PROJECT SUMMARY/ABSTRACT Diabetes has become a major public health crisis, afflicting nearly 30 million people in the United States, and these numbers continue to rise at an alarming rate. Both type 1 and type 2 diabetes result from insulin insufficiency, in large part due to loss of functional beta-cells. Significant research efforts are currently focused on understanding beta-cell failure in diabetes, and developing effective therapeutic approaches to replenishing the beta-cell deficit in diabetes. Despite significant advances in these aspects, challenges remain in development of effective beta-cell therapies, primarily due to gaps in our current understanding of mechanisms that regulate normal beta-cell development, function, and growth. Our recent work has identified DNA methylation as a pivotal epigenetic mechanism that regulates beta-cell identity and function. Moreover, we found that DNA methylation patterns defining functional beta-cell phenotype are disrupted in the diabetic beta-cells, suggesting dynamic nature of DNA methylation. Our preliminary studies indicate that dynamic remodeling of DNA methylation (5- methylcytosine; 5mC) via its conversion to a hydroxylated form (5-hydroxymethylcytosine; 5hmC) is essential for beta-cell differentiation, function, and adaptive response. We hypothesize that stage-specific, appropriate patterning of 5mC and 5hmC is critical for beta-cell homeostasis, and is disrupted in diabetes leading to beta- cell failure. Thus, we seek to determine how enzymatic regulation of the balance between 5mC and 5hmC governs functional beta-cell mass and affects diabetes susceptibility. We will employ mouse genetics, disease models, human islet studies, and state-of-the-art genome wide epigenetic profiling methods to address the following aims: In Specific Aim 1, we aim to establish the requirement of 5mC and 5hmC patterning in differentiation of beta-cells from progenitors. Specific Aim 2 seeks to define the contribution of dynamic remodeling of 5mC and 5hmC patterns in beta-cell replication and adaptive capacity. In Specific Aim 3, we address if and how environmental factors like oxidative stress and metabolite variation can disrupt the beta-cell 5mC 5hmC landscape to drive beta-cell failure, and diabetes. The proposed studies will delineate a novel regulatory module that governs beta-cell development and growth, and establish a fundamental regulatory paradigm that link beta-cell environment, metabolism and epigenome. Our work is likely to have a broad and significant impact by providing novel clues to promote beta- cell differentiation, function, and expansion towards strategies aimed at beta-cell rejuvenation and replacement for diabetes therapy.