cis-requirement for the maintenance of round spermatid-specific transcription.

Maintenance of strict developmental stage- and cell type-specific gene expression is critical for the progression of spermatogenesis. However, the mechanisms which sustain the spatiotemporal order of gene transcription within the seminiferous epithelium are poorly understood. Previous work has established that the proximal promoter of the mouse SP-10 gene was sufficient to maintain round spermatid-specific expression (Reddi, P.P., Shore, A.N., Shapiro, J.A., Anderson, A., Stoler, M.H., Acharya, K.K., 2003b. Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells. Dev. Biol. 262, 173-182). The present study addressed the cis-requirement for this regulation and sought to identify the cognate transcription factor(s). We found that mutation of two 5'-ACACAC motifs (at -172 and -160) within the -186/+28 SP-10 promoter led to premature and indiscriminate expression of a reporter gene in the seminiferous epithelium of transgenic mice, whereas the wild-type -186/+28 promoter retained spermatid specificity. Neither promoter showed ectopic expression in the somatic tissues. Expression cloning using the -186/-148 portion of the promoter yielded transcriptional repressors TDP-43 and Puralpha of which TDP-43 required the complementary 5'-GTGTGT elements located on the opposite strand for binding in vitro. Further, Northern analysis and immunohistochemistry of mouse testis showed the presence of TDP-43 in cell-types where the SP-10 gene remains repressed. Taken together, our results demonstrate that 5'-GTGTGT motifs on the complementary strand are required to prevent premature expression of SP-10 during spermatogenesis and implicate TDP-43 as the putative regulatory factor. The study also implied that additional level(s) of regulation keep the SP-10 gene silent in the somatic tissues.