Purification and characterization of a transcription factor which appears to regulate cAMP responsiveness of the human CYP21B gene

J Biol Chem. 1992 Dec 15;267(35):25213-9.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • Cell Line
  • Cyclic AMP / metabolism*
  • Deoxyribonuclease I
  • Gene Expression Regulation, Enzymologic*
  • Genes*
  • HeLa Cells
  • Humans
  • Kinetics
  • Mice
  • Molecular Sequence Data
  • Nuclear Proteins / isolation & purification
  • Nuclear Proteins / metabolism*
  • Oligodeoxyribonucleotides
  • RNA, Messenger / biosynthesis
  • Regulatory Sequences, Nucleic Acid*
  • Steroid 21-Hydroxylase / genetics*
  • Transcription Factors / isolation & purification
  • Transcription Factors / metabolism*

Substances

  • Nuclear Proteins
  • Oligodeoxyribonucleotides
  • RNA, Messenger
  • Transcription Factors
  • Cyclic AMP
  • Steroid 21-Hydroxylase
  • Deoxyribonuclease I