scholarly journals DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells

2007 ◽  
Vol 176 (5) ◽  
pp. 565-571 ◽  
Author(s):  
Fabio Spada ◽  
Andrea Haemmer ◽  
David Kuch ◽  
Ulrich Rothbauer ◽  
Lothar Schermelleh ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Regulation Of Gene Expression ◽  
Human Cells ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
Catalytically Active ◽  
Methylation Patterns ◽  
Proliferating Cell

DNA methylation plays a central role in the epigenetic regulation of gene expression in vertebrates. Genetic and biochemical data indicated that DNA methyltransferase 1 (Dnmt1) is indispensable for the maintenance of DNA methylation patterns in mice, but targeting of the DNMT1 locus in human HCT116 tumor cells had only minor effects on genomic methylation and cell viability. In this study, we identified an alternative splicing in these cells that bypasses the disrupting selective marker and results in a catalytically active DNMT1 protein lacking the proliferating cell nuclear antigen–binding domain required for association with the replication machinery. Using a mechanism-based trapping assay, we show that this truncated DNMT1 protein displays only twofold reduced postreplicative DNA methylation maintenance activity in vivo. RNA interference–mediated knockdown of this truncated DNMT1 results in global genomic hypomethylation and cell death. These results indicate that DNMT1 is essential in mouse and human cells, but direct coupling of the replication of genetic and epigenetic information is not strictly required.

scholarly journals Selective Anchoring of DNA Methyltransferases 3A and 3B to Nucleosomes Containing Methylated DNA

2009 ◽  
Vol 29 (19) ◽  
pp. 5366-5376 ◽  
Author(s):  
Shinwu Jeong ◽  
Gangning Liang ◽  
Shikhar Sharma ◽  
Joy C. Lin ◽  
Si Ho Choi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Molecular Mechanisms ◽  
Dna Methyltransferases ◽  
Micrococcal Nuclease ◽  
Nuclear Antigen ◽  
Heterochromatin Protein 1 ◽  
Histone Deacetylase 1 ◽  
Methylation Patterns

ABSTRACT Proper DNA methylation patterns are essential for mammalian development and differentiation. DNA methyltransferases (DNMTs) primarily establish and maintain global DNA methylation patterns; however, the molecular mechanisms for the generation and inheritance of methylation patterns are still poorly understood. We used sucrose density gradients of nucleosomes prepared by partial and maximum micrococcal nuclease digestion, coupled with Western blot analysis to probe for the interactions between DNMTs and native nucleosomes. This method allows for analysis of the in vivo interactions between the chromatin modification enzymes and their actual nucleosomal substrates in the native state. We show that little free DNA methyltransferase 3A and 3B (DNMT3A/3B) exist in the nucleus and that almost all of the cellular contents of DNMT3A/3B, but not DNMT1, are strongly anchored to a subset of nucleosomes. This binding of DNMT3A/3B does not require the presence of other well-known chromatin-modifying enzymes or proteins, such as proliferating cell nuclear antigen, heterochromatin protein 1, methyl-CpG binding protein 2, Enhancer of Zeste homolog 2, histone deacetylase 1, and UHRF1, but it does require an intact nucleosomal structure. We also show that nucleosomes containing methylated SINE and LINE elements and CpG islands are the main sites of DNMT3A/3B binding. These data suggest that inheritance of DNA methylation requires cues from the chromatin component in addition to hemimethylation.

Suppression of clonogenicity by mammalian Dnmt1 mediated by the PCNA-binding domain

2004 ◽  
Vol 82 (5) ◽  
pp. 589-596 ◽  
Author(s):  
Simeon Santourlidis ◽  
Fumihiro Kimura ◽  
Johannes Fischer ◽  
Wolfgang A Schulz
Keyword(s):  
Dna Methylation ◽  
Cell Lines ◽  
Dna Methyltransferase ◽  
Nuclear Antigen ◽  
Wild Type ◽  
Acid Domain ◽  
Safe Mechanism ◽  
Fail Safe ◽  
Proliferating Cell ◽  
Amino Acid Domain

Overexpression of the major DNA methyltransferase Dnmt1 is cytotoxic and has been hypothesized to result in aberrant hypermethylation of genes required for cell survival. Indeed, overexpression of mouse or human Dnmt1 in murine and human cell lines decreased clonogenicity. By frame-shift and deletion constructs, this effect of mouse Dnmt1 was localized at the N-terminal 124 amino acid domain, which mediates interaction with proliferating cell nuclear antigen (PCNA). Mutation of the PCNA-binding site restored normal cloning efficiencies. Overexpression of Dnmt3A or Dnmt3B, which do not interact with PCNA, yielded weaker effects on clonogenicity. Following introduction of the toxic domain, no significant effects on apoptosis, replication, or overall DNA methylation were observed for up to 3 d. Suppression of clonogenicity by Dnmt1 was also observed in cell lines lacking wild-type p53, p21CIP1, or p16INK4A. Suppression of clonogenicity by Dnmt1 overexpression may act as a fail-safe mechanism against carcinogenicity of sustained Dnmt1 overexpression.Key words: carcinogenesis, DNA methyltransferase, DNA methylation, p53, PCNA.

scholarly journals Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase η in translesion DNA synthesis

2008 ◽  
Vol 105 (46) ◽  
pp. 17724-17729 ◽  
Author(s):  
Narottam Acharya ◽  
Jung-Hoon Yoon ◽  
Himabindu Gali ◽  
Ildiko Unk ◽  
Lajos Haracska ◽  
...  
Keyword(s):  
Human Cells ◽  
Nuclear Antigen ◽  
Interacting Protein ◽  
Translesion Dna Synthesis ◽  
Binding Domains ◽  
Dna Damaging Agents ◽  
Direct Binding ◽  
Replication Foci ◽  
Proliferating Cell

Treatment of yeast and human cells with DNA-damaging agents elicits Rad6–Rad18-mediated monoubiquitination of proliferating cell nuclear antigen (PCNA) at its Lys-164 residue [ubiquitin (Ub)-PCNA], and this PCNA modification is indispensable for promoting the access of translesion synthesis (TLS) polymerases (Pols) to PCNA. However, the means by which K164-linked Ub modulates the proficiency of TLS Pols to bind PCNA and take over synthesis from the replicative Pol has remained unclear. One model that has gained considerable credence is that the TLS Pols bind PCNA at 2 sites, to the interdomain connector loop via their PCNA-interacting protein (PIP) domain and to the K164-linked Ub moiety via their Ub-binding domain (UBD). Specifically, this model postulates that the UBD-mediated binding of TLS Pols to the Ub moiety on PCNA is necessary for TLS. To test the validity of this model, we examine the contributions that the PIP and Ub-binding zinc finger (UBZ) domains of human Polη make to its functional interaction with PCNA, its colocalization with PCNA in replication foci, and its role in TLS in vivo. We conclude from these studies that the binding to PCNA via its PIP domain is a prerequisite for Polη's ability to function in TLS in human cells and that the direct binding of the Ub moiety on PCNA via its UBZ domain is not required. We discuss the possible role of the Ub moiety on PCNA in TLS.

Structure of mammalian DNA methyltransferase as deduced from the inferred amino acid sequence and direct studies of the protein

1988 ◽  
Vol 16 (6) ◽  
pp. 944-947 ◽  
Author(s):  
TIMOTHY BESTOR
Keyword(s):  
Amino Acid ◽  
Metal Binding ◽  
Dna Methyltransferase ◽  
Active Species ◽  
Metal Binding Sites ◽  
Terminal Domain ◽  
Catalytically Active ◽  
Methylation Patterns

Abstract DNA (cytosine-5)-methyltransferase (DNA MeTase) establishes and maintains methylation patterns in the genome of higher eukaryotes. This enzyme has been purified, and the cDNA which encodes it has been cloned and sequenced. DNA MeTase appears to contain a large (1000 amino acid) N-terminal domain that contains potential metal-binding sites. This domain appears to contain a series of five to seven structural units of Mr about 20 000, since post-translational processing in vivo or partial proteolysis of the purified protein in vitro leads to the production of a series of catalytically active species differing in Mr by units of 20 000. The N-terminal domain is fused to a smaller (570 amino acid) C-terminal domain that is related to bacterial type II cytosine methyltransferases. The relevance of these findings for the biological function of DNA MeTase is discussed.

Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jessilyn Dunn ◽  
Haiwei Qiu ◽  
Soyeon Kim ◽  
Daudi Jjingo ◽  
Ryan Hoffman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Western Diet ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

scholarly journals TET2 coactivates gene expression through demethylation of enhancers

2018 ◽  
Vol 4 (11) ◽  
pp. eaau6986 ◽  
Author(s):  
Lu Wang ◽  
Patrick A. Ozark ◽  
Edwin R. Smith ◽  
Zibo Zhao ◽  
Stacy A. Marshall ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Regulation Of Gene Expression ◽  
Estrogen Receptor Α ◽  
Dna Demethylation ◽  
Estrogen Response ◽  
Dna Base ◽  
Modified Dna ◽  
Global Increase ◽  
Methylation Patterns

The tet methylcytosine dioxygenase 2 (TET2) enzyme catalyzes the conversion of the modified DNA base 5-methylcytosine to 5-hydroxymethylcytosine. TET2 is frequently mutated or dysregulated in multiple human cancers, and loss of TET2 is associated with changes in DNA methylation patterns. Here, using newly developed TET2-specific antibodies and the estrogen response as a model system for studying the regulation of gene expression, we demonstrate that endogenous TET2 occupies active enhancers and facilitates the proper recruitment of estrogen receptor α (ERα). Knockout of TET2 by CRISPR-CAS9 leads to a global increase of DNA methylation at enhancers, resulting in attenuation of the estrogen response. We further identified a positive feedback loop between TET2 and ERα, which further requires MLL3 COMPASS at these enhancers. Together, this study reveals an epigenetic axis coordinating a transcriptional program through enhancer activation via DNA demethylation.

scholarly journals Interactions between Telomerase and Primase Physically Link the Telomere and Chromosome Replication Machinery

2002 ◽  
Vol 22 (16) ◽  
pp. 5859-5868 ◽  
Author(s):  
Saugata Ray ◽  
Zemfira Karamysheva ◽  
Libin Wang ◽  
Dorothy E. Shippen ◽  
Carolyn M. Price
Keyword(s):  
De Novo ◽  
Telomerase Activity ◽  
Nuclear Antigen ◽  
Replication Machinery ◽  
Developmentally Regulated ◽  
Coordinate Regulation ◽  
Euplotes Crassus ◽  
Order Complexes ◽  
Proliferating Cell ◽  
Lagging Strand

ABSTRACT In the ciliate Euplotes crassus, millions of new telomeres are synthesized by telomerase and polymerase α-primase during macronuclear development in mated cells. Concomitant with de novo telomere formation, telomerase assembles into higher-order complexes of 550 kDa, 1,600 kDa, and 5 MDa. We show here that telomerase is physically associated with the lagging-strand replication machinery in these complexes. Antibodies against DNA primase precipitated telomerase activity from all three complexes from mated cells but not the 280-kDa telomerase complex from vegetatively growing cells. Moreover, when telomerase was affinity purified, primase copurified with enzyme from mated cells but not with the 280-kDa vegetative complex. Thus, the association of telomerase and primase is developmentally regulated. Intriguingly, PCNA (proliferating cell nuclear antigen) was also found in the 5-MDa complex from mated cells. We therefore speculate that this complex is a complete telomere synthesis machine, while the smaller complexes are assembly intermediates. The physical association of telomerase and primase explains the coordinate regulation of telomeric G- and C-strand synthesis and the efficiency of telomere addition in E. crassus.

scholarly journals Fetal and Neonatal Exposure to the Endocrine Disruptor Methoxychlor Causes Epigenetic Alterations in Adult Ovarian Genes

Endocrinology ◽  
2009 ◽  
Vol 150 (10) ◽  
pp. 4681-4691 ◽  
Author(s):  
Aparna Mahakali Zama ◽  
Mehmet Uzumcu
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Ovarian Function ◽  
Endocrine Disrupting Chemicals ◽  
Neonatal Exposure ◽  
Dna Hypermethylation ◽  
Promoter Regions ◽  
Altered Expression ◽  
Arbitrarily Primed Pcr ◽  
Methylation Patterns

Abstract Exposure to endocrine-disrupting chemicals during development could alter the epigenetic programming of the genome and result in adult-onset disease. Methoxychlor (MXC) and its metabolites possess estrogenic, antiestrogenic, and antiandrogenic activities. Previous studies showed that fetal/neonatal exposure to MXC caused adult ovarian dysfunction due to altered expression of key ovarian genes including estrogen receptor (ER)-β, which was down-regulated, whereas ERα was unaffected. The objective of the current study was to evaluate changes in global and gene-specific methylation patterns in adult ovaries associated with the observed defects. Rats were exposed to MXC (20 μg/kg·d or 100 mg/kg·d) between embryonic d 19 and postnatal d 7. We performed DNA methylation analysis of the known promoters of ERα and ERβ genes in postnatal d 50–60 ovaries using bisulfite sequencing and methylation-specific PCRs. Developmental exposure to MXC led to significant hypermethylation in the ERβ promoter regions (P < 0.05), whereas the ERα promoter was unaffected. We assessed global DNA methylation changes using methylation-sensitive arbitrarily primed PCR and identified 10 genes that were hypermethylated in ovaries from exposed rats. To determine whether the MXC-induced methylation changes were associated with increased DNA methyltransferase (DNMT) levels, we measured the expression levels of Dnmt3a, Dnmt3b, and Dnmt3l using semiquantitative RT-PCR. Whereas Dnmt3a and Dnmt3l were unchanged, Dnmt3b expression was stimulated in ovaries of the 100 mg/kg MXC group (P < 0.05), suggesting that increased DNMT3B may cause DNA hypermethylation in the ovary. Overall, these data suggest that transient exposure to MXC during fetal and neonatal development affects adult ovarian function via altered methylation patterns.

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