Alterations in DNA methylation patterns are a hallmark of cancer development demonstrated as both global hypomethylation and gene specific hypermethylation. This results in genomic instability as well as tumor suppressor gene silencing. Methylation is catalyzed by the DNA methyltransferases (DNMTs) DNMT1, DNMT3A, and DNMT3B and is assisted by DNMT3L. In order to understand the role of the DNMTs in aberrant methylation and cancer, a better understanding of how they function and interact with one another is essential. All of the DNMTs, but especially DNMT3B, are alternatively spliced generating many isoforms of unknown function. Immunodeficiency, centromere instability, and facial anomalies syndrome (ICF), which is caused by mutations in the de novo methyltransferase DNMT3B and results in altered DNA methylation patterns, is a model disease that can be studied to better understand the function of DNMT3B. We have successfully expressed each of the DNMTs using baculovirus and E. coli expression systems along with several of their common splice variants, mutations associated with ICF syndrome, and deletion constructs of Dnmt3b. A novel system was developed for these recombinant enzymes using a plasmid containing a satellite 2 sequence as a template in order to analyze both the amount of methylation and site specific methylation with bisulfite genomic sequencing and pyrosequencing. The effects of common splice variants and mutations/deletions on protein-protein interactions are being ascertained using GST pull downs and effects on their interaction with DNA examined using electrophoretic mobility shift assays. Preliminary analyses suggest that the splice variants and mutations/deletions significantly affect DNA methylation and also alter their interaction with DNMT3L and DNA. For example, Dnmt3b2 demonstrates less DNA methylation activity than Dnmt3b1, but both are enhanced by the addition of DNMT3L. Both Dnmt3b1 and Dnmt3b2 bind DNMT3L in pull down assays, but unlike Dnmt3b1 the interaction between Dnmt3b2 and DNMT3L is lost at higher salt concentrations suggesting a weaker association. Ongoing studies will provide additional insights into how interaction between Dnmt3a and Dnmt3b splice variants influences methylation and what impact mutations or deletions can have on this. We anticipate that our results will allow for an improved understanding of the functional interactions of DNMTs which will, in turn, enhance our understanding of how and why methylation patterns go awry in cancer. (Supported by NCI grants F32CA132327 and R01CA114229)

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 145.