suggest that patient-specific iPS cells provide a potential to dissecting the complex cardiac malformations in human. The epigenetic and transcriptional regulation of NKX2-5, HAND1, and NOTCH1 may mutually participate in the development of myocardial growth, patterning, and morphogenesis in HLHS.

The role of nuclear pore proteins in epigenetic regulation and spatial organization of the genome (471.2)

In yeast and metazoa, active genes interact with nuclear pore proteins, which promotes stronger transcription. Targeting to the NPC in yeast is mediated by cis‐acting “DNA zip codes” that are necessary and sufficient to confer targeting to the nuclear periphery. Loci with the same zip codes cluster together. Some inducible genes remain associated with the NPC for several generations after being repressed, a phenomenon called epigenetic transcriptional memory. This interaction poises genes for future reactivation and requires the nuclear pore protein Nup100, a homologue of human Nup98. A similar phenomenon occurs in human cells; for at least four generations after treatment with interferon gamma (IFNg), many IFNg‐inducible genes are induced more rapidly and more strongly than in cells that have not previously been exposed to IFNg. In both yeast and human cells, the recently expressed promoters of genes with memory exhibit persistent dimethylation of histone H3 lysine 4 (H3K4me2) and physically interact with Nups and a poised form of RNA polymerase II. Transcriptional memory in yeast requires several chromatin modifications: H2B ubiquitination, H3K4 methylation and the SET3C histone deacetylase. In human cells transiently depleted of Nup98 or yeast cells lacking Nup100, transcriptional memory is lost; RNA polymerase II does not remain associated with promoters, H3K4me2 is lost, and the rate of transcriptional reactivation is reduced. Thus, Nup binding to active genes plays a conserved role in promoting stronger transcription and Nup binding to recently expressed promoters plays a conserved role in promoting epigenetic transcriptional memory.

Genistein‐induced histone modifications on ATF3 may contribute to cell cycle arrest and apoptosis in human colon cancer cell line SW620 (800.8)

Genistein, a soy‐derived isoflavone, has been shown to suppress colon cancer progression by regulating different cellular properties. The aim of the study is to investigate the role of Activating Transcription Factor 3 (ATF3), an immediate‐early response gene, in genistein‐modulated cell cycle arrest and apoptosis in a human colon cancer cell line SW620. Results showed that genistein induced G2 phase arrest and increased late apoptosis in SW620. Genistein also increased the expression of ATF3 mRNA and protein, as well as its transcriptional activity in SW620. mRNA expression of downstream genes of ATF3, such as cell cycle inhibitors p53, p21, CyclinD1, and apoptotic enzymes Casp8, and Casp9, were all induced by genistein. Chromatin immunoprecipitation (ChIP) showed decreased phosphorylated histone H3 serine 10 (H3S10p) at both the promoter and coding region of ATF3. In addition, genistein increased trimethylated histone H3 lysine 36 (H3K36me3) level at a coding region and dimethylated histone H3 lysine 4 (H3K4me2) level at the promoter region. Methylation sensitive PCR showed that genistein did not change the level of DNA methylation at various regions of ATF3 gene. In conclusion, genistein‐mediated histone modifications of ATF3 may lead to G2 phase arrest and apoptosis in SW620.