CpG Sites Preferentially Methylated by Dnmt3a in Vivo

Abstract

Dnmt3a and Dnmt3b are two major de novo DNA methyltransferases essential for embryonic development in mammals. It has been shown that Dnmt3a and Dnmt3b have distinct substrate preferences for certain genomic loci, including major and minor satellite repeats. However, the exact target CpG sites where Dnmt3a and Dnmt3b catalyze DNA methylation remains largely unknown. To identify a CpG site that is specifically methylated by Dnmt3a or Dnmt3b, we screened methylated genomic loci by methylation sensitive restriction fingerprinting using genomic DNA from wildtype, Dnmt3a null, Dnmt3b null, and Dnmt3a-Dnmt3b double null ES cells. Interestingly, one of the CpG sites was preferentially methylated in wild-type and Dnmt3b null ES cells but not in Dnmt3a null or Dnmt3a-Dnmt3b double null ES cells, suggesting that the sitespecific methylation was Dnmt3a-dependent. Sequencing results revealed that the isolated CpG site is located within the 1st exon of the G isoform of fibroblast growth factor (Fgf-1.G) on mouse chromosome 18. Exogenous expression of Dnmt3a but not Dnmt3b in the double null ES cells restored DNA methylation of this CpG site. When we examined alternative transcription initiation sites, we determined that another CpG site in the 5-flanking region of the Fgf-1.A isoform was also methylated specifically by Dnmt3a. Using chimeric constructs between Dnmt3a and Dnmt3b, we further determined that the NH 2 -terminal regulatory domain of Dnmt3a was responsible for establishing its substrate specificity. These results indicate that certain CpG sites within the Fgf-1 gene locus are preferentially methylated by Dnmt3a but not by Dnmt3b. Selective methylation by a specific member of Dnmt3 may therefore play a role in the orchestration of gene expression during embryonic development.In mammals, global DNA methylation is catalyzed mainly by three DNA methyltransferases, Dnmt1, Dnmt3a, and Dnmt3b. Dnmt1 has a high preference for hemimethylated DNA and is essential for maintaining methylation patterns during DNA replication (1, 2). On the other hand, Dnmt3a and Dnmt3b have a preference for both unmethylated and hemimethylated DNA (3, 4) and are responsible for de novo methylation during early development. Although Dnmt3a and Dnmt3b contain highly conserved amino acid sequences in their PWWP domains, cysteine-rich domains, and carboxyl-terminal catalytic domains, the overall homology of these proteins is relatively low (3, 5). Gene disruption studies revealed that Dnmt3a null mice and Dnmt3b null mice showed different developmental defects (6). Conditional knock-outs of Dnmt3a or Dnmt3b in germ cells also exhibited distinct phenotypes (7). Dnmt3a disruption in male germ cells showed impaired spermatogenesis, while offspring from the Dnmt3a conditional mutant females died in utero. In both cases, Dnmt3a null cells displayed a lack of methylation at imprinting loci (7). In contrast, no apparent phenotype was observed in germ cell development when Dnmt3b was conditionally disrupted (7). In in vitro cell culture, a disruption of D...

Genome-wide DNA methylation profiling reveals novel epigenetically regulated genes and non-coding RNAs in human testicular cancer

“…The other differentially methylated CpG islands resided either inside genes or in nongenic regions. For promoter-associated CpG islands, a number of them, including those of NTF3, FGF, OSR1, HOXA6, NPY and WT1, have previously been reported as differentially methylated in other cancer types (Mares et al, 2001;Bibikova et al, 2006;Oka et al, 2006;Houshdaran et al, 2007;Illingworth et al, 2008). Our study also identified many CpG islands that were not previously shown to be differentially methylated, such as CXCL5, EID1 and TRHDE.…”

Cigarette smoke induces demethylation of prometastatic oncogene synuclein-γ in lung cancer cells by downregulation of DNMT3B

“…Altered expressions of DNMTs have been linked to the cancer-associated hypermethylation of TSGs (Gius et al, 2004;Suzuki et al, 2004;Pruitt et al, 2005;Oka et al, 2006), as well as the hypomethylation of melanoma antigen gene MAGE-A1 (Loriot et al, 2006). We sought to determine which DNMT was involved in CSE-induced changes in the methylation pattern of SNCG.…”

“…It was shown that inactivation of DNMT3B in mouse embryonic fibroblasts resulted in DNA hypomethylation, chromosomal instability and spontaneous immortalization, but these changes were not observed by inactivating DNMT3A (Dodge et al, 2005). Another recent study reported that the first exon of the G isoform of fibroblast growth factor is the preferred substrate for DNMT3A but not for DNMT3B (Oka et al, 2006). Our results in this study further show that these two enzymes are under different regulation by CSE, and that the CpG island of SNCG is a specific substrate of DNMT3B not DNMT3A.…”

Involvement of DNMT3B in the pathogenesis of Hirschsprung disease and its possible role as a regulator of neurogenesis in the human enteric nervous system

“…14,15 DNA methylation patterns are established by three independent DNA methyltransferases-DNMT1, DNMT3A, and DNMT3B 16,17 -known to be necessary during neural progenitor cell development. [18][19][20] DNMT1 is mainly implicated in Purpose: Hirschsprung disease (OMIM 142623) is a neurocristopathy attributed to a failure of cell proliferation or migration and/or failure of the enteric precursors along the gut to differentiate during embryonic development. Although some genes involved in this pathology are well characterized, many aspects remain poorly understood.…”