Date of Graduation
Master of Science in Biology
College of Arts and Sciences
Epigenetic modifications influence gene expression and thereby play a pivotal role in development and disease. Misregulation and mutations in the DAX-1 gene, or Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X chromosome, gene 1, have been implicated in Adrenal Hypoplasia Congenita (AHC) and Dosage Sensitive Sex Reversal (DSS). The orphan nuclear hormone receptor DAX-1 is expressed predominantly in tissues such as the testes, ovaries, breast, adrenal cortex, and lung. Critically, DAX-1 may serve as an indicator of aberrant growth in these tissues. Here we hypothesize that DAX-1 is epigenetically regulated, specifically in cancer cells, thereby reducing its expression. In a survey of several human cancer cell lines, the methylation status of the promoter region of DAX-1 was investigated in order to determine whether epigenetic control played a role in repressing DAX-1 gene expression. Through molecular techniques such as qPCR and western blots, differential expression of DAX-1 in human cell lines was confirmed. Additionally, methylation specific restriction enzyme analysis and bisulfite sequencing identified the location of methylation in breast, adrenal, lung, liver, and kidney cancer cell lines. Following these experiments, a correlation of the methylation status of the DAX-1 promoter and DAX-1 expression is evident. In tandem with bisulfite sequence analysis, chromatin immunoprecipitation experiments elucidated a primary region of interest in which methylation may be critical to the silencing of DAX-1 gene expression. Centered around the transcriptional start site, a stark difference in methyl binding protein occupancy between cancerous and noncancerous breast tissue was identified and likely plays a critical role in gene repression via methylation. Ultimately, this research aims to elucidate the role of epigenetic regulation in gene expression as well as further our understanding of the role of DAX-1 in human cancers.
Riedstra, Caroline P., "Examination of Methylation Status and Occupancy of DNA Methylation Modifying Proteins on Regulatory Regions of the DAX-1 Gene" (2021). Master's Theses. 1393.