Defining the regulation of UHRF1 and DNMT1 for maintenance of the epigenome

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PROJECT SUMMARY In multicellular organisms, methylation of DNA on the 5’ carbon of cytosine helps ensure proper gene expression. Methylation occurs symmetrically on both DNA strands, and the maintenance methyltransferase DNMT1 is responsible for copying the methylation pattern from the parent strand to the daughter strand during DNA replication. Maintenance methylation by DNMT1 requires the chromatin-associated ubiquitin ligase UHRF1, which catalyzes ubiquitination of histone H3 (H3ub). Many questions remain about how the various domain functions of DNMT1 and UHRF1 are regulated and which are necessary to maintain DNA methylation. The objective of this proposal is to define the mechanistic relationship between UHRF1, DNMT1, and H3 ubiquitination in the maintenance of DNA methylation. The first aim investigates the hypothesis that ubiquitination of histone H3 by UHRF1 regulates the catalytic activity of DNMT1 and that maintenance methylation by DNMT1 depends on both UHRF1 ubiquitin ligase-dependent and independent mechanisms. Aim 1 will use in vitro biochemical assays to characterize point mutations in DNMT1 and UHRF1 that compromise individual domains of these proteins. Transgenes encoding these mutant enzymes will then be tested in cancer cells using western blot analysis of H3ub and EPIC array analysis of DNA methylation. These experiments will define the contribution of individual functions of DNMT1 and UHRF1 to H3 ubiquitination and DNA methylation in vitro and during DNA maintenance methylation in cancer cells. The second aim tests the hypothesis that allosteric activation of UHRF1’s ubiquitin ligase activity by hemimethylated DNA (heDNA) operates in cells and regulates genome architecture in cooperation with CTCF/cohesin. Aim 2 will use UHRF1 mutant transgenes to test whether heDNA stimulates UHRF1’s enzymatic activity in cancer cells using western blot analysis of H3ub and hairpin bisulfite sequencing to determine levels of heDNA. The contribution of UHRF1 function to genome architecture through recently identified stable heDNA at CTCF/cohesin sites will also be defined using CTCF ChIP-seq and Hi-C. These experiments will determine if heDNA regulates the activity of UHRF1 in cancer cells and test the contribution of stable heDNA at CTCF/cohesin sites to genome architecture through UHRF1. Misregulation of DNA methylation is a driving force in many cancers. Thus, insight into how DNA methylation is synthesized and propagated is central to understanding how cancer begins and progresses. The studies proposed here will advance our knowledge of the mechanisms involved in methylation maintenance to allow the rational design of therapies that halt or reverse the formation of oncogenic DNA methylation patterns.