PARENT SESSION
Sex Determination

IN VITRO CHARACTERIZATION OF THE PIG SRY PROMOTER.

Pilon, Nicolas¹, Daneau, Isabelle ¹, Behdjani, Ramin ¹, Viger, Robert ², Lussier, Jacques¹, Silversides, David ¹, ^{1 2}

ABSTRACT- Since the discovery of the SRY gene more than ten years ago, many studies have demonstrated that this single gene is the genetic switch responsible for determination of the mammalian testis from the bipotential gonad. Unfortunately, very little is currently known about the potential target genes of SRY or how SRY expression is regulated. To further our understanding of SRY transcription regulation, we cloned 4.5 kb of pig SRY 5' flanking sequences. To analyze the promoter activity of this fragment in vitro, we created porcine genital ridge cell lines (PGR cell lines) by immortalization of pig e22-23 genital ridge cells with SV40 Large T antigen. By RT-PCR analysis, SRY expression was detected (as well as SF-1, DAX-1 and SOX9) in one (PGR 9E11) out of the four cell lines obtained. Using RNA from PGR 9E11 cells, a primer extension analysis was performed which allowed the identification of a transcription start site at -664 bp from the translation initiation site. Despite this, deletion studies of the SRY promoter in PGR 9E11 cells demonstrated that sequences between this transcription start site and the translation start site are required for full promoter activity. Furthermore, by analysis of the sequences in this region and upstream, the presence of potential binding sites (BS) for SF-1, Sp1, SOX9 and SRY itself was noted. Mutagenesis of these potential BS demonstrated a strong decrease in promoter activity with the single mutation of the Sp1 potential BS and with the mutation of one out of the two SRY potential BS or one out of the two SF-1 potential BS. Band shift experiments demonstrated that this SF-1 potential BS can be bound by SF-1 and that SOX9 can bind the SOX9 potential BS. Finally, to identify factors responsible for the cell specific expression of SRY, cotransfection studies were performed in an heterologous system (CV-1 cells); Lhx9, SF-1, and SOX9 were each able to transactivate the SRY promoter.

KEY WORDS: SRY promoter, sex determination, pig, porcine genital ridge cell lines