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At least 325 records · Page 18Linked to original sources
PubMed2006

The orphan nuclear receptor GCNF recruits DNA methyltransferase for Oct-3/4 silencing.

Somatic DNA methylation patterns are determined in part by the de novo methylation that occurs after early embryonic demethylation. Oct-3/4, a pluripotency gene, is unmethylated in the blastocyst, but undergoes de novo methylation and silencing during gastrulation. Here we show that the transcriptional repressor GCNF recruits DNA methyltransferase to the Oct-3/4 promoter and facilitates its methylation. Although acetylation of histone H3 at lysine 9 (K9) and/or 14 (K14) and methylation of H3 at lysine 4 (K4) decrease during this period, as do Oct-3/4 transcript levels, H3K9 and H3K27 methylation levels remain constant, indicating that DNA methylation does not require repressive histone modifications. We found that GCNF interacts directly with Dnmt3 molecule(s) and verified that this interaction induces the methylation of the Oct-3/4 promoter. Our finding suggests a model in which differentiation-induced GCNF recruits de novo DNA methyltransferase and facilitates the silencing of a pluripotency gene.

Animals↗
PubMed2004

5-Aza-2'-deoxycytidine stimulates inducible nitric oxide synthase induction in C6 astrocytoma cells.

The influence of a nucleoside analog 5-aza-2'-deoxycytidine (5-AzadC) on inducible nitric oxide synthase (iNOS)-dependent nitric oxide (NO) production in various rat cell types was investigated. In C6 astrocytoma cell line and primary astrocytes, 5-AzadC enhanced proinflammatory cytokine (IFN-gamma, TNF-alpha, IL-1)-triggered NO synthesis in a time- and dose-dependent manner. In contrast, 5-AzadC did not potentiate NO production in IFN-gamma-stimulated macrophages, fibroblasts, or endothelial cells. Blockade of transcription or translation in C6 cells abolished the observed effect, suggesting the iNOS gene expression, rather than its catalytic activity, as a target for the drug action. Accordingly, 5-AzadC upregulated IFN-gamma-induced expression of iNOS mRNA in C6 astrocytes. The effect of 5-AzadC on astrocyte NO release was blocked by the inhibitor of p44/42 mitogen activated protein kinase-dependent signaling. Finally, the observed stimulatory effect of 5-AzadC on iNOS expression was apparently independent of DNA demethylation, as DNA digestion with methylation-sensitive restriction enzyme HpaII showed that 5-AzadC failed to demethylate cellular DNA in conditions used for iNOS induction.

Animals↗
PubMed1999

LXIII Cold Spring Harbor Symposium on Quantitative Biology: Mechanisms of Transcription, 3-8 June 1998.

A new perspective is emerging in the transcription field towards understanding gene regulation not only at its most fundamental level but also in the context of chromatin, nuclear compartmentalization, and physiological processes. This direction is being fueled by several key observations. Among them is the discovery of multi-protein complexes whose components reveal a link between gene activity, nuclear structure, and cellular signaling pathways. This information will no doubt be extended by identifying expanded regulatory circuitry using the microchip oligonucleotide array technology. In addition to elucidating the regulatory consequences of these intricate connections, another frontier will be to analyze gene expression within chromosomes. This requires deciphering the mechanism of action of a variety of DNA elements that create a genetic domain such as locus control regions, distal enhancers, insulators, silencers, and matrix attachment regions. Hopefully, with the development of new assays these elements can be as rigorously defined as promoters have been. We can also look forward to capturing critical transcriptional processes by increasingly refined structural analyses. Thus, the scope of problems being addressed in gene regulation has been greatly expanded and the opportunity exists to answer very sophisticated questions in the future.

Archaea↗
PubMed2003

DNA methylation in animal development.

Nuclear transfer experiments have demonstrated that epigenetic mechanisms operate to limit gene expression during animal development. In somatic cells, silenced genes are associated with defined chromatin states which are characterised by hypermethylation of DNA, hypoacetylation of histones and specific patterns of methylation at distinct residues of the N-terminal tails of histone H3 and H4. This review describes the role of the DNA methylation-mediated repression system (Dnmt1's, MeCPs and MBDs and associated chromatin remodelling activities) in animal development. DNA methylation is essential for normal vertebrate development but has distinct regulatory roles in non-mammalian and mammalian vertebrates. In mammals, DNA methylation has an additional role in regulating imprinting. This suggests that epigenetic regulation is plastic in its application and should be considered in a developmental context that may be species specific.

Animals↗
PubMed2003

Active recruitment of DNA methyltransferases regulates interleukin 4 in thymocytes and T cells.

How T cells regulate interleukin 4 (IL-4) expression is not completely understood. We show here that single-positive thymocytes express IL-4, but attenuate GATA-3 expression, recruit DNA methyltransferases (Dnmts) to the Il4-Il13 locus and downregulate IL-4 expression as they mature into T cells. Type 2 polarization blocks Dnmt1 recruitment, enhances histone H3 Lys4 methylation (indicative of accessible chromatin) and initiates DNA demethylation of the locus. Dnmt1-/- CD4 and CD8 T cells derepress IL-4 expression considerably, demethylate DNA and increase H3 Lys4 methylation without affecting GATA-3 expression, demonstrating that Dnmt1 and DNA methylation are essential for proper Il4 regulation. These results indicate that Dnmts, DNA and histone methylation, and transcription factors 'collaborate' to determine appropriate Il4 expression patterns.

Animals↗
PubMed2003

Downregulation of ID4 by promoter hypermethylation in gastric adenocarcinoma.

Promoter hypermethylation has become apparent as a common mechanism of gene silencing in cancer. Based on our published microarray expression data, we noticed a prominent downregulation of ID4 in gastric adenocarcinoma. The dense 5' CpG island covering the previously mapped upstream promoter of ID4 has prompted us to relate its downregulation to promoter hypermethylation. ID proteins are distinct members in the helix-loop-helix family of transcriptional regulators, which modulate various key developmental processes. Emerging data have suggested the involvement of ID genes in tumorigenesis. In this study using bisulfite genomic sequencing, we have found hypermethylation of ID4 promoter in most gastric cancer cell lines and 30% of primary tumors. This correlated with decreased level of ID4 expression. Restoration of ID4 expression in various gastric cancer cell lines was achieved by treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine, which at times required the synergistic action of the histone deacetylase inhibitor trichostatin A, but not with trichostatin A alone. Re-expression was accompanied by the corresponding ID4 promoter demethylation. Furthermore, we have found significant association of ID4 promoter methylation with hMLH1 promoter methylation (P=0.008) and microsatellite instability (P=0.006). Overall, our results have shown that transcriptional silencing of ID4 is related to the aberrant methylation of its promoter in gastric cancer. The significant association of ID4 and hMLH1 promoter hypermethylation suggested that ID4 may also be among the genes being targeted in the CpG island methylator phenotype tumorigenic pathway.

Adenocarcinoma↗
PubMed2005

STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes.

Expression of SHP-1 phosphatase, a key negative regulator of cell signaling, is lost in T cell lymphomas and other malignancies due to DNA methylation of the SHP-1 promoter by a currently undefined mechanism. We demonstrate that malignant T cells express DNA methyltransferase (DNMT) 1 and that constantly activated signal transducer and activator of transcription (STAT) 3 is capable of binding in vitro to DNA oligonucleotides corresponding to four STAT3 SIE/GAS binding sites identified in the SHP-1 promoter. STAT3, DNMT1, and histone deacetylase 1 form complexes and bind to the SHP-1 promoter in vivo. Treatment with pharmacologic grade DNMT1 anti-sense oligonucleotides and STAT3 small-interfering RNA induces in the malignant T cells DNA demethylation and expression of SHP-1 gene. These data indicate that STAT3 may, in part, transform cells by inducing epigenetic silencing of SHP-1 in cooperation with DNMT1 and, apparently, histone deacetylase 1. Reversal of such gene silencing represents an attractive aim for novel anticancer therapies.

Base Sequence↗
PubMed1996

Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer.

The association between increased DNA-methyltransferase (DNA-MTase) activity and tumor development suggest a fundamental role for this enzyme in the initiation and progression of cancer. A true functional role for DNA-MTase in the neoplastic process would be further substantiated if the target cells affected by the initiating carcinogen exhibit changes in enzyme activity. This hypothesis was addressed by examining DNA-MTase activity in alveolar type II (target) and Clara (nontarget) cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity was found only in the target alveolar type II cells of the susceptible A/J mouse and caused a marked increase in overall DNA methylation in these cells. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. Increased gene expression was also detected by RNA in situ hybridization in hypertrophic alveolar type II cells of carcinogen-treated A/J mice, indicating that elevated levels of expression may be a biomarker for premalignancy. Enzyme activity increased incrementally during lung cancer progression and coincided with increased expression of the DNA-MTase activity are strongly associated with neoplastic development and constitute a key step in carcinogenesis. The detection of premalignant lung disease through increased DNA-MTase expression and the possibility of blocking the deleterious effects of this change with specific inhibitors will offer new intervention strategies for lung cancer.

Animals↗
PubMed1999

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells.

Inhibitors of DNA methyltransferase, typified by 5-aza-2'-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-alpha. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT's limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-alpha2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

Antimetabolites, Antineoplastic↗
PubMed1995

Specific Alu binding protein from human sperm chromatin prevents DNA methylation.

A protein from human sperm nuclei that specifically binds to Alu DNA repeats has been purified. The specific DNA binding site of this protein within the Alu sequence has been mapped by methylation interference and electrophoretic mobility shift assays. This sperm Alu binding protein selectively protects Alu elements from methylation in vitro and may be responsible for the unmethylated state of Alu sequences in the male germ line resulting in a parent-specific differential inheritance of Alu methylation.

Amino Acid Sequence↗
PubMed1999

Modified oligonucleotides as bona fide antagonists of proteins interacting with DNA. Hairpin antagonists of the human DNA methyltransferase.

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report describes the design of potent DNA based antagonists of DNA MeTase and their utilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary structure, hemimethylation, and phosphorothioate modification strongly affect the antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cellular transformation, human lung non-small carcinoma cells were treated with the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTase, cell growth, and anchorage independent growth (an indicator of tumorigenesis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antagonists of critical cellular proteins.

Base Sequence↗
PubMed2004

Components of the DNA methylation system of chromatin control are RNA-binding proteins.

The view that autosomal gene expression is controlled exclusively by protein trans-acting factors has been challenged recently by the identification of RNA molecules that regulate chromatin. In the majority of cases where RNA molecules are implicated in DNA control, the molecular mechanisms are unknown, in large part because the RNA.protein complexes are uncharacterized. Here, we identify a novel set of RNA-binding proteins that are well known for their function in chromatin regulation. The RNA-interacting proteins are components of the mammalian DNA methylation system. Genomic methylation controls chromatin in the context of transposon silencing, imprinting, and X chromosome dosage compensation. DNA methyltransferases (DNMTs) catalyze methylation of cytosines in CGs. The methyl-CGs are recognized by methyl-DNA-binding domain (MBD) proteins, which recruit histone deacetylases and chromatin remodeling proteins to effect silencing. We show that a subset of the DNMTs and MBD proteins can form RNA.protein complexes. We characterize the MBD protein RNA-binding activity and show that it is distinct from the methyl-CG-binding domain and mediates a high affinity interaction with RNA. The RNA and methyl-CG binding properties of the MBD proteins are mutually exclusive. We speculate that DNMTs and MBD proteins allow RNA molecules to participate in DNA methylation-mediated chromatin control.

Animals↗
PubMed2004

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase.

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.

Animals↗
PubMed2026

A pragmatic adaptation of the RANO clinical risk score for IDH-Wildtype glioblastoma in the absence of MGMT promoter methylation testing.

BACKGROUND: The novel RANO risk score provides prognostic stratification for patients with IDH-wildtype glioblastoma(GBM) and includes age, Karnofsky Performance Scale(KPS), RANO resection class(RRC), and MGMT promoter methylation(MGMTm). However, MGMTm testing is unavailable in many countries. We aimed to explore the prognostic performance of a RANO-adapted clinical score excluding MGMTm. METHODS: We applied the same scoring system established by original RANO score, excluding MGMTm. Three risk classes were defined as numerical scores derived through tertiles. The primary endpoint was overall survival(OS), and the secondary exploratory endpoint was progression-free survival(PFS). RESULTS: One-hundred twenty patients were included. Three risk classes were identified:low-risk(0-1 points,n:47, 39.2%), intermediate-risk(2-3 points,n:27, 22.5%), and high-risk(&#x2265;4 points,n:46, 38.3%). The median OS was 35.5&#x2009;months(95% CI:21.1-49.9) for the low-risk group, 16&#x2009;months(95% CI:9.9-22.1) for the intermediate-risk group, and 5&#x2009;months(95% CI:3.6-6.3) for the high-risk group(p&#x2009;<&#x2009;0.001). Similarly, the median PFS was 16.3&#x2009;months(95% CI:12.3-20.3) for the low-risk group, 9.9&#x2009;months(95% CI:7.2-12.6) for the intermediate-risk group, and 4.1&#x2009;months(95% CI:3.5-4.7) for the high-risk group(p&#x2009;<&#x2009;0.001). CONCLUSION: This simplified RANO-adapted score demonstrated promising prognostic stratification using basic clinical parameters. As a pragmatic adaptation study, external validation is required before clinical application, particularly in settings where MGMTm testing is unavailable.

Humans↗
PubMed1999

Mechanisms of spiroplasma genome variation associated with SpV1-like viral DNA inferred from sequence comparisons.

Genomes of Spiroplasma citri strains have rearranged frequently during their evolution, partly due to multiple integrated sequences of spiroplasma viruses. To understand better the role of viral sequences in genome evolution, we examined available nucleotide sequences of viruslike elements in the S. citri chromosome. Comparison of integrated and nonintegrated sequences of spiroplasma virus SpV1-C74 DNA suggested that it is an encapsidated form of the circular transposition intermediate belonging to an insertion sequence (IS3) family member. One SpV1-C74 viral DNA fragment was identified as interrupting the remains of a DNA adenine modification methylase gene. A viral DNA insertion of SpV1-R8A2 B DNA had hallmarks of having suffered an internal deletion by a site-specific recombination system. Homologous recombination likely was responsible for several deletions within viral DNA. A homologous recombination event was inferred between part of a viral DNA insertion and a similar chromosomal sequence. Dispersed sequences from SpV1-like C4 open reading frames (ORFs) were identified as involved in a complex deletion-inversion event. Thus, SpV1-like sequences likely have altered spiroplasma genomes by inserting within active genes, destroying their function, by providing targets for site-specific recombination, by mediating deletions of sequences adjacent to their integration sites, and by providing targets for homologous recombination, leading to inversions.

Amino Acid Sequence↗
PubMed2003

Global effects of histone modifications.

The relationship between chromatin structure and transcriptional regulation has been the focus of many research publications over the past few years. A lot of progress has been made in understanding the role of histone modifications. The interdependence of histone methylation and DNA methylation has been revealed. Methylation, acetylation, phosphorylation and ubiquitination of specific histone residues are all able to affect the DNA binding of transcription factors and to change the structure of chromatin on a genome-wide scale. This review aims to summarise some of the experiments that demonstrated the global effects of the modifications of the five histone proteins, H1, H2A, H2B, H3 and H4.

Acetylation↗
PubMed1995

DNA adduct 8-hydroxyl-2'-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase.

8-Hydroxyl-2'-deoxyguanosine (also referred to as 8-hydroxyguanine [8-OH-dG] or 7,8-dihydro-8-oxoguanine), a common DNA adduct resulting from injury to DNA via reactive oxygen species, affects the in vitro methylation of nearby cytosine moieties by the human DNA methyltransferase. The exact position of 8-OH-deoxyguanosine relative to a CpG dinucleotide appears important to this effect. Our data indicate that 8-OH-deoxyguanosine diminishes the ability of the methyltransferase to methylate a target cytosine when the 8-OH-deoxyguanosine is one or two nucleotides 3' from the cytosine, on the same strand. On the other hand 8-OH-deoxyguanosine does not diminish the ability of the enzyme to respond to a methyl director (5-methylcytosine) when the 8-OH-deoxyguanosine is on the same strand but one or two nucleotides 3' from the methyl director. Differences in methylation rates as great as 13-fold have been detected using various 8-OH-deoxyguanosine-containing oligonucleotides as substrates in methylation assays. Our findings suggest that oxidative damage of parental strand guanines would permit normal copying of methylation patterns through maintenance methylation, while oxidative damage of guanines in the nascent strand DNA would inhibit such methylation.

Base Sequence↗