Effects of chromatin structure on the enzymatic and DNA binding functions of DNA methyltransferases DNMT1 and Dnmt3a in vitro

Robertson, Andrea K.; Geiman, Theresa M.; Sankpal, Umesh T.; Hager, Gordon L.; Robertson, Keith D.

doi:10.1016/j.bbrc.2004.07.083

Cited by 65 publications

(46 citation statements)

References 44 publications

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“…Thus far our preliminary experiments indicate that LSH binds to DNA and to linker DNA regions of reconstituted chromatin more efficiently than either DNMT3B or DNMT1 (data not shown) (27,31). It is possible to envision that the binding properties of the LSH complex as a whole are determined by the additive affinities of individual proteins, i.e., LSH, DNMTs, and HDACs, for DNA and/or histones.…”

Section: Discussionmentioning

confidence: 86%

“…Dnmt1, Dnmt3b, and Dnmt3a have also been shown to interact with each other and to coimmunoprecipitate and copurify with HDAC1 and HDAC2 (Fig. 3A) (9,17,(31)(32)(33). Given all these complex interactions, we next investigated whether DNMTs contribute to LSH-mediated transcriptional repression.…”

Section: Resultsmentioning

confidence: 99%

“…Given the homology of DDM1 and Lsh with chromatin-remodeling ATPases of the SNF2 family, it has been suggested that chromatin remodeling by DDM1 and Lsh may facilitate the processivity of DNMT enzymes on nucleosomal DNA templates (20,28). In agreement with the requirement for SNF2-like proteins to support DNA methylation in vivo, several studies have demonstrated that in vitro DNMT enzymes methylate DNA assembled into chromatin 10-to 20-fold less efficiently than naked DNA (11,25,31). Whether DDM1 or Lsh is able to stimulate DNA methylation on nucleosomal templates in vitro has not been demonstrated.…”

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confidence: 94%

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LSH Cooperates with DNA Methyltransferases To Repress Transcription

Myant

Stancheva

2008

Molecular and Cellular Biology

122

129

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LSH, a protein related to the SNF2 family of chromatin-remodeling ATPases, is required for efficient DNA methylation in mammals. How LSH functions to support DNA methylation and whether it associates with a large protein complex containing DNA methyltransferase (DNMT) enzymes is currently unclear. Here we show that, unlike many other chromatin-remodeling ATPases, native LSH is present mostly as a monomeric protein in nuclear extracts of mammalian cells and cannot be detected in a large multisubunit complex. However, when targeted to a promoter of a reporter gene, LSH acts as an efficient transcriptional repressor. Using this as an assay to identify proteins that are required for LSH-mediated repression we found that LSH cooperates with the DNMTs DNMT1 and DNMT3B and with the histone deacetylases (HDACs) HDAC1 and HDAC2 to silence transcription. We show that transcriptional repression by LSH and interactions with HDACs are lost in DNMT1 and DNMT3B knockout cells but that the enzymatic activities of DNMTs are not required for LSH-mediated silencing. Our data suggest that LSH serves as a recruiting factor for DNMTs and HDACs to establish transcriptionally repressive chromatin which is perhaps further stabilized by DNA methylation at targeted loci.In vertebrate genomes, DNA methylation patterns are established during gametogenesis, embryo development, and cell differentiation by enzymes of the DNA cytosine methyltransferase family, which includes the maintenance DNA methyltransferase (DNMT) DNMT1 and the de novo methyltransferases DNMT3A and DNMT3B (1,24,45). DNMT1 binds to PCNA and functions primarily during S phase to restore fully methylated CpGs on hemimethylated daughter DNA strands generated during DNA replication (3, 4). DNMT3A and -3B are able to methylate unmethylated DNA and in mouse embryogenesis are required during gastrulation, when DNA methylation patterns are established in differentiating cell lineages of the embryo (24). Mice lacking DNMT proteins die early during embryogenesis and display aberrant expression of retrotransposons and various imprinted and nonimprinted genes (14,18,24). Genetic studies with plants and mammals have revealed that additional factors besides DNMTs are required for the establishment of DNA methylation patterns in vivo. Loss-of-function mutations in SNF2 family-related putative chromatin-remodeling ATPases such as the Arabidopsis thaliana DDM1 (decrease in DNA methylation 1) protein and its murine homolog Lsh (lymphoid-specific helicase) lead to dramatic hypomethylation of the genome in Arabidopsis thaliana and mice, respectively (5, 16). Unlike animals that are null for DNMTs, Lsh-deficient mice develop to term but die soon after birth with symptoms of renal failure (5). Interestingly, mice expressing a hypomorph allele of Lsh with targeted disruption of the SNF2 domain survive much longer and display modest hypomethylation of DNA and premature aging (39). Collectively, these studies indicate that the low levels of DNA methylation (ϳ30 to 35% of the wild-type level) in Lsh...

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Section: Discussionmentioning

confidence: 86%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 94%

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LSH Cooperates with DNA Methyltransferases To Repress Transcription

Myant

Stancheva

2008

Molecular and Cellular Biology

122

129

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“…Interestingly, the GAD 65 promoter is embedded in CpG islands and therefore, an increased expression of DNMT1 in GABAergic neurons would predict GAD 65 promoter hypermethylation and GAD 65 mRNA and protein expression downregulation. Based on this possibility, we can speculate that the unexpected lack of GAD 65 downregulation in the same neurons in which epigenetic promoter hypermethylation likely induces reelin and GAD 67 expression downregulation: a) the GAD 65 promoter, unlike RELN and GAD 67 promoters, is already hypermethylated in NPS and therefore in SZ and BDP patients its expression cannot be further downregulated by DNMT1 overexpression; and b) DNMT1 fails to target GAD 65 promoters due to the inhibitory role of specific/local chromatin factors, such as histone modifying enzymes, chromatin remodeling proteins, and the protein complexes, including the repressor MeCP2 proteins, which alter the affinity of DNMT1 for Sadenosylmethionine and CpG island promoters (Jenuwein and Allis 2001,Robertson et al, 2004,Hong et al, 2005. This consideration suggests study of whether the expression of other gene promoters rich in CpG islands is also downregulated in GABAergic neurons showing elevated levels of DNMT1.…”

Section: The Ove Rexpression Of Dnmt1 In Ba 9 and Basal Ganglia Gabaementioning

confidence: 99%

Epigenetic mechanisms expressed in basal ganglia GABAergic neurons differentiate schizophrenia from bipolar disorder

Veldić

Kadriu

Maloku

et al. 2007

Schizophrenia Research

135

108

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“…88 One component of this complex, the chromatin remodeler hSNF2H, increases the binding affinity of DNMT1 to mononucleosomes. 89 The interaction with these chromatin remodeling factors may help DNMT1 to access substrate sites in heterochromatic regions.…”

Section: Nucleus Volume 2 Issuementioning

confidence: 99%