BBB seminar: Barbara J. Clark

Barbara J. Clark
Department of Biochemistry and Molecular Biology and The Center for Genetics and Molecular Medicine, University of Louisville, School of Medicine, Louisville, USA

The steroidogenic acute regulatory protein (StAR) facilitates cholesterol transport into the mitochondria where the first enzymatic step in steroid hormone biosynthesis occurs. The transfer of cholesterol into mitochondria is the rate-limiting step in steroidogenesis and this step is controlled by regulating StAR expression levels. Our functional studies on StAR transcriptional control in MA-10 mouse Leydig tumor cells have identified both a positive cAMP-responsive region as well as a negative regulatory region localized within the StAR proximal promoter at -87bp/-64bp and -180bp/-150bp from the transcriptional start site, respectively. We have now characterized the protein complexes associated with these functional promoter regions. The cAMP-responsive region has an AP-1-like element that binds the cAMP-responsive element binding protein (CREB) family members and we have investigated the identity of the CREB family member and the role of CREB phosphorylation and recruitment of the co-activator CREB binding protein (CBP) to the promoter in StAR transcription. Western blot analyses demonstrated CREB, CREM, and ATF-1 are all expressed in MA-10 cells and DNA-affinity chromatography showed that CREB and ATF-1, but not CREM, interact with the AP-1 element within this region of the StAR promoter. Furthermore, (Bu)₂cAMP treatment increased only CREB phosphorylation (P-CREB) and increased P-CREB association with the StAR promoter but did not affect total CREB binding to the StAR promoter. In vivo chromatin immunoprecipitation (ChIP) assays confirmed that total CREB binding to the StAR proximal promoter is independent of (Bu)₂cAMP-treatment, but that P-CREB and CBP interaction with the StAR promoter occurred only after (Bu)₂cAMP-treatment. These data demonstrate for the first time that cAMP stimulation of StAR transcription follows the classical mechanism for increased P-CREB:DNA interactions resulting in CBP recruitment to the StAR proximal promoter and activation of gene transcription.

Several factors have been identified in the transcriptional repression of the StAR gene promoter; yet, no associating co-repressor complexes have been characterized. We have identified a unique repressor region within the StAR proximal promoter and have demonstrated that mutations in both a CAGA and a Sp element within this region resulted in a four-fold increase in StAR promoter activity in the absence of hormonal stimulation. Electrophoretic mobility shift assays identified Sp3 and specific CAGA element binding protein(s) associate with their respective elements. Coimmunoprecipitation analysis revealed the co-repressor complex consisting of mSin3A, HDAC1, and HDAC2 was present in MA-10 cells. HDAC inhibition by trichostatin A treatment of MA-10 cells resulted in a dose-dependent activation of a wild-type StAR reporter construct, while mutations of the CAGA and Sp3 elements diminished this effect by 44%. Lastly, ChIP assays revealed Sp3, mSin3A, and HDAC1/2 associate with the proximal region of the StAR promoter in vivo. Together these data support Sp3 and CAGA element binding proteins bind to the StAR proximal promoter and are associated with the Sin3 co-repressor complex containing histone deacetylase activity thereby contributing to maintaining basal StAR transcription in MA-10 cells.