Identification of Ube2b as a Novel Target of Androgen Receptor in Mouse Sertoli Cells 1 Short title: Ube2b as a Novel Target of Androgen Receptor

Many genes are regulated by androgen and its receptor (AR), but the direct target genes of AR, especially those involved in spermatogenesis and male infertility, remain unclear. Here, we identified ubiquitin-conjugating enzyme E2B (Ube2b) as a critical target gene of AR. The expression of Ube2b was decreased in the testes of Sertoli cell AR knockout (S-AR -/y ) mice analyzed by qRT-PCR and immunofluorescence. The upregulation of Ube2b gene by testosterone was further demonstrated by Western blot, qRT-PCR in TM4 cells, a mouse Sertoli cell line. Moreover, luciferase assay, EMSA and ChIP assay validated that the ligand-bound AR activated Ube2b transcription via direct binding to the androgen responsive element of the Ube2b promoter. In vitro analyses showed that testosterone increased Ube2b expression and activated H2A ubiquitylation, while downregulation of Ube2b blocked the testosterone-induced H2A ubiquitylation. The ubiquitylation of H2A was markedly decreased in the testes of S-AR -/y mice by immunohistochemistry. Digital gene expression analysis showed that 113 genes were significantly downregulated and 71 were upregulated by UBE2B in TM4 cells. These results suggest that Ube2b as a direct AR transcriptional target in Sertoli cells mediates the function of AR in spermatogenesis by promoting H2A ubiquitylation. Summary sentence: Androgen and its receptor directly upregulates Ube2b gene expression via the potential ARE element.