Analysis of novel Steroidogenic Factor-1 targets in the human adrenal gland

Steroidogenic Factor-1 (SF-1, NR5A1) is a nuclear receptor transcription factor that plays a central role in adrenal and reproductive biology. In humans, SF-1 regulates adrenal development and disruption of SF-1 or its known targets is associated with impaired adrenal function. Therefore, the identification of novel SF-1 targets could reveal important new mechanisms in adrenal development and disease. This thesis describes three approaches to identifying SF-1 targets in NCI-H295R human adrenocortical cells. SF-1-dependent regulation of CITED2 and PBX1 was investigated since these factors regulate adrenal development in mice through pathways shared with Sf-1. Expression of CITED2 and PBX1 was confirmed in the developing human adrenal, and SF-1 was found to bind to and activate the CITED2 promoter and to cooperate with DAX1 to activate the PBX1 promoter. SF-1 binding was investigated using chromatin immunoprecipitation microarrays (ChIP-on-chip). These studies revealed that SF-1 binds to the extended promoter of 445 genes, including factors involved in angiogenesis. Angiopoietin 2 (ANGPT2) emerged as a key novel SF-1 target, confirmed by transactivational studies, suggesting that regulation of angiogenesis might be an important additional action of SF-1 during adrenal development and tumorigenesis. Global gene expression analysis following SF-1 overexpression revealed differential expression of 1058 genes, many of which are involved in steroidogenesis, lipid metabolism and cell proliferation. Bidirectional manipulation of SF-1 revealed a subset of positively regulated genes, including the known targets STAR and CYP11A1 and novel target SOAT1, a regulator of cholesterol esterification. Considering that defects in several SF-1 targets have been associated with adrenal disorders, mutational analysis of SOAT1 was performed in forty-three subjects with unexplained adrenal insufficiency but failed to reveal potentially disease-causing variants. Taken together, manipulation of SF-1 in human adrenal cells has expanded our knowledge of the many potential actions of SF-1 in the human adrenal gland.