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与mab-3相关转录因子，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，兼具体细胞雄性化与生殖细胞发育两项截然不同的生物学功能。然而，其转录调控机制至今仍未阐明。本研究采用抗DMRT1抗体进行免疫沉淀结合质谱（MS/MS）分析，从非洲爪蟾睾丸组织中筛选鉴定DMRT1互作蛋白，最终获得三种候选蛋白，其中包括源自睾丸的PACT/PRKRA（干扰素诱导型双链RNA依赖蛋白激酶激活因子A，Interferon-inducible double-stranded RNA dependent protein kinase activator A）。随后，我们探究了PACT/PRKRA和/或p53对DMRT1转录活性的调控作用。在转染后的293T细胞中，PACT/PRKRA与p53分别可显著增强与抑制DMRT1介导的荧光素酶活性。我们同时观察到，PACT/PRKRA所介导的活性增强效应可被p53强力削弱。此外，对蝌蚪性腺中Pact/Prkra mRNA的原位杂交分析结果显示，该基因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，进而参与生殖系干细胞的命运决定过程。