Characterization of a human TSPY promoter.

Human TSPY is a candidate oncogene and is supposed to function as a proliferation factor during spermatogenesis. It is the only mammalian protein-coding gene known to be organized as a tandem repeat gene family. It is expressed at highest level in spermatogonia and to a lower amount in primary spermatocytes. To characterize the human TSPY promoter we used the luciferase reporter system in a mouse spermatogonia derived cell line (GC-1 spg) and in a GC-4 spc cell line, that harbour prophase spermatocytes of the preleptotene and early pachytene stage. We isolated a 1303 bp fragment of the 5'-flanking region of exon 1 that shows significant promoter activity in GC-1 spg and reduced activity in GC-4 spc cells. In order to gain further insight into the organization of the TSPY-promoter, stepwise truncations of the putative promoter sequence were performed. The resulting fragments were cloned into the pGL 3-vector and analysed for reporter gene activity in the murine germ cell lines GC-1 spg and GC-4 spc, leading to the characterization of a core promoter (--159 to--1), an enhancing region (--673 to--364) and a silencing region (--1262 to--669). Database research for cis-active elements yielded two putative SOX-like binding sites in the enhancing region and reporter gene activity was drastically reduced when three nucleotides of the AACAAT SOX core sequence were mutated. Our findings strongly suggest that testis-specific expression of human TSPY is mediated by Sox proteins.