A GC-box within the proximal promoter region of the rat cathepsin L gene activates transcription in Sertoli cells of sexually mature rats.
It has been proposed that stage-specific gene expression in Sertoli cells results from sequential activation and repression of transcription. However, the exact molecular mechanisms are unknown. As a first step in addressing this fundamental issue, we recently demonstrated that a 3-kilobase (kb) genomic fragment immediately upstream of the rat cathepsin L translation start site directed stage-specific expression of a reporter gene only in Sertoli cells of transgenic mice in a manner comparable to that of the endogenous gene (predominantly in stages VI-VIII tubules). Supporting the activation/repression model of regulation, an upstream domain that mediated an inhibitory effect by male germ cells was identified within this 3-kb promoter region. In the present study, we localized and characterized the regulatory elements that activate transcription. Analyses of a series of 5' deletion constructs demonstrated that a 120-base pair (bp) region that spans the transcription start site of the rat cathepsin L gene was sufficient to activate transcription in Sertoli cells isolated from sexually mature rats. Within this region, electrophoretic mobility shift assays showed that one member of the Sp/XKLF family of factors, Sp3, specifically bound to a GC-box. Furthermore, Sp1-binding activity was not detected in nuclear extracts from Sertoli cells of sexually mature rats. Finally, the GC-box was shown to be essential for promoter activity since mutating this binding motif abolished promoter activity. Collectively, these results suggest that the GC-box is a critical regulatory element for the cathepsin L promoter in mature Sertoli cells.
Charron
Martin
M
Division of Reproductive Biology, Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, The Johns Hopkins University, Baltimore, Maryland 21205, USA.
DeCerbo
Joshua N
JN
Wright
William W
WW
D000818:Animals; D056668:Cathepsin L; D002403:Cathepsins / Q000235:genetics*; D002467:Cell Nucleus / Q000235:genetics; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases; D004268:DNA-Binding Proteins / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D024202:Electrophoretic Mobility Shift Assay; D006151:Guanosine / Q000235:genetics; D008156:Luciferases / Q000235:genetics; D008297:Male; D009841:Oligonucleotides / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D017207:Rats, Sprague-Dawley; D012708:Sertoli Cells / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic / Q000502:physiology*; D014162:Transfection
D004268:DNA-Binding Proteins; D009841:Oligonucleotides; C497123:Sp3 protein, rat; D014157:Transcription Factors; D006151:Guanosine; D051705:Sp3 Transcription Factor; D008156:Luciferases; D002403:Cathepsins; D003546:Cysteine Endopeptidases; D056668:Cathepsin L; C534278:Ctsl protein, mouse; C534280:Ctsl protein, rat