Involvement of DLX Genes in the Regulation of Insulin Gene(s) Transcription

• Author / Creator

  Moradipoor,Sara

• Even though extensive research over recent decades has increased our understanding of the key steps in pancreatic development and gene regulatory networks involved in pancreatic cell specification and maturation, it is well accepted that a more detailed understanding of pancreatic development is needed for successful early diagnosis and treatment of pancreatic diseases such as diabetes mellitus and pancreatic cancer. The endocrine portion of the pancreas, known as the islets of Langerhans, consist of 5 main cell types: α cells, β cells, δ cells, PP cells and Epsilon cells. The α and β cells each secrete a regulatory hormone into the bloodstream. Insulin and glucagon are the main pancreatic hormones secreted by β- and α- cells, respectively. Several homeodomain-containing transcription factors, such as PDX1, NKX2.2 and PAX4, have been identified that not only regulate the expression of insulin in β-cells, but also participate in pancreatic development and pancreatic islet cell differentiation. Some of these transcription factors have been associated with the pathogenesis of diabetes. The Dlx1 and Dlx2 homeodomain-containing transcription factors are highly expressed in the developing pancreas as early as E14.5 and are co-expressed with pancreatic hormones and transcription factors such as PDX1. Previously, the Eisenstat lab showed that neonatal pancreas from Dlx1/2 double knockout mice shows reduced expression of glucagon and insulin compared with wild‐type littermates at P0. Based on these observations, I hypothesized that DLX2 plays a role in pancreatic islet cell development by direct transcriptional regulation of insulin and glucagon gene expression and that loss of function of Dlx1/Dlx2 results in islet-cell-specific defects. ChIP-qPCR of mouse E18 pancreas showed DLX2 enrichment at putative DLX2 binding sites on INS1, INS2 and proglucagon 5’-regulatory regions. The direct binding of DLX2 to the INS2 regulatory region was confirmed by electrophoretic mobility shift assays in vitro. Co-transfection of Dlx2 expression plasmids with PGL3-INS2 promoter regionsiiiresulted in a significant increase in luciferase reporter gene activity. qRT-PCR showed a reduction in mRNA levels of INS1 and INS2 in the Dlx1/2 DKO pancreas tissue compared to wild-type littermates at E18.5, supporting the activating effects of DLX2 on INS1 and INS2 transcription. However, INS1 and INS2 mRNA levels were significantly elevated in Dlx2-siRNA treated Beta-TC 6 cells, which was in contrast with our luciferase reporter assay results, where DLX2 activated the expression of INS1and INS2 genes in HEK923 cells, and also our in vivo results where a decrease in mRNA levels of INS1 and INS2 in the Dlx1/2 DKO pancreas tissue was observed. Overall, this project provides more information about pancreatic islet cell development and gene regulation, and contributes to our understanding of pancreatic islet cell function in health and disease, including diabetes.

• Subjects / Keywords

  • DLX Genes
  • Insulin Gene
  • transcription factor

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-0yjm-g992

• License

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