Monomethylation Of Histone H4 Lysine Research Articles

KMT2D-mediated H3K4me1 recruits YBX1 to facilitate triple-negative breast cancer progression through epigenetic activation of c-Myc.

Lysine methyltransferase 2D (KMT2D) mediates mono-methylation of histone H3 lysine 4 (H3K4me1) in mammals. H3K4me1 mark is involved in establishing an active chromatin structure to promote gene transcription. However, the precise molecular mechanism underlying the KMT2D-mediated H3K4me1 mark modulates gene expression in triple-negative breast cancer (TNBC) progression is unresolved. We recognized Y-box-binding protein 1 (YBX1) as a "reader" of the H3K4me1 mark, and a point mutation of YBX1 (E121A) disrupted this interaction. We found that KMT2D and YBX1 cooperatively promoted cell growth and metastasis of TNBC cells in vitro and in vivo. The expression levels of KMT2D and YBX1 were both upregulated in tumour tissues and correlated with poor prognosis for breast cancer patients. Combined analyses of ChIP-seq and RNA-seq data indicated that YBX1 was co-localized with KMT2D-mediated H3K4me1 in the promoter regions of c-Myc and SENP1, thereby activating their expressions in TNBC cells. Moreover, we demonstrated that YBX1 activated the expressions of c-Myc and SENP1 in a KMT2D-dependent manner. Our results suggest that KMT2D-mediated H3K4me1 recruits YBX1 to facilitate TNBC progression through epigenetic activation of c-Myc and SENP1. These results together unveil a crucial interplay between histone mark and gene regulation in TNBC progression, thus providing novel insights into targeting the KMT2D-H3K4me1-YBX1 axis for TNBC treatment. YBX1 is a KMT2D-mediated H3K4me1-binding effector protein and mutation of YBX1 (E121A) disrupts its binding to H3K4me1. KMT2D and YBX1 cooperatively promote TNBC proliferation and metastasis by activating c-Myc and SENP1 expression in vitro and in vivo. YBX1 is colocalized with H3K4me1 in the c-Myc and SENP1 promoter regions in TNBC cells and increased YBX1 expression predicts a poor prognosis in breast cancer patients.

Distinct developmental phenotypes result from mutation of Set8/KMT5A and histone H4 lysine 20 in Drosophila melanogaster.

Mono-methylation of histone H4 lysine 20 (H4K20me1) is catalyzed by Set8/KMT5A and regulates numerous aspects of genome organization and function. Loss-of-function mutations in Drosophila melanogaster Set8 or mammalian KMT5A prevent H4K20me1 and disrupt development. Set8/KMT5A also has non-histone substrates, making it difficult to determine which developmental functions of Set8/KMT5A are attributable to H4K20me1 and which to other substrates or to non-catalytic roles. Here, we show that human KMT5A can functionally substitute for Set8 during Drosophila development and that the catalytic SET domains of the two enzymes are fully interchangeable. We also uncovered a role in eye development for the N-terminal domain of Set8 that cannot be complemented by human KMT5A. Whereas Set820/20 null mutants are inviable, we found that an R634G mutation in Set8 predicted from in vitro experiments to ablate catalytic activity resulted in viable adults. Additionally, Set8(R634G) mutants retain significant, albeit reduced, H4K20me1, indicating that the R634G mutation does not eliminate catalytic activity in vivo and is functionally hypomorphic rather than null. Flies engineered to express only unmodifiable H4 histones (H4K20A) can also complete development, but are phenotypically distinct from H4K20R, Set820/20 null, and Set8R634G mutants. Taken together, our results demonstrate functional conservation of KMT5A and Set8 enzymes, as well as distinct roles for Set8 and H4K20me1 in Drosophila development.

SMYD5 is a histone H3-specific methyltransferase mediating mono-methylation of histone H3 lysine 36 and 37

Although post-translational modifications (-PTMs) of some histone H3 lysine residues are well studied, the PTMs of histone H3 lysine 37 in mammalian cells remain largely unknown. In this study, we provide evidence to show that SMYD family member 5 (SMYD5) is a histone H3-specfic methyltransferase that catalyzes mono-methylation of H3 lysine 36 and 37 (H3K36/K37me1) in vitro. The site-mutagenesis analysis shows that a species-conserved histidine in its catalytic SET domain is required for its histone methyltransferase activity. Genetic deletion of Smyd5 in murine embryonic stem cells (mESCs) partially reduces the global histone H3K37me1 level in cells, suggesting SMYD5 is one of histone methyltransferases catalyzing histone H3K37me1 in vivo. Hence, our study reveals that SMYD5 is a histone H3-specific methyltransferase that mediates histone H3K36/K37me1, which provides a biochemical basis for further studying its functions in mammalian cells.

The MLL3/4 H3K4 methyltransferase complex in establishing an active enhancer landscape.

Enhancers are cis-regulatory elements that play essential roles in tissue-specific gene expression during development. Enhancer function in the expression of developmental genes requires precise regulation, while deregulation of enhancer function could be the main cause of tissue-specific cancer development. MLL3/KMT2C and MLL4/KMT2D are two paralogous histone modifiers that belong to the SET1/MLL (also named COMPASS) family of lysine methyltransferases and play critical roles in enhancer-regulated gene activation. Importantly, large-scale DNA sequencing studies have revealed that they are amongst the most frequently mutated genes associated with human cancers. MLL3 and MLL4 form identical multi-protein complexes for modifying mono-methylation of histone H3 lysine 4 (H3K4) at enhancers, which together with the p300/CBP-mediated H3K27 acetylation can generate an active enhancer landscape for long-range target gene activation. Recent studies have provided a better understanding of the possible mechanisms underlying the roles of MLL3/MLL4 complexes in enhancer regulation. Moreover, accumulating studies offer new insights into our knowledge of the potential role of MLL3/MLL4 in cancer development. In this review, we summarize recent evidence on the molecular mechanisms of MLL3/MLL4 in the regulation of active enhancer landscape and long-range gene expression, and discuss their clinical implications in human cancers.

KDM1A maintains genome-wide homeostasis of transcriptional enhancers.

Transcriptional enhancers enable exquisite spatiotemporal control of gene expression in metazoans. Enrichment of monomethylation of histone H3 lysine 4 (H3K4me1) is a major chromatin signature of transcriptional enhancers. Lysine (K)-specific demethylase 1A (KDM1A, also known as LSD1), an H3K4me2/me1 demethylase, inactivates stem-cell enhancers during the differentiation of mouse embryonic stem cells (mESCs). However, its role in undifferentiated mESCs remains obscure. Here, we show that KDM1A actively maintains the optimal enhancer status in both undifferentiated and lineage-committed cells. KDM1A occupies a majority of enhancers in undifferentiated mESCs. KDM1A levels at enhancers exhibit clear positive correlations with its substrate H3K4me2, H3K27ac, and transcription at enhancers. In Kdm1a-deficient mESCs, a large fraction of these enhancers gains additional H3K4 methylation, which is accompanied by increases in H3K27 acetylation and increased expression of both enhancer RNAs (eRNAs) and target genes. In postmitotic neurons, loss of KDM1A leads to premature activation of neuronal activity-dependent enhancers and genes. Taken together, these results suggest that KDM1A is a versatile regulator of enhancers and acts as a rheostat to maintain optimal enhancer activity by counterbalancing H3K4 methylation at enhancers.

Distinct kinetic mechanisms of H3K4 methylation catalyzed by MLL3 and MLL4 core complexes

The methyltransferases MLL3 and MLL4 primarily catalyze the monomethylation of histone H3 lysine 4 (H3K4) on enhancers to regulate cell-type-specific gene expression and cell fate transition. MLL3 and MLL4 share almost identical binding partners and biochemical activities, but perform specific and nonredundant functions. The features and functions that distinguish MLL3 and MLL4 remain elusive. Here, we characterize the kinetic mechanisms of MLL3 and MLL4 ternary complexes containing the catalytic SET domain from MLL3 or MLL4 (MLL3SET or MLL4SET), the SPRY domain of ASH2L (ASH2LSPRY), and a short fragment of RBBP5 (RBBP5AS-ABM) to search for possible explanations. Steady-state kinetic analyses and inhibition studies reveal that the MLL3 complex catalyzes methylation in a random sequential bi–bi mechanism. In contrast, the MLL4 complex adopts an ordered sequential bi–bi mechanism, in which the cofactor S-adenosylmethionine (AdoMet) binds to the enzyme prior to the H3 peptide, and the methylated H3 peptide dissociates from the enzyme before S-adenosylhomocysteine (AdoHcy) detaches after methylation. Substrate-binding assays using fluorescence polarization (FP) confirm that AdoMet binding is a prerequisite for H3 binding for the MLL4 complex but not for the MLL3 complex. Molecular dynamic simulations reveal that the binding of AdoMet exclusively induces conformational constraints on the AdoMet-binding groove and the H3 substrate-binding pocket of MLL4, therefore stabilizing a specific active conformation to ease entry of the substrate H3. The distinct kinetic mechanisms and conformational plasticities provide important insights into the differential functions of MLL3 and MLL4 and may also guide the development of selective inhibitors targeting MLL3 or MLL4.

The histone lysine methyltransferase SETD8 regulates angiogenesis through HES-1 in human umbilical vein endothelial cells

Histone modifications, including histone lysine methylation, regulate gene expression in the vasculature, and targeting tumor blood vessels through histone modification decreases tumor growth. SETD8, a methyltransferase that catalyzes the mono-methylation of histone H4 lysine 20 is known to promote tumorigenesis in various cancers and its high levels of expression are related to poor prognosis. However, the detailed mechanisms by which SETD8 stimulates tumor progression and angiogenesis are still not well understood. Recent studies have demonstrated that, in vitro, BVT-948 efficiently and selectively suppresses SETD8 activity and histone methylation levels. In this study, we showed that BVT-948-mediated SETD8 inhibition in HUVECs results in an inhibition of angiogenesis. Inhibition of SETD8 not only inhibited angiogenesis but also disrupted actin stress fiber formation and induced cell cycle arrest at S phase. These effects were accompanied by increased HES-1 expression levels, decreased osteopontin levels, and a decreased differentiation of human induced pluripotent stem cells into endothelial cells. Interestingly, BVT-948 treatment reduced pathological angiogenesis in mouse OIR model. These data illustrate the mechanisms by which SETD8 regulates angiogenesis and may enable the use of a SETD8 inhibitor to treat various pathological conditions that are known to be associated with excessive angiogenesis, including and tumor growth.

Downregulation of histone methyltransferase SET8 inhibits progression of hepatocellular carcinoma

The expression of lysine methyltransferase SET8, which is involved in carcinogenesis of many types of human cancers through monomethylation of histone H4 lysine 20 (H4K20), is associated with the prognosis of hepatocellular carcinoma (HCC). We performed a functional analysis for SET8 to assess its effect on HCC progression. SET8 knockdown inhibited proliferation, migration and invasion of HCC cells. SET8 knockdown also inhibited tumour growth in a human xenograft mouse model. Overexpression of SET8 displayed the reverse effect, while treatment with the SET8 inhibitor UNC0379 produced an effect similar to SET8 knockdown. In addition, drug sensitivity testing in SET8-siRNA transfected HCC cells indicated that docetaxel inhibited cell growth dramatically, as demonstrated by the Cell Counting Kit-8 (CCK-8) assay. Furthermore, gene expression microarray analysis showed that genes altered after SET8 knockdown were clustered in pathways related to tumorigenesis and metastasis. Our data suggests that targeting SET8 for HCC therapy can inhibit the proliferation and invasion of HCC cells as well as increase their sensitivity to chemotherapy.

Mutant p53 regulates enhancer-associated H3K4 monomethylation through interactions with the methyltransferase MLL4

Monomethylation of histone H3 lysine 4 (H3K4me1) is enriched at enhancers that are primed for activation and the levels of this histone mark are frequently altered in various human cancers. Yet, how alterations in H3K4me1 are established and the consequences of these epigenetic changes in tumorigenesis are not well understood. Using ChIP-Seq in human colon cancer cells, we demonstrate that mutant p53 depletion results in decreased H3K4me1 levels at active enhancers that reveal a striking colocalization of mutant p53 and the H3K4 monomethyltransferase MLL4 following chronic tumor necrosis factor alpha (TNFα) signaling. We further reveal that mutant p53 forms physiological associations and direct interactions with MLL4 and promotes the enhancer binding of MLL4, which is required for TNFα-inducible H3K4me1 and histone H3 lysine 27 acetylation (H3K27ac) levels, enhancer-derived transcript (eRNA) synthesis, and mutant p53-dependent target gene activation. Complementary in vitro studies with recombinant chromatin and purified proteins demonstrate that binding of the MLL3/4 complex and H3K4me1 deposition is enhanced by mutant p53 and p300-mediated acetylation, which in turn reflects a MLL3/4-dependent enhancement of mutant p53 and p300-dependent transcriptional activation. Collectively, our findings establish a mechanism in which mutant p53 cooperates with MLL4 to regulate aberrant enhancer activity and tumor-promoting gene expression in response to chronic immune signaling.

Fanconi Anemia FANCM/FNCM-1 and FANCD2/FCD-2 Are Required for Maintaining Histone Methylation Levels and Interact with the Histone Demethylase LSD1/SPR-5 in Caenorhabditis elegans.

The histone demethylase LSD1 was originally discovered by removing methyl groups from di- and monomethylated histone H3 lysine 4 (H3K4me2/1). Several studies suggest that LSD1 plays roles in meiosis as well as in the epigenetic regulation of fertility given that, in its absence, there is evidence of a progressive accumulation of H3K4me2 and increased sterility through generations. In addition to the progressive sterility phenotype observed in the mutants, growing evidence for the importance of histone methylation in the regulation of DNA damage repair has attracted more attention to the field in recent years. However, we are still far from understanding the mechanisms by which histone methylation is involved in DNA damage repair, and only a few studies have focused on the roles of histone demethylases in germline maintenance. Here, we show that the histone demethylase LSD1/CeSPR-5 interacts with the Fanconi anemia (FA) protein FANCM/CeFNCM-1 using biochemical, cytological, and genetic analyses. LSD1/CeSPR-5 is required for replication stress-induced S phase-checkpoint activation, and its absence suppresses the embryonic lethality and larval arrest observed in fncm-1 mutants. FANCM/CeFNCM-1 relocalizes upon hydroxyurea exposure and colocalizes with FANCD2/CeFCD-2 and LSD1/CeSPR-5, suggesting coordination between this histone demethylase and FA components to resolve replication stress. Surprisingly, the FA pathway is required for H3K4me2 maintenance, regardless of the presence of replication stress. Our study reveals a connection between FA and epigenetic maintenance and therefore provides new mechanistic insight into the regulation of histone methylation in DNA repair.

MiR-502-mediated histone methyltransferase SET8 expression is associated with clear cell renal cell carcinoma risk.

Genetic variants may affect the interactions between microRNAs (miRNAs/miRs) and their target genes by modulating their binding affinity or by creating, or destroying a miRNA-binding site. SET domain containing (lysine methyltransferase) 8 (SET8) is the sole lysine methyltransferase that catalyzes the monomethylation of histone H4 lysine 20, and is associated with tumor growth, invasion and metastasis. In the present study, the rs16917496 polymorphism within the miR-502 binding site of the SET8 mRNA 3′ untranslated region (3′UTR) in patients with clear cell renal cell carcinoma (ccRCC) and healthy controls was genotyped. The SET8 CC genotype was associated with a decreased ccRCC risk compared with the CT [P=0.003; odds ratio (OR)=0.318; 95% confidence interval (CI), 0.146–0.691], TT (P=0.011; OR=0.402; 95% CI, 0.197–0.819) and CT+TT (P=0.004; OR=0.370; 95% CI, 0.186–0.736) genotypes. The SET8 CC genotype was associated with reduced SET8 expression based on immunostaining of ccRCC tissue. Low SET8 protein levels were negatively associated with tumor-node-metastasis staging in patients with ccRCC according to the size of tumor and lymph node metastases. SET8-knockdown inhibited renal carcinoma 786-O cell proliferation, migration and invasion. c-Myc and matrix metalloproteinase-7 mRNA expression were downregulated upon SET8-knockdown in renal carcinoma 786-O cells. These data indicated that SET8 may be a functional tumor promoter and that its activation, which is partially regulated by changing the miR-502 and SET8 3′UTR binding affinity, may serve an important role in ccRCC development.

H3K23me1 is an evolutionarily conserved histone modification associated with CG DNA methylation in Arabidopsis.

Amino-terminal tails of histones are targets for diverse post-translational modifications whose combinatorial action may constitute a code that will be read and interpreted by cellular proteins to define particular transcriptional states. Here, we describe monomethylation of histone H3 lysine 23 (H3K23me1) as a histone modification not previously described in plants. H3K23me1 is an evolutionarily conserved mark in diverse species of flowering plants. Chromatin immunoprecipitation followed by high-throughput sequencing in Arabidopsis thaliana showed that H3K23me1 was highly enriched in pericentromeric regions and depleted from chromosome arms. In transposable elements it co-localized with CG, CHG and CHH DNA methylation as well as with the heterochromatic histone mark H3K9me2. Transposable elements are often rich in H3K23me1 but different families vary in their enrichment: LTR-Gypsy elements are most enriched and RC/Helitron elements are least enriched. The histone methyltransferase KRYPTONITE and normal DNA methylation were required for normal levels of H3K23me1 on transposable elements. Immunostaining experiments confirmed the pericentromeric localization and also showed mild enrichment in less condensed regions. Accordingly, gene bodies of protein-coding genes had intermediate H3K23me1 levels, which coexisted with CG DNA methylation. Enrichment of H3K23me1 along gene bodies did not correlate with transcription levels. Together, this work establishes H3K23me1 as a so far undescribed component of the plant histone code.

Chromatin priming of genes in development: Concepts, mechanisms and consequences.

During ontogeny, cells progress through multiple alternate differentiation states by activating distinct gene regulatory networks. In this review, we highlight the important role of chromatin priming in facilitating gene activation during lineage specification and in maintaining an epigenetic memory of previous gene activation. We show that chromatin priming is part of a hugely diverse repertoire of regulatory mechanisms that genes use to ensure that they are expressed at the correct time, in the correct cell type, and at the correct level, but also that they react to signals. We also emphasize how increasing our knowledge of these principles could inform our understanding of developmental failure and disease.

Small Cell Lung Cancer Exhibits Frequent Inactivating Mutations in the Histone Methyltransferase KMT2D/MLL2: CALGB 151111 (Alliance)

IntroductionSCLC is a lethal neuroendocrine tumor type that is highly prone to metastasis. There is an urgency to understand the mutated genes that promote SCLC, as there are no approved targeted therapies yet available. SCLC is rarely resected, limiting the number of samples available for genomic analyses of somatic mutations. MethodsTo identify potential driver mutations in human SCLC we sequenced the whole exomes of 18 primary SCLCs and seven cell lines along with matched normal controls. We extended these data by resequencing a panel of genes across 40 primary SCLCs and 48 cell lines. ResultsWe report frequent mutations in the lysine methyltransferase 2D gene (KMT2D) (also known as MLL2), a key regulator of transcriptional enhancer function. KMT2D exhibited truncating nonsense/frameshift/splice site mutations in 8% of SCLC tumors and 17% of SCLC cell lines. We found that KMT2D mutation in human SCLC cell lines was associated with reduced lysine methyltransferase 2D protein levels and reduced monomethylation of histone H3 lysine 4, a mark associated with transcriptional enhancers. We also found mutations in other genes associated with transcriptional enhancer control, including CREB binding protein gene (CREBBP), E1A binding protein p300 gene (EP300), and chromodomain helicase DNA binding protein 7 gene (CHD7), and we report mutations in additional chromatin remodeling genes such as polybromo 1 gene (PBRM1). ConclusionsThese data indicate that KMT2D is one of the major mutated genes in SCLC, and they point to perturbation of transcriptional enhancer control as potentially contributing to SCLC.

ZMYND8 Reads the Dual Histone Mark H3K4me1-H3K14ac to Antagonize the Expression of Metastasis-Linked Genes

Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating functions of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness invitro and invivo. The planthomeodomain (PHD) and Bromodomain cassette inZMYND8 mediated the combinatorial recognition ofH3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a metastasis-suppressive epigenetic mechanism in which ZMYND8's PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes.

Set7 mediated interactions regulate transcriptional networks in embryonic stem cells.

Histone methylation by lysine methyltransferase enzymes regulate the expression of genes implicated in lineage specificity and cellular differentiation. While it is known that Set7 catalyzes mono-methylation of histone and non-histone proteins, the functional importance of this enzyme in stem cell differentiation remains poorly understood. We show Set7 expression is increased during mouse embryonic stem cell (mESC) differentiation and is regulated by the pluripotency factors, Oct4 and Sox2. Transcriptional network analyses reveal smooth muscle (SM) associated genes are subject to Set7-mediated regulation. Furthermore, pharmacological inhibition of Set7 activity confirms this regulation. We observe Set7-mediated modification of serum response factor (SRF) and mono-methylation of histone H4 lysine 4 (H3K4me1) regulate gene expression. We conclude the broad substrate specificity of Set7 serves to control key transcriptional networks in embryonic stem cells.

Abstract 4521: Substrate profiling for SETD8 reveals novel protein targets involved in DNA replication and DNA damage response

Abstract Protein lysine methyltransferases utilize S-adenosyl-L-methionine (SAM) to catalyze the addition of methyl groups to specific lysine residues in both histones and non-histone proteins. Recent evidence indicates that their dysregulation is closely linked to the development and progression of cancer, making them potential therapeutic targets. SETD8 is the sole methyltransferase responsible for mono-methylation of histone H4 lysine 20 (H4K20me1). Its activity is essential for cell viability and has been associated with many essential cellular processes including transcriptional regulation, heterochromatin formation, genomic stability and cell-cycle progression. SETD8 is overexpressed in several types of cancer and may play important roles on malignancy through its trans-methylation activity or by direct interaction with its binding partners. It has been reported that the association between SETD8 and TWIST, a regulator of epithelial-mesenchymal transition, enhances the invasiveness of breast cancer cells. In addition, it was shown that SETD8-mediated methylation of p53 suppresses p53 dependent transcription activation in cancer cells. Despite the identification of a few non-histone proteins such as p53, PCNA and Numb as SETD8's substrates, a comprehensive investigation of new potential substrates is lacking to date. Here we used Bioorthogonal Profiling of Protein Methylation (BPPM) with engineered SETD8 and synthetic SAM analogues to profile novel substrates for SETD8. We identified SETD8 mutants amenable to accommodate non-native SAM analogues containing a terminal alkyne moiety for click chemistry. The engineered SETD8 can transfer this distinct chemical moiety into target proteins for subsequent pulldown and identification of the modified substrates. The BBPM technology applied here revealed previously known substrates of SETD8 as well as novel SETD8 targets. Among the main targets of our interest are those involved in DNA replication and DNA damage response. Our finding is expected to bring new perspectives on the biological importance of SETD8 as well as on its role in carcinogenesis. Citation Format: Fabio Pittella Silva, Gil Blum, Chamara Senevirathne, Minkui Luo. Substrate profiling for SETD8 reveals novel protein targets involved in DNA replication and DNA damage response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4521.

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.

ChREBP binding and histone modifications modulate hepatic expression of the Fasn gene in a metabolic syndrome rat model

ObjectiveAlthough the expression of hepatic lipogenic genes is enhanced in insulin resistance, the underlying mechanism is unclear. To reveal the details, the aim of this study was to investigate whether the expression of hepatic lipogenic genes are mediated by epigenetic regulation and specific transcription factors in an insulin resistance model of rats. MethodsUsing a rat model of insulin resistance (SHR/NDmc-cp), we investigated the relationship between hepatic expression of the lipogenic gene fatty-acid synthase (Fasn), binding of the transcription factor carbohydrate-responsive element-binding protein (ChREBP) to the Fasn gene, and histone modifications in the region of the Fasn gene by real-time reverse transcriptase polymerase chain reaction, immunoblotting, and chromatin immunoprecipitation assay. ResultsCompared with control rats, Fasn mRNA expression and protein levels were higher in the livers of SHR/NDmc-cp rats, as were protein expression levels and Fasn binding of ChREBP and RNA polymerase II. Moreover, compared with the livers of control rats, levels of mono-methylated histone H3 lysine (K) 4 and acetylated histone H4 were higher in the promoter/enhancer region of the Fasn gene in the livers of SHR/NDmc-cp rats. Levels of trimethylated histone H3K4 and acetylated histone H3 were higher in the transcribed region. ConclusionThe results of this study indicate that expression of the Fasn gene in the livers of insulin-resistant rats is associated with increased H3K4 methylation, increased histone H3 acetylation, and increased H4 acetylation, and also, binding levels of ChREBP to promoter/enhancer region of Fasn gene is involved in the Fasn gene expression caused by hyperglycemia.

Condensin-mediated chromosome organization and gene regulation.

In many organisms sexual fate is determined by a chromosome-based method which entails a difference in sex chromosome-linked gene dosage. Consequently, a gene regulatory mechanism called dosage compensation equalizes X-linked gene expression between the sexes. Dosage compensation initiates as cells transition from pluripotency to differentiation. In Caenorhabditis elegans, dosage compensation is achieved by the dosage compensation complex (DCC) binding to both X chromosomes in hermaphrodites to downregulate gene expression by twofold. The DCC contains a subcomplex (condensin IDC) similar to the evolutionarily conserved condensin complexes which play a fundamental role in chromosome dynamics during mitosis. Therefore, mechanisms related to mitotic chromosome condensation are hypothesized to mediate dosage compensation. Consistent with this hypothesis, monomethylation of histone H4 lysine 20 is increased, whereas acetylation of histone H4 lysine 16 is decreased, both on mitotic chromosomes and on interphase dosage compensated X chromosomes in worms. These observations suggest that interphase dosage compensated X chromosomes maintain some characteristics associated with condensed mitotic chromosome. This chromosome state is stably propagated from one cell generation to the next. In this review we will speculate on how the biochemical activities of condensin can achieve both mitotic chromosome compaction and gene repression.