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Jhaldeman Dissertation Creation of Versatile Cloning Platforms for Transgene Expression and Epigenome Editing and Their Application to Pancreatic Islet Biology by Jonathan Mark Haldeman Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ Christopher Newgard, Supervisor ___________________________ Deborah Muoio ___________________________ Donald McDonnell ___________________________ Qi-Jing Li ___________________________ Praveen Sethupathy Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2018 i v ABSTRACT Creation of Versatile Cloning Platforms for Transgene Expression and Epigenome Editing and Their Application to Pancreatic Islet Biology by Jonathan Mark Haldeman Department of Pharmacology and Cancer Biology Duke University Date:_______________________ Approved: ___________________________ Christopher Newgard, Supervisor ___________________________ Deborah Muoio ___________________________ Donald McDonnell ___________________________ Qi-Jing Li ___________________________ Praveen Sethupathy An abstract of a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Pharmacology and Cancer Biology in the Graduate School of Duke University 2018 Copyright by Jonathan Mark Haldeman 2018 Abstract Insulin secreting β-cells within the pancreatic islets of Langerhans are vital to maintaining glycemic control. β-cell functional mass is lost during the progression to both Type 1 and Type 2 diabetes mellitus, resulting in hyperglycemia. Therefore, a major goal of diabetes research is to uncover pathways that can be exploited to induce β-cell replication while simultaneously maintaining β-cell function. We previously reported that adenovirus-mediated overexpression of the transcription factor PDX1 is sufficient to induce β-cell replication, but underlying mechanisms remain to be resolved. Using statistical modeling, we identified the miR-17 family, a member of the miR17~92 miRNA cluster, as a candidate regulator of the PDX1-gene network. We show that PDX1 can directly regulate the MIR17HG promoter, the first example of β-cell specific regulation for this important miRNA cluster. Furthermore, the miR17~92 target PTEN is reduced in PDX1-overexpressing β-cells, and chemical inhibition of PTEN potentiates PDX1-mediated β-cell replication, supportive of the presence of a PDX1/miR17~92/PTEN regulatory node. Recombinant adenovirus approaches pioneered by our laboratory have been the main method of genetic manipulation of primary islets in culture since 1994. Whereas adenovirus vectors have proved useful in an otherwise difficult model system, virus construction, especially for cell-type specific applications, is still laborious and time- iv consuming. To overcome this, we have created a new modular cloning system (pMVP) that allows a gene of interest to be rapidly recombined in the context of an array of promoters, N- or C-terminal epitope tags, inducible gene expression modalities, and/or fluorescent reporters, into 18 custom destination vectors, including adenovirus, expression plasmid, lentivirus, and Sleeping Beauty transposon, thus, permitting the creation of > 8000 unique vector permutations. Multiple features of this new vector platform, including cell type-specific and inducible control of gene expression, were validated in the setting of pancreatic islets and other cellular contexts. Furthermore, using pMVP as a foundation, we also developed an S. aureus dCas9 epigenetic engineering platform, pMAGIC, that enables the packaging of 3 guide RNAs with Sa- dCas9 fused to one of five epigenetic modifiers into a single vector. Using pMAGIC- derived adenoviruses, we functionally validated the regulation of PDX1 by Area IV, a cross-species conserved enhancer, through LSD1-mediated epigenetic modification in both INS1 832/13 cells and primary rat pancreatic islets. In sum, my work has uncovered novel information about the role of PDX1 in regulation of the miR17~92 miRNA cluster in pancreatic islet cells. In an effort to contribute more broadly to our laboratory’s pancreatic islet research efforts, I also designed and built the pMVP and pMAGIC systems for efficient generation of purpose- built, customized vectors for manipulation of gene expression in islets and other cell types, including via targeted epigenetic modification of putative regulatory elements v within their native chromatin context. Development of this novel vector platform facilitated additional discoveries about the role of Area IV in control of PDX1 expression in islet β-cells. vi Dedication To my late grandfather Albert, who was a continual example of self-motivation, perseverance, unconditional love, and the pursuit of knowledge. vii Contents Abstract ......................................................................................................................................... iv List of Tables ................................................................................................................................ xii List of Figures ............................................................................................................................ xiii List of Abbreviations .................................................................................................................xvi Acknowledgements ................................................................................................................... xix 1. Introduction ............................................................................................................................... 1 1.1 The Pancreatic Islets of Langerhans ............................................................................... 2 1.2 Pancreatic Islet Dysfunction in Diabetes ....................................................................... 5 1.3 PDX1 (MODY4) is a Master Transcription Factor Required for β-Cell Development and Identity .................................................................................................... 7 1.4 PDX1 Overexpression is Sufficient to Induce β-cell Replication ............................. 15 1.5 Recombinant Adenovirus Vectors Permit Genetic Manipulation of Primary Islets ................................................................................................................................................. 17 1.6 Cas9-Based Epigenetic Engineering Permits Interrogation of Disease-Associated Regulatory Elements Within Native Chromatin Contexts ............................................. 19 1.7 Goals of the Dissertation ............................................................................................... 23 2. Methods ................................................................................................................................... 27 3. A PDX1/miR17~92/PTEN Axis Regulates β-Cell Replication ........................................... 46 3.1 Introduction ..................................................................................................................... 46 3.2 Results .............................................................................................................................. 49 3.2.1 miR-17 Target Sites are Enriched in the PDX1 Gene Network ........................... 49 3.2.2 PDX1 Directly Regulates the Human MIR17HG Promoter ................................ 54 viii 3.2.3 The PDX1 DNA Binding Motif in the MIR17HG Promoter is Conserved in the Rat Genome ......................................................................................................................... 58 3.2.4 Identification of Putative Human Islet MIR17HG Enhancers............................. 60 3.2.5 miR17~92 miRNAs are Induced by PDX1 Overexpression ................................. 67 3.2.6 Peptide-Nucleic Acids as a Tool for Islet Research .............................................. 70 3.2.7 Regulation of PDX1-Mediated Replication by miR17~92 miRNAs ................... 73 3.2.8 miR17~92 miRNAs are Not Sufficient to Drive β-cell Replication ..................... 74 3.2.9 The miR-17~92 Target PTEN Regulates PDX1-Mediated Replication ............... 78 3.3 Discussion ........................................................................................................................ 80 4. Creation of pMVP, a Versatile Cloning Platform for Transgene Expression ................. 85 4.1 Introduction ..................................................................................................................... 85 4.2 Results .............................................................................................................................. 88 4.2.1 Application of MultiSite Gateway® Pro to Enable Rapid Assembly of Multicomponent Vectors ................................................................................................... 88 4.2.2 Generation of pMVP Entry Plasmids ..................................................................... 91 4.2.2.1 Promoters ............................................................................................................ 91 4.2.2.2 Genes of Interest and Control Genes ............................................................... 94 4.2.2.3 shRNA Expression ............................................................................................. 95 4.2.2.4 Epitope
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