| DNA methylation is an epigenetic mark that is required for transcriptional repression in mammalian development, imprinting, and in the maintenance of genome stability. Genome-wide methylation patterns are established and maintained by three DNA methyltransferases (DNMTs)---DNMT1, DNMT3A, and DNMT3B. DNMT3B is specifically involved in silencing the satellite repeats at the centromeric and pericentromeric regions. The role of DNMT3B at the centromeric region is also emphasized by mitotic defects arising due to chromosome instability observed in ICF syndrome, a disease caused by germline mutations in DNMT3B. Although the mechanism of DNA methylation is well known, the targeting of DNA methylation and DNMT3B to certain genomic loci remains poorly understood. DNMT3B is also regulated by alternative splicing. Several DNMT3B splice variants are overexpressed in tumor cells and negatively regulate normal DNMT3B mediated DNA methylation. Therefore it is important to understand the significance of DNMT3B splice variants in development and tumorigenesis.;In the present study, a yeast two-hybrid screening was performed and several novel DNMT3B protein interactions were identified. Of the several proteins identified, the interaction of DNMT3B with the mammalian chromatin associated factor MCAF and the chromodomain helicase DNA binding protein CHD3 were confirmed, and need further characterization. The interaction between DNMT3B and the constitutive centromeric protein CENP-C was confirmed in mammalian cells. Results from siRNA knock downs, bisulfite genomic sequencing and ChIP, demonstrate that CENP-C recruits DNA methylation and DNMT3B to both centromeric and pericentromeric satellite repeats. CENP-C and DNMT3B influence the histone modifications in satellite repeat regions, including marks characteristic of centromeric chromatin and disruption of this interaction causes elevated transcription of centromeric repeats. Loss of CENP-C or DNMT3B leads to elevated chromosome misalignment and segregation defects during mitosis. Taken together, the interaction between CENP-C and DNMT3B suggests a novel mechanism by which DNA methylation is targeted to discrete regions of the genome and contributes to chromosomal stability.;In another study, a novel alternatively spliced form of DNMT3B lacking exon 5 was identified and characterized. This variant was termed DNMT3B3Delta5 because of its close resemblance with the ubiquitously expressed DNMT3B3 isoform. The novel splice variant lacking exon 5 is highly expressed in pluripotent cells and neural tissues, and is conserved in the mouse and is re-expressed on converting differentiated mEFs into pluripotent iPS cells. DNMT3B3Delta5 also displays altered expression in human tumor cell lines as well as an altered subcellular localization. Ectopic overexpression of DNMT3B3Delta5 resulted in repetitive element hypomethylation. Taken together, these results demonstrate that alternative splicing of exon 5 may play an important role in stem cell maintenance or differentiation and exon 5 could influence the functional properties of DNMT3B. |
