GATA-4 and GATA-6 modulate tissue-specific transcription of the human gene for P450c17 by direct interaction with Sp1.
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.
Department of Pediatrics, Building MR IV, Room 207, University of California San Francisco, San Francisco, California 94143-0978, USA.
Christa E
CE
Flück
Walter L
WL
Miller
D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics* / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D005260:Female; D050991:GATA4 Transcription Factor; D050993:GATA6 Transcription Factor; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D008745:Methylation; D025542:Neurofibromin 1 / Q000378:metabolism; D010920:Placenta / Q000378:metabolism; D011401:Promoter Regions, Genetic / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics* / Q000378:metabolism; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation / Q000235:genetics*
D004268:DNA-Binding Proteins; D050991:GATA4 Transcription Factor; C484170:GATA4 protein, human; D050993:GATA6 Transcription Factor; C494759:GATA6 protein, human; C515057:NR5A1 protein, human; D025542:Neurofibromin 1; D016329:Sp1 Transcription Factor; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D003577:Cytochrome P-450 Enzyme System