Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.1.1.37 (DNA methyltransferase)
4,983 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epigenetic drugs are in use in clinical trials of various human cancers and are potent at reactivating genes silenced by DNA methylation and chromatin modifications. We report here the analysis of a set of normal fibroblast and cancer cell lines after combination treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-CdR) and the histone deacetylase inhibitor 4-phenylbutyric acid (PBA). Low doses of the drug combination caused cell cycle arrest, whereas high doses induced apoptosis in T24 bladder carcinoma cells. Both p16 (CDKN2A/INK4) and p21 (CIP1/SDI1/WAF1) expression were induced to similar levels in normal and cancer cells in a dose-dependent fashion after combination treatments. We detected a distinct increase of histone H3 acetylation at lysine 9/14 near the transcription start sites, in both LD419 normal fibroblasts and T24 bladder carcinoma cells, whereas the acetylation changes in the p21 locus were less apparent. Interestingly, the levels of trimethylation of histone H3 on lysine 9, which usually marks inactive chromatin regions and was associated with the p16 promoter in silenced T24 cells, did not change after drug treatments. Furthermore, we provide evidence that the remethylation of the p16 promoter CpG island in T24 cells after 5-aza-CdR treatment cannot be halted by subsequent continuous PBA treatment. The p16 gene is resilenced with kinetics similar to 5-aza-CdR only-treated cells, which is also marked by a localized loss of histone acetylation at the transcription start site. Altogether, our data provide new insights into the mechanism of epigenetic drugs and have important implications for epigenetic therapy.
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PMID:Inhibition of histone deacetylation does not block resilencing of p16 after 5-aza-2'-deoxycytidine treatment. 1721 Jul 17

Ovine leukemia/lymphoma resulting from bovine leukemia virus infection of sheep offers a large animal model for studying mechanisms underlying leukemogenesis. Silencing of viral information including Tax, the major contributor to the oncogenic potential of the virus, is critical if not mandatory for tumor progression. In this study, we have identified epigenetic mechanisms that govern the complete suppression of viral expression, using a lymphoma-derived B-cell clone carrying a silent provirus. Silencing was not relieved by injection of the malignant B cells into sheep. However, exogenous expression of Tax or treatment with either the DNA methyltransferase inhibitor 5'azacytidine or the histone deacetylase (HDAC) inhibitor trichostatin A rescued viral expression, as demonstrated by in vivo infectivity trials. Comparing silent and reactivated provirus, we found mechanistic connections between chromatin conformation and tumor-associated transcriptional repression. Silencing is associated with DNA methylation and decreased accessibility of promoter sequences. HDAC1 and the transcriptional corepressor mSin3A are associated with the inactive but not the reactivated promoter. Silencing correlates with a repressed chromatin structure marked by histone H3 and H4 hypoacetylation, a loss of methylation at H3 lysine 4, and an increase of H3 lysine 9 methylation. These observations point to the critical role of epigenetic mechanisms in tumor-specific virus/oncogene silencing, a potential strategy to evade immune response and favor the propagation of the transformed cell.
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PMID:Suppression of viral gene expression in bovine leukemia virus-associated B-cell malignancy: interplay of epigenetic modifications leading to chromatin with a repressive histone code. 1739 71

Evidence indicates that failure of nuclear transfer (NT) embryos to develop normally can be attributed, at least partially, to the use of a differentiated cell nucleus as the donor karyoplast. It has been hypothesized that blastocyst production and development to term of cloned embryos may differ between population doublings (PDs) of the same cell line as a consequence of changes in DNA methylation and histone acetylation patterns during in vitro culture. The objective of this study was to determine gene expression patterns of the chromatin remodeling proteins DNA methyltransferase-1 (Dnmt1), methyl CpG binding protein-2 (MeCP2), and histone deacetyltransferse-1 (HDAC1), in addition, to measuring levels of DNA methylation and histone acetylation of bovine fibroblast cells at different PDs. Bovine fibroblast cell lines were established from four 50-day fetuses. Relative levels of Dnmt1, MeCP2, HDAC1, methylated DNA, and acetylated histone were analyzed at PDs 2, 7, 15, 30, 45, and 70. RNA levels of Dnmt1, HDAC1, and MeCP2 were examined using Q-PCR. Global levels of methylated DNA and acetylated histone were determined by incubation of fixed cells with an anti-5-methylcytidine and anti-acetyl-histone H3 antibody, respectively. Cells were labeled with a second antibody, counter-stained with propidium iodide and analyzed by flow cytometry. These data demonstrate that chromatin remodeling protein mRNAs involved in epigenetic modifications are altered during in vitro culture. Methylated DNA and acetylated histone patterns of in vitro cells change with time in culture. Subsequent use of these cells for NT will provide insight as to how these epigenetic modifications affect reprogramming.
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PMID:DNA methylation and histone acetylation patterns in cultured bovine fibroblasts for nuclear transfer. 1744 Sep 41

DNA methylation and histone modifications have emerged as key mechanisms in transcriptional regulation. The target of methylation-induced silencing 1 (TMS1) is a bipartite protein. Recent studies have indicated that methylation-associated silencing of TMS1 occurs in many cancers. However, whether and how TMS1 is regulated by epigenetic mechanisms in cancers remains unknown. In this study we showed that methylation of the TMS1 promoter occurred in five of six hepatocellular carcinoma (HCC) cell lines. TMS1 expression was reduced in four HCC cell lines and correlated with methylation status. Furthermore, the TMS1 promoter was completely methylated and mRNA expression was undetectable. TMS1 expression could be restored by 5-aza-2'-deoxycitidine (5-Aza-dC) (a DNA methyltransferase inhibitor) or trichostatin A (TSA) (a histone deacetylase inhibitor) alone and the promoter methylation was partially reversible. TSA was more efficient than 5-Aza-dC in inducing TMS1 expression, and the combination of 5-Aza-dC and TSA resulted in markedly synergistic reactivation of the gene and completely reversed promoter methylation. Interestingly, TMS1 promoter methylation-associated gene silencing was accompanied by histone H3 Lysine 9 (H3K9) hypoacetylation and trimethylation. 5-Aza-dC and/or TSA also had some effect on conversion of methylated to acetylated H3K9 in restoring TMS1. This conversion was dynamic at the TMS1 promoter and a decrease in H3K9 trimethylation preceded an increase in H3K9 acetylation after 5-Aza-dC and/or TSA treatment. Our results thus suggest that epigenetic inactivation of TMS1 expression is regulated by promoter hypermethylation and H3K9 modifications in a coordinated way.
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PMID:Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells. 1747 63

Tumor angiogenesis requires intricate regulation of gene expression in endothelial cells. We recently showed that DNA methyltransferase (DNMT) and histone deacetylase (HDAC) inhibitors directly repress endothelial cell growth and tumor angiogenesis, suggesting that epigenetic modifications mediated by DNMTs and HDAC are involved in regulation of endothelial cell gene expression during tumor angiogenesis. To understand the mechanisms behind the epigenetic regulation of tumor angiogenesis, we used microarray analysis to perform a comprehensive screen to identify genes down-regulated in tumor-conditioned versus quiescent endothelial cells, and reexpressed by 5-aza-2'-deoxycytidine (DAC) and trichostatin A (TSA). Among the 81 genes identified, 77% harbored a promoter CpG island. Validation of mRNA levels of a subset of genes confirmed significant down-regulation in tumor-conditioned endothelial cells and reactivation by treatment with a combination of DAC and TSA, as well as by both compounds separately. Silencing of these genes in tumor-conditioned endothelial cells correlated with promoter histone H3 deacetylation and loss of H3 lysine 4 methylation, but did not involve DNA methylation of promoter CpG islands. For six genes, down-regulation in microdissected human tumor endothelium was confirmed. Functional validation by RNA interference revealed that clusterin, fibrillin 1, and quiescin Q6 are negative regulators of endothelial cell growth and angiogenesis. In summary, our data identify novel angiogenesis-suppressing genes that become silenced in tumor-conditioned endothelial cells in association with promoter histone modifications and reactivated by DNMT and HDAC inhibitors through reversal of these epigenetic modifications, providing a mechanism for epigenetic regulation of tumor angiogenesis.
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PMID:Identification of epigenetically silenced genes in tumor endothelial cells. 1748 24

We recently reported that a large proportion of aggressive squamous cell carcinomas of humans and mice express markedly reduced IKKalpha. However, the role of IKKalpha in maintaining genomic stability is unknown. Here we reported that IKKalpha-deficient keratinocytes had a defect in the G(2)/M cell-cycle arrest in response to DNA damage due to downregulated 14-3-3sigma, a cell cycle checkpoint protein. Trimethylated histone H3 lysine 9 (H3-K9) was found to associate with the histone trimethyltransferase Suv39h1 and DNA methyltransferase Dnmt3a in the methylated 14-3-3sigma locus. Reintroduction of IKKalpha restored the expression of 14-3-3sigma. IKKalpha was found to associate with H3 in 14-3-3sigma, which prevented access of Suv39h1 to H3, thereby preventing hypermethylation of 14-3-3sigma. IKKalpha mutants that failed to bind to H3 did not restore the expression of 14-3-3sigma. Thus, IKKalpha protects the 14-3-3sigma locus from hypermethylation, which serves as a mechanism of maintaining genomic stability in keratinocytes.
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PMID:IKKalpha shields 14-3-3sigma, a G(2)/M cell cycle checkpoint gene, from hypermethylation, preventing its silencing. 1764 71

We reported previously that homocysteine (Hcy) inhibits endothelial cell (EC) growth by transcriptional inhibition of the cyclin A gene via a hypomethylation-related mechanism. In this study, we examined the effect of Hcy on epigenetic modification of the cyclin A gene and its biologic role in human ECs. Cyclin A mRNA levels were significantly suppressed by Hcy and a DNA methyltransferase inhibitor. The cyclin A promoter contains a CpG island spanning a 477-bp region (-277/200). Bisulfite sequencing followed by polymerase chain reaction (PCR) amplification of the cyclin A promoter (-267/37) showed that Hcy eliminated methylation at 2 CpG sites in the cyclin A promoter, one of which is located on the cycle-dependent element (CDE). Mutation of CG sequence on the CDE leads to a 6-fold increase in promoter activity. Hcy inhibited DNA methyltransferase 1 (DNMT1) activity by 30%, and reduced the binding of methyl CpG binding protein 2 (MeCP2) and increased the bindings of acetylated histone H3 and H4 in the cyclin A promoter. Finally, adenovirus-transduced DNMT1 gene expression reversed the inhibitory effect of Hcy on cyclin A expression and EC growth inhibition. In conclusion, Hcy inhibits cyclin A transcription and cell growth by inhibiting DNA methylation through suppression of DNMT1 in ECs.
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PMID:Homocysteine inhibits endothelial cell growth via DNA hypomethylation of the cyclin A gene. 1769 32

Genetic imprinting, found in flowering plants and placental mammals, uses DNA methylation to yield gene expression that is dependent on the parent of origin. DNA methyltransferase 3a (Dnmt3a) and its regulatory factor, DNA methyltransferase 3-like protein (Dnmt3L), are both required for the de novo DNA methylation of imprinted genes in mammalian germ cells. Dnmt3L interacts specifically with unmethylated lysine 4 of histone H3 through its amino-terminal PHD (plant homeodomain)-like domain. Here we show, with the use of crystallography, that the carboxy-terminal domain of human Dnmt3L interacts with the catalytic domain of Dnmt3a, demonstrating that Dnmt3L has dual functions of binding the unmethylated histone tail and activating DNA methyltransferase. The complexed C-terminal domains of Dnmt3a and Dnmt3L showed further dimerization through Dnmt3a-Dnmt3a interaction, forming a tetrameric complex with two active sites. Substitution of key non-catalytic residues at the Dnmt3a-Dnmt3L interface or the Dnmt3a-Dnmt3a interface eliminated enzymatic activity. Molecular modelling of a DNA-Dnmt3a dimer indicated that the two active sites are separated by about one DNA helical turn. The C-terminal domain of Dnmt3a oligomerizes on DNA to form a nucleoprotein filament. A periodicity in the activity of Dnmt3a on long DNA revealed a correlation of methylated CpG sites at distances of eight to ten base pairs, indicating that oligomerization leads Dnmt3a to methylate DNA in a periodic pattern. A similar periodicity is observed for the frequency of CpG sites in the differentially methylated regions of 12 maternally imprinted mouse genes. These results suggest a basis for the recognition and methylation of differentially methylated regions in imprinted genes, involving the detection of both nucleosome modification and CpG spacing.
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PMID:Structure of Dnmt3a bound to Dnmt3L suggests a model for de novo DNA methylation. 1785 1

Dysregulation of DNA methyltransferase (DNMT)1 expression is associated with cellular transformation, and inhibition of DNMT1 exerts antitumorigenic effects. Here, we report that DNMT1 abnormally expressed in HeLa cells is downregulated by a histone deacetylase (HDAC) inhibitor apicidin, which is correlated with induction of repressive histone modifications on the promoter site. Apicidin selectively represses the expression of DNMT1 among DNMTs in HeLa cells, independent of cell cycle arrest at G0/G1. Furthermore, apicidin causes a significant reduction in the recruitment of RNA polymerase II into the promoter. Chromatin immunoprecipitation analysis shows that even though apicidin causes global hyperacetylation of histone H3 and H4, localized deacetylation of histone H3 and H4 occurs at the E2F binding site, which is accompanied by the recruitment of pRB and the replacement of P/CAF with HDAC1 into the sites. In addition, K4-trimethylated H3 on nucleosomes associated with the transcriptional start site is depleted following apicidin treatment, whereas repressive markers, K9- and K27-trimethylation of H3 are enriched on the site. The downregulation of DNMT1 expression seems to require de novo protein synthesis, because the apicidin effect is antagonized by cycloheximide treatment. Moreover, knock down of DNMT1 with siRNA induces the apoptosis of HeLa cells, indicating that downregulation of DNMT1 might be a good strategy for therapeutics of human cervix cancer. Collectively, our findings will provide a mechanistic rationale for the use of HDAC inhibitors in cancer therapeutics.
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PMID:Histone deacetylase inhibitor apicidin downregulates DNA methyltransferase 1 expression and induces repressive histone modifications via recruitment of corepressor complex to promoter region in human cervix cancer cells. 1782 6

The claudin (CLDN) genes encode a family of proteins involved in the formation and function of tight junctions. CLDN gene expression is frequently altered in several human cancers, and in particular, CLDN3 and CLDN4 are commonly overexpressed in ovarian cancer. However, the mechanisms leading to the deregulation of these genes in cancer remain unclear. In the present study, we have examined the CLDN3 promoter and have identified a minimal region containing an Sp1 site crucial for its activity. In addition, we find that the CLDN3 promoter is regulated through epigenetic processes. Cells that express high levels of CLDN3 exhibit low DNA methylation and high histone H3 acetylation of the critical CLDN3 promoter region, and the reverse is observed in cells that do not express this gene. CLDN3-negative cells can be induced to express CLDN3 through treatment with DNA methyltransferase or histone deacetylase inhibitors. Interestingly, in vitro binding experiments, as well as chip assays show that Sp1 binds the unmethylated promoter much more efficiently, providing a mechanism for CLDN3 silencing in non-expressing cells. Finally, siRNA-mediated knockdown of Sp1 led to a significant decrease of CLDN3 expression at both the mRNA and protein levels, demonstrating a crucial role for this transcription factor in the regulation of CLDN3. Our data provide a basis for CLDN3 expression in ovarian cancer cells, as well as a mechanism for the silencing of this promoter in tumors lacking expression of claudin-3.
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PMID:Regulation of the CLDN3 gene in ovarian cancer cells. 1798 52


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