CCAAT enhancer-binding protein beta and GATA-4 binding regions within the promoter of the steroidogenic acute regulatory protein (StAR) gene are required for transcription in rat ovarian cells.
Steroidogenic acute regulatory protein (StAR) is a vital accessory protein required for biosynthesis of steroid hormones from cholesterol. The present study shows that in primary granulosa cells from prepubertal rat ovary, StAR transcript and protein are acutely induced by gonadotropin (FSH). To determine the sequence elements required for hormone inducibility of the StAR promoter, truncated regions of the -1002/+6 sequence of the mouse gene were ligated to pCAT-Basic plasmid and transfected by electroporation to freshly prepared cells. FSH inducibility determined over a 6-h incubation was 10-40-fold above basal levels of chloramphenicol acetyltransferase activity. These functional studies, supported by electrophoretic mobility shift assays indicated that two sites were sufficient for transcription of the StAR promoter constructs: a non-consensus binding sequence (-81/-72) for CCAAT enhancer-binding protein beta (C/EBPbeta) and a consensus motif for GATA-4 binding (-61/-66). Western analyses showed that GATA-4 is constitutively expressed in the granulosa cells, while all isoforms of C/EBPbeta were markedly inducible by FSH. Site-directed mutations of both binding sequences practically ablated both basal and hormone-driven chloramphenicol acetyltransferase activities to less than 5% of the parental -96/+6 construct. Unlike earlier notions, elimination of potential binding sites for steroidogenic factor-1, a well known tissue-specific transcription factor, did not impair StAR transcription. Consequently, we propose that C/EBPbeta and GATA-4 represent a novel combination of transcription factors capable of conferring an acute response to hormones upon their concomitant binding to the StAR promoter.
Silverman
E
E
Department of Biological Chemistry, Alexander Silberman Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
Eimerl
S
S
Orly
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005260:Female; D005640:Follicle Stimulating Hormone / Q000494:pharmacology; D051717:Fushi Tarazu Transcription Factors; D050991:GATA4 Transcription Factor; D005786:Gene Expression Regulation / Q000235:genetics*; D017930:Genes, Reporter; D006107:Granulosa Cells / Q000378:metabolism; D018398:Homeodomain Proteins; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins / Q000235:genetics*; D010053:Ovary / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor / Q000235:genetics; D054339:Steroidogenic Factor 1; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic; D000098682:Steroidogenic Acute Regulatory Protein
D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D051717:Fushi Tarazu Transcription Factors; D050991:GATA4 Transcription Factor; D018398:Homeodomain Proteins; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D018160:Receptors, Cytoplasmic and Nuclear; D016329:Sp1 Transcription Factor; D054339:Steroidogenic Factor 1; D014157:Transcription Factors; D000098682:Steroidogenic Acute Regulatory Protein; C515058:steroidogenic factor 1, mouse; C515060:steroidogenic factor 1, rat; D005640:Follicle Stimulating Hormone; D015500:Chloramphenicol O-Acetyltransferase