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 a 4.5-kilo-base (kb) pig SRY 5′ flanking sequence. To facilitate the in vitro analysis of these sequences, we have generated a porcine genital ridge (PGR) cell line (9E11) that expresses SRY as well as SOX9, steroidogenic factor-1 (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 base pairs (bps) 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 with the -4.5 kb fragment, but that transcriptional activity fell when further deletions were made. Sequences down-stream of the transcriptional start site are important for promoter activity, because 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. To address the role of SF-1 transactivation in SRY promoter activity, mutagenesis studies of the potential SF-1 binding sites were performed and revealed that these sites were indeed important for SRY promoter activity. Cotransfection 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 bp and at -290 bp, and that the site at -1369 bp is quantitatively the most important.