Creation of Versatile Cloning Platforms for Transgene Expression and Epigenome Editing and Their Application to Pancreatic Islet Biology

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-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 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.