Transcriptional activation of human CYP17 in H295R adrenocortical cells depends on complex formation among p54(nrb)/NonO, protein-associated splicing factor, and SF-1, a complex that also participates in repression of transcription.
The first 57 bp upstream of the transcription initiation site of the human CYP17 (hCYP17) gene are essential for both basal and cAMP-dependent transcription. EMSA carried out by incubating H295R adrenocortical cell nuclear extracts with radiolabeled -57/-38 probe from the hCYP17 promoter showed the formation of three DNA-protein complexes. The fastest complex contained steroidogenic factor (SF-1) and p54(nrb)/NonO, the intermediate complex contained p54(nrb)/NonO and polypyrimidine tract-binding protein-associated splicing factor (PSF), and the slowest complex contained an SF-1/PSF/p54(nrb)/NonO complex. (Bu)(2)cAMP treatment resulted in a cAMP-inducible increase in the binding intensity of only the upper complex and also activated hCYP17 gene transcription. SF-1 coimmunoprecipitated with p54(nrb)/NonO, indicating direct interaction between these proteins. Functional assays revealed that PSF represses basal transcription. Further, the repression of hCYP17 promoter-reporter construct luciferase activity resulted from PSF interacting with the corepressor mSin3A. Trichostatin A attenuated the inhibition of basal transcription, suggesting that a histone deacetylase interacts with the SF-1/PSF/p54(nrb)/NonO/mSin3A complex. Our studies lend support to the idea that the balance between transcriptional activation and repression is essential in the control of adrenocortical steroid hormone biosynthesis.
Sewer
Marion B
MB
Department of Biochemistry and Center in Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, USA. marion@toxicology.mc.vanderbilt.edu
Nguyen
Viet Q
VQ
Huang
Ching-Jung
CJ
Tucker
Philip W
PW
Kagawa
Norio
N
Waterman
Michael R
MR
D000302:Adrenal Cortex / Q000201:enzymology*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D003513:Cycloheximide / Q000494:pharmacology; D004268:DNA-Binding Proteins / Q000502:physiology*; D051717:Fushi Tarazu Transcription Factors; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics; D006655:Histone Deacetylases / Q000235:genetics; D018398:Homeodomain Proteins; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear; D000068103:Serine-Arginine Splicing Factors; D013254:Steroid 17-alpha-Hydroxylase / Q000235:genetics* / Q000378:metabolism*; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics* / Q000502:physiology*
D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D011500:Protein Synthesis Inhibitors; D018160:Receptors, Cytoplasmic and Nuclear; C101745:Sfrs11 protein; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D000068103:Serine-Arginine Splicing Factors; D003513:Cycloheximide; D013254:Steroid 17-alpha-Hydroxylase; D006655:Histone Deacetylases