Purification and characterization of a transcription factor which appears to regulate cAMP responsiveness of the human CYP21B gene.
A unique cAMP regulatory sequence, -129/-96 base pairs (bp), associated with the gene encoding human cytochrome P450C21 (CYP21B) binds a nuclear protein designated ASP, as described previously (Kagawa, N., and Waterman, M. R. (1991) J. Biol. Chem. 266, 11199-11204). This putative transcription factor required for cAMP-dependent transcription of the human CYP21B gene has been purified from the nuclear extracts of mouse Y1 cells by using sequence-specific DNA-affinity chromatography. The purified ASP is 78 kDa as estimated by SDS-polyacrylamide gel electrophoresis and binds to its specific recognition site, -126/-113-bp CACTCTGTGGGCGG, which has been demonstrated to be the minimum cAMP regulatory sequence of the human CYP21B gene. To characterize ASP more precisely, an antibody was raised against the 78-kDa protein. This antibody led to a supershift of the DNA.ASP complex on gel shift analysis and inhibition of in vitro transcription promoted by the ASP binding sequence, thereby indicating that ASP is a 78-kDa transcription factor. Upon DNase I footprinting experiments, ASP showed a characteristic footprint which very closely resembles but is distinct from that of Sp1 which also occupies a binding site within -129/-96 bp. Furthermore, the addition of purified ASP enhanced the mRNA synthesis promoted by the minimum cAMP regulatory sequence in a cell-free transcription system using HeLa cell extracts, whereas added Sp1 does not. These results indicate that ASP is a primary transcription factor for the cAMP-dependent regulation of the human CYP21B gene.
Kagawa
N
N
Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
Waterman
M R
MR
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism*; D003850:Deoxyribonuclease I; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D013255:Steroid 21-Hydroxylase / Q000235:genetics*; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D009687:Nuclear Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D014157:Transcription Factors; D000242:Cyclic AMP; D013255:Steroid 21-Hydroxylase; D003850:Deoxyribonuclease I