GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1

Mol Endocrinol. 2004 May;18(5):1144-57. doi: 10.1210/me.2003-0342. Epub 2004 Feb 26.


Cytochrome P450c17 catalyzes steroidogenic 17alpha-hydroxylase and 17,20 lyase activities. Expression of the gene for P450c17 is cAMP dependent, tissue specific, developmentally programmed, and varies among species. Binding of Sp1, Sp3, and NF1-C (nuclear factor 1-C) to the first 227 bp of 5'flanking DNA (-227/LUC) is crucial for basal transcription in human NCI-H295A adrenal cells. Human placental JEG-3 cells contain Sp1, Sp3, and NF1, but do not express -227/LUC, even when transfected with a vector expressing steroidogenic factor 1 (SF-1). Therefore, other factors are essential for basal expression of P450c17. Deoxyribonuclease I footprinting and EMSAs identified a GATA consensus site at -64/-58 and an SF-1 site at -58/-50. RT-PCR identified GATA-4, GATA-6, and SF-1 in NCI-H295A cells and GATA-2 and GATA-3, but not GATA-4, GATA-6, or SF-1 in JEG-3 cells. Cotransfection of either GATA-4 or GATA-6 without SF-1 activated -227/LUC in JEG-3 cells, but cotransfection of GATA-2 or GATA-3 with or without SF-1 did not. Surprisingly, mutation of the GATA binding site in -227/LUC increased GATA-4 or GATA-6 induced activity, whereas mutation of the Sp1/Sp3 site decreased it. Furthermore, promoter constructs including the GATA site, but excluding the Sp1/Sp3 site at -196/-188, were not activated by GATA-4 or GATA-6, suggesting an interaction between Sp1/Sp3 and GATA-4 or GATA-6. Glutathione-S-transferase pull-down experiments and coimmunoprecipitation demonstrated interaction between GATA-4 or GATA-6 and Sp1, but not Sp3. Chromatin immunoprecipitation assays confirmed that this GATA-4/6 interaction with Sp1 occurred at the Sp site in the P450c17 promoter in NCI-H295A cells. Demethylation with 5-aza-2-deoxycytidine permitted JEG-3 cells to express endogenous P450c17, SF-1, GATA-4, GATA-6, and transfected -227/LUC. Thus, GATA-4 or GATA-6 and Sp1 together regulate expression of P450c17 in adrenal NCI-H295A cells and methylation of P450c17, GATA-4 and GATA-6 silence the expression of P450c17 in placental JEG-3 cells.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Binding Sites
  • Cells, Cultured
  • Cytochrome P-450 Enzyme System / genetics*
  • Cytochrome P-450 Enzyme System / metabolism
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Drosophila / genetics
  • Female
  • GATA4 Transcription Factor
  • GATA6 Transcription Factor
  • Gene Expression Regulation / genetics
  • Humans
  • Methylation
  • Neurofibromin 1 / metabolism
  • Placenta / metabolism
  • Promoter Regions, Genetic / genetics
  • Sp1 Transcription Factor / genetics*
  • Sp1 Transcription Factor / metabolism
  • Steroidogenic Factor 1
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism
  • Transcriptional Activation / genetics*


  • DNA-Binding Proteins
  • GATA4 Transcription Factor
  • GATA4 protein, human
  • GATA6 Transcription Factor
  • GATA6 protein, human
  • NR5A1 protein, human
  • Neurofibromin 1
  • Sp1 Transcription Factor
  • Steroidogenic Factor 1
  • Transcription Factors
  • Cytochrome P-450 Enzyme System