Targeted epigenetic editing to increase adult pancreatic beta-cell proliferation

β-cell replacement therapy is potentially a curative approach in treating diabetes, as demonstrated by the success of pancreatic islet transplantation in type 1 diabetes. However, there are an insufficient number of organ donors to meet the demand of this disease, which is increasing in prevalence. One strategy to increase the supply of human β-cells for transplantation in type 1 diabetics, or to increase residual β-cell mass in type 2 diabetics, is to induce human β-cell replication. This strategy has not been implemented clinically because adult human β-cells are largely quiescent and the capacity for proliferation decreases with age. I hypothesized that changes in DNA methylation contribute to the age-related decline in proliferative capacity in human βcells, and that altering the DNA methylome in a targeted manner could improve proliferative capacity. To investigate this hypothesis, I sought to profile the β-cell across the human lifespan, and to develop tools that permit targeted DNA methylation modifications and efficiency in measuring DNA methylation. I conducted RNA-Seq and whole-genome bisulfite sequencing (WGBS) to profile the aging human β-cell transcriptome and DNA methylome. I found that there are significant changes in gene expression with age, and in DNA methylation, particularly at islet-specific active enhancers. Further, I developed transcription activator-like effector (TALE) fusion proteins conjugated to DNA methyltransferases (DNMTs) and demonstrated that targeting TALE-DNMTs to the promoter of the CDKN2A locus, encoding the cell cycle inhibitor p16, increases proliferation in primary human fibroblasts. Finally, I developed BisPCR2, a novel technique for preparing targeted bisulfite next-generation sequencing libraries, which greatly improves the efficiency in which DNA methylation can be measured at target regions. I demonstrated the utility of this tool to validate genome-wide findings of type 2 diabetes CpG risk loci. Together, these novel datasets and epigenetic tools poise the β-cell regeneration field to investigate targeted epigenetic modifications as a strategy to improve proliferative capacity of adult human β-cells. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Pharmacology First Advisor Klaus H. Kaestner Subject Categories Pharmacology This dissertation is available at ScholarlyCommons: http://repository.upenn. edu/edissertations/1610 TARGETED EPIGENETIC EDITING TO INCREASE ADULT PANCREATIC β-CELL PROLIFERATION Diana L. Bernstein A DISSERTATION in Pharmacology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2016 Supervisor of Dissertation _________________________ Klaus Kaestner, Ph.D., M.S., Thomas and Evelyn Suor Butterworth Professor in Genetics Graduate Group Chairperson _________________________ Julie Blendy, Ph.D., Professor of Pharmacology Dissertation Committee Mitchell A. Lazar, M.D., Ph.D., Sylvan Eisman Professor of Medicine Marisa Bartolomei, Ph.D., Professor of Cell & Developmental Biology Gerd Blobel, M.D., Ph.D., Frank E. Weise III Professor of Pediatrics, Children's Hospital of Philadelphia Benjamin F. Voight, Ph.D., Assistant Professor, Departments of Pharmacology and Genetics TARGETED EPIGENETIC EDITING TO INCREASE ADULT PANCREATIC β-CELL PROLIFERATION COPYRIGHT 2016 Diana Lynne Bernstein This work is licensed under the Creative Commons AttributionNonCommercial-ShareAlike 3.0 License To view a copy of this license, visit http://creativecommons.org/licenses/by-ny-sa/2.0/