Male Reproductive Tract

Porcine SRY Promoter Is a Target for Steroidogenic Factor 1

Abstract

To study the process of mammalian sex determination and in particular to further understand the mechanisms of transcriptional regulation of the SRY gene, we have isolated 4.5 kb of pig SRY 5' flanking sequences. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge cell line (PGR 9E11) that expresses SRY as well as SOX9, SF-1 and DAX1. Via primer extension analysis on RNA from this cell line, a transcription start site for porcine SRY was identified at -661 bp 5' from the translation initiation site. Deletion studies of the SRY 5' flanking sequences in PGR 9E11 cells demonstrated that -1.4 kb of 5' flanking sequences retained full transcriptional activity compared to the -4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences downstream of the transcriptional start site are important for promoter activity, since deleting transcribed but not translated sequences eliminated promoter activity. Sequence analysis of the -1.4 kb fragment identified two potential binding sites for SF-1, at -1369 and at -290 from the ATG. Mutagenesis studies showed that both these sites are indeed important for SRY promoter activity. Co-transfection studies in a heterologous cell system (mouse CV-1 cells) demonstrated that pig SF-1 was able to transactivate the pig SRY promoter. Gel shift assays confirmed that the upstream site was recognized by mouse Sf-1 protein. We conclude that two sites for SF-1 transactivation exist within the pig SRY promoter, at -1369 pb and at -290 pb, and that the site at -1369 bp is quantitatively the most important.