Mechanisms of DNA methylation and its regulation by epigenetic histone modifications

Project Summary A hallmark in cancer is genome-wide hypomethylation and promoter specific hypermethylation. A genome- wide reduction in DNA methylation is also observed in ageing, linking increased risk for cancer and dementia with age. This may become increasingly prevalent since the global population is aging and life expectancies are rising. Thus, managing and monitoring DNA methylations has become a priority for scientists and clinicians. Current managing strategies often target the chromatin modifiers that establish and maintain DNA methylation. Central to maintaining DNA methylation in mammals is the DNA Methyltransferase 1 (DNMT1), which is overexpression in a range of cancers such as gastric, pancreatic, colorectal, and lung cancer. However, clinical strategies to specifically inhibit DNMT1 are limited. The most common FDA-approved inhibitor lacks specificity, leading to undesirable off-target effects. It’s desirable to have specific inhibitors against DNMT1. However, developing these inhibitors may halt from missing mechanistic knowledge on DNMT1 methylation. Specifically, we are missing explanations for DNMT1’s selectivity and regulation by chromatin modification. I hypothesize the molecular interactions between DNMT1 and chromatin explains this missing mechanistic information. AIM 1 of my proposed research is to determining the cryo-EM structure of DNMT1 with specifically modified nucleosomes, revealing insights into these mechanisms. This will be followed by thorough validation through in vitro and in vivo experiments. Another strategy for managing DNA methylation is by targeting the enzymes regulating DNMT1. A significant histone posttranslational modification regulating DNMT1, mono-ubiquitinated H3 (H3Ub), currently has no identified deubiquitinating enzyme. Our research has identified a deubiquitinase that can remove H3Ub in vitro, and preliminary structural studies revealed that it exhibits a unique mechanism. AIM 2 of my proposed research involves characterizing this H3Ub deubiquitinase in cells, understanding how it regulates DNA methylation, and resolving the deubiquitination mechanism using cryo-EM. In summary, my research focuses on uncovering the mechanisms of DNA methylation and it regulation, aiming to open therapeutic avenues for age-related diseases and cancer.