Cyclic adenosine 3',5'-monophosphate (cAMP) enhances cAMP-responsive element binding (CREB) protein phosphorylation and phospho-CREB interaction with the mouse steroidogenic acute regulatory protein gene promoter.
Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.
Clem
Brian F
BF
Department of Biochemistry and Molecular Biology and The Center for Genetics and Molecular Medicine, University of Louisville School of Medicine, Louisville, Kentucky 40292, USA.
Hudson
Elizabeth A
EA
Clark
Barbara J
BJ
D000107:Acetylation; D051697:Activating Transcription Factor 1; D000818:Animals; D050882:CREB-Binding Protein; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D002845:Chromatography; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism; D015151:Immunoblotting; D007985:Leydig Cells; D008297:Male; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D015398:Signal Transduction; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
D051697:Activating Transcription Factor 1; C497100:Atf1 protein, mouse; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D006657:Histones; D009687:Nuclear Proteins; D010750:Phosphoproteins; D015534:Trans-Activators; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; D004247:DNA; D000242:Cyclic AMP; D050882:CREB-Binding Protein; C494364:Crebbp protein, mouse