Yan Z, Jetten A M
Cell Biology Section, Laboratory of Pulmonary Pathobiology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
J Biol Chem. 2000 Nov 10;275(45):35077-85. doi: 10.1074/jbc.M005566200.
Retinoid receptor-related testis-associated receptor (RTR)/germ cell nuclear factor is a nuclear orphan receptor that plays an important role in the control of gene expression during early embryonic development and gametogenesis. It has been shown to repress transcriptional activation. In this study, we further characterize this repressor function. We demonstrate that RTR can suppress the transcriptional activation induced by the estrogen receptor related-receptor alpha1 through its response element. The latter is at least in part due to competition for binding to the same response element. In addition, RTR inhibits basal transcriptional activation, indicating that it functions as an active repressor. Mammalian two-hybrid analyses showed that RTR interacts with the co-repressor nuclear co-repressor (N-CoR) but is unable to interact with the co-repressor SMRT or RIP140. Pull-down analyses with glutathione S-transferase-RTR fusion protein demonstrated that RTR physically interacts with N-CoR in vitro, suggesting a potential role for N-CoR in the transcriptional repression by RTR. To identify the regions in RTR essential for the binding of RTR to N-CoR, the effect of various deletion and point mutations on this interaction was examined. This analysis revealed that this interaction requires the hinge domain, helix 3 as well as the helix 12 region of RTR. The residues Ser(246)-Tyr(247) in the hinge domain, Lys(318) in helix 3, and Lys(489)-Thr(490) in helix 12 are identified as being critical in this interaction. Our results demonstrate that RTR can function as an active transcriptional repressor and that this repression can be mediated through interactions with the co-repressor N-CoR. We show that this interaction exhibits several characteristics unique to RTR. Through its repressor function, RTR can suppress the induction of transcriptional activation by other nuclear receptors. These repressor activities may provide important mechanisms by which RTR regulates gene expression during development and gametogenesis.
类视黄醇受体相关的睾丸关联受体（RTR）/生殖细胞核因子是一种核孤儿受体，在早期胚胎发育和配子发生过程中的基因表达调控中发挥重要作用。已有研究表明它能抑制转录激活。在本研究中，我们进一步对这种抑制功能进行了表征。我们证明RTR可通过其反应元件抑制雌激素受体相关受体α1诱导的转录激活。后者至少部分是由于对相同反应元件结合的竞争。此外，RTR抑制基础转录激活，表明它作为一种活性阻遏物发挥作用。哺乳动物双杂交分析表明，RTR与共抑制因子核共抑制因子（N-CoR）相互作用，但不能与共抑制因子SMRT或RIP140相互作用。用谷胱甘肽S-转移酶-RTR融合蛋白进行的下拉分析表明，RTR在体外与N-CoR发生物理相互作用，提示N-CoR在RTR的转录抑制中可能发挥潜在作用。为了确定RTR与N-CoR结合所必需的区域，研究了各种缺失和点突变对这种相互作用的影响。该分析揭示，这种相互作用需要RTR的铰链区、螺旋3以及螺旋12区域。铰链区的Ser(246)-Tyr(247)残基、螺旋3中的Lys(318)以及螺旋12中的Lys(489)-Thr(490)被确定为在这种相互作用中至关重要。我们的结果表明，RTR可作为一种活性转录阻遏物发挥作用，且这种抑制可通过与共抑制因子N-CoR的相互作用介导。我们表明这种相互作用展现出RTR特有的几个特征。通过其抑制功能，RTR可抑制其他核受体诱导的转录激活。这些抑制活性可能为RTR在发育和配子发生过程中调节基因表达提供重要机制。
