DNA Sequence-Dependent Regulation of SF-1–Mediated Transcription

Abstract

Rat P450c17 gene transcription is regulated by several nuclear factors, including steroidogenic factor-1 (SF-1), nerve growth factor-inducible protein B (NGF-IB, Nurr77), COUP-TF, SET, and Ku autoimmune antigen. A region of this gene, -447/-419, that mediates both basal and cAMP-stimulated transcription, contains two binding sites for orphan nuclear receptors. While SF-1 activates transcription through a single binding site, we show that both binding sites at -447/-419 are required for transcriptional activation by SF-1 and cAMP. Both SF-1 and a novel factor, Steroidogenic Factor-Inducer of Transcription-2 (StF-IT-2) bind to this region, suggesting that a DNA-dependent interaction between StF-IT-2 and SF-1 may be required for full transcriptional activity. Each of the two orphan nuclear receptor sites -429/-424 and at -444/-439 are sufficient for SF-1 binding but are insufficient for SF-1-mediated transcription. Increasing the distance between or changing the orientation of these two sites does not affect basal or SF-1-stimulated activity. Circular permutation analysis, which measures the degree of DNA bending caused by protein binding, indicates that SF-1 binding to -447/-419 induces a different degree of DNA bending than it does at another SF-1-responsive site. However, similar domains of the SF-1 protein are required for its actions at these two regions. Southwestern blots suggest that StF-IT-2 is a approximately 33 kDa protein, and gel shift assays suggest it is expressed primarily in the gonad and brain early in rodent development. These data suggest that the mechanism by which SF-1 stimulates transcription is DNA sequence dependent, and may require additional proteins, such as StF-IT-2, for activation at specific regions of DNA.