GATA-4 and GATA-6 Modulate Tissue-Specific Transcription of the Human Gene for P450c17 by Direct Interaction with Sp1

• Published in: Molecular endocrinology (Baltimore, Md.) Md.), 2004-05, Vol.18 (5), p.1144-1157
• Main Authors: Flück, Christa E, Miller, Walter L
• Format: Article
• Language: English
• Subjects: 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
• ISSN: 0888-8809; 1944-9917
• DOI: 10.1210/me.2003-0342

Cytochrome P450c17 catalyzes steroidogenic 17α-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.