PACT/PRKRA and p53 regulate transcriptional activity of DMRT1

Abstract The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.

摘要 转录因子DMRT1（doublesex and mab-3 related transcription factor）在包括小鼠与非洲爪蟾（Xenopus laevis）在内的部分脊椎动物物种中，具备两项截然不同的功能：体细胞雄性化与生殖细胞发育。然而，其转录调控机制目前仍未明确。本研究通过抗DMRT1抗体进行免疫沉淀结合串联质谱（MS/MS）分析，从非洲爪蟾睾丸中筛选DMRT1互作蛋白，并获得了包括PACT/PRKRA（Interferon-inducible double-stranded RNA dependent protein kinase activator A）在内的三种蛋白，其中PACT/PRKRA源自睾丸组织。随后，我们探究了PACT/PRKRA及/或p53对DMRT1转录活性的影响。在转染后的293T细胞中，PACT/PRKRA与p53分别显著增强与抑制了DMRT1介导的荧光素酶活性。我们同时观察到，p53可显著削弱PACT/PRKRA介导的活性增强效应。此外，对蝌蚪性腺中Pact/Prkra mRNA的原位杂交（in situ hybridization）分析显示，其在雌性与雄性生殖系干细胞中均呈现高表达水平。综上，上述研究结果表明，PACT/PRKRA与p53可能分别通过正向与负向调控DMRT1的活性，进而影响生殖系干细胞的命运决定。