Investigation of Epigenetic Control of DAX-1 Expression in Human Cell Lines

Dosage-Sensitive Sex Reversal, Adrenal Hypoplasia Congenita, Critical Region on the X chromosome, gene 1 (DAX-1 or NR0B1) is an orphan nuclear hormone receptor implicated in Adrenal Hypoplasia Congenita (AHC) and Dosage Sensitive Sex Reversal (DSS). In both instances, DAX-1 plays a key role in growth and development by modulating hormone function. In DSS, mutations on the X-chromosome lead to duplication of the region containing DAX-1, resulting in sex reversal, and in AHC, mutations in the DAX-1 gene diminish development of adrenal tissue which leads to a reduction in adrenal hormone production. Expressed predominantly in tissues such as the testes, ovaries, breast, adrenal cortex, and lung, DAX-1 may serve as an indicator of aberrant growth. Here we hypothesize that DAX-1 is epigenetically regulated, specifically in cancer cells, thereby reducing its expression. We surveyed various human cancer cells in order to determine whether inhibiting DNA methylating enzymes released epigenetic control of the DAX-1 gene, resulting in an increase in expression. By implementing molecular techniques, such as bisulfite sequencing, we determined the precise methylation sites in the DAX-1 gene. Additionally, we carried out methylation specific restriction enzyme analysis to differentiate degrees of methylation between lung, breast, liver, cervical, and adrenal carcinoma cell lines. Following confirmation of the precise methylation sites, we utilized chromatin immunoprecipitation (ChIP) in order to identify the modifying proteins present on the DAX-1 CpG islands. In conjunction with these experimental techniques, we implemented a bioinformatics approach to analyze methylation in the promoter region of the DAX-1 gene across tissue sample data acquired from The Cancer Genome Atlas Program. The results of this research could lead to a translational application of understanding where this orphan NHR fits into the development and progression of cancer. As a quickly growing field, cancer epigenetics is a key player in the ongoing pursuit for identifying biomarkers that may be pertinent in future therapeutic applications.