REGULATION OF THE BMI1 AND RET PROTO-ONCOGENES BY THE DLX2 TRANSCRIPTION FACTOR IN THE DEVELOPING GASTRO-INTESTINAL TRACT

• Author / Creator

  McColl, Hunter D

• The Dlx1/Dlx2 double knockout (DKO) mouse dies at P0 with a cleft palate and a distended abdomen. Within the intestinal crypts of Leiberkuhn a stable, non-dividing stem cell group is marked by the oncogene BMI1. Unpublished data from the Eisenstat laboratory demonstrates co-expression of BMI1 and the homeobox transcription factor DLX2 in the intestinal crypts and also suggests a role for DLX2 in the regulation of expression of the Ret proto-oncogene. Ret is responsible for enteric nervous system (ENS) development. Over-expression of RET induces Multiple Endocrine Neoplasia syndrome type cancers whereas RET loss-of-function is associated with Hirschsprung’s Disease (HD) with partial or complete loss of intestinal innervation. We investigated the potential regulatory effect of DLX2 on Bmi1 and Ret. Our hypothesis was that DLX2 suppresses Bmi1 while promoting Ret expression through direct binding of the gene targets’ promoters during intestinal and ENS development. Methods: Chromatin Immunoprecipitation (ChIP) assays using a polyclonal DLX2 antibody and embryonic intestinal tissues studied candidate in vivo interactions between DLX2 and target promoter regions of genomic DNA. Electrophoretic mobility shift assays (EMSAs) and site directed mutagenesis of DLX2 binding sites was used to determine if there was direct binding by DLX2 of the Bmi1/ Ret regulatory regions identified by ChIP in vitro. Reporter gene assays were applied to assess the effect of Dlx2 co-expression on Bmi1/ Ret expression in vitro. Quantitative PCR analysis of the embryonic gastrointestinal tract demonstrated the in vivo effect of loss of Dlx1/Dlx2 expression on Ret and Bmi1 expression when comparing wild-type to Dlx1/Dlx2 DKO mouse tissues. Results: DLX2 interaction with specific regions of the Bmi1 and Ret’s promoters in vivo was demonstrated using ChIP. The potential for specific binding of DLX2 to only a subset of the promoters identified by ChIP assays in vitro was demonstrated by EMSA assay. The in vitro regulatory effects of DLX2 on the expression of these genes were revealed through luciferase gene reporter assays: Ret expression was repressed whereas Bmi1 expression was activated. In vivo analysis shows a significant change in ret expression in the Dlx1/Dlx2 DKO when measured by qPCR. Ret expression was decreased in the small intestine and increased in the large intestine at E13 in the DKO. However, Bmi1 shows no change in intestinal expression at E18 with the loss of Dlx1/Dlx2 gene function. Conclusion: Occupancy of the Bmi1 and Ret promoters by DLX2 in vivo occurs at specific time points, while EMSAs demonstrate direct binding of DLX2 to specific promoter regions in vitro. While reporter gene assays show the ability of DLX2 to repress ret expression in vitro, in vivo this regulation may be influenced by proximal/distal patterning which may also underlie the severity of the various phenotypes of HD. While DLX2 shows in vitro regulatory potential over Bmi1expression, in vivo assays did not support a regulatory interaction during embryonic development. Ongoing expression studies of cell-type specific markers for epithelial and ganglion cell markers in the intestine and ENS in the Dlx1/Dlx2 DKO mouse as well as in human HD biopsies will further explore the biological significance of DLX2 on intestinal and ENS development.

• Subjects / Keywords

  • Bmi1
  • Ret
  • DLX

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3R49GR80

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.