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 ∼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.