Castet Audrey, Boulahtouf Abdelhay, Versini Gwennaëlle, Bonnet Sandrine, Augereau Patrick, Vignon Françoise, Khochbin Saadi, Jalaguier Stéphan, Cavaillès Vincent
INSERM U540, Endocrinologie Moléculaire et Cellulaire des Cancers and Université de Montpellier I, 60 rue de Navacelles, 34090 Montpellier, France.
Nucleic Acids Res. 2004 Apr 1;32(6):1957-66. doi: 10.1093/nar/gkh524. Print 2004.
In this study, we have investigated the role of C-terminal binding proteins (CtBPs) and histone deacetylases (HDACs) in the repressive activity of the nuclear receptor cofactor Receptor-Interacting Protein 140 (RIP140). We have defined the interaction of both CtBP1 and CtBP2 with RIP140 and delineated two motifs (PIDLS and PINLS) differentially required for in vitro interaction. Using different approaches (titration of endogenous CtBPs, mutagenesis and transfection in CtBP knock-out cells), we find that recruitment of CtBPs only partially explains the negative regulation exerted by RIP140. We then demonstrate that RIP140 associates in vitro not only with class I HDACs but also with class II enzymes such as HDAC5. This interaction mainly involves the N-terminus of RIP140 (residues 27-199) and two domains of HDAC5. Moreover, the two proteins functionally interfere in transfection experiments, and confocal microscopy indicates that they co-localize in the nucleus. Interestingly, using the specific HDAC inhibitor trichostatin A, we show that HDAC activity is dispensable for active transrepression by RIP140. Finally, we demonstrate that the C-terminal region of RIP140 contains two additional silencing domains and confers strong active transrepression independently of HDAC activity and CtBPs. Altogether, these data indicate that transcriptional inhibition by the cofactor RIP140 involves complex mechanisms relying on multiple domains and partners.
在本研究中，我们探究了C末端结合蛋白（CtBPs）和组蛋白去乙酰化酶（HDACs）在核受体辅因子相互作用蛋白140（RIP140）的抑制活性中的作用。我们确定了CtBP1和CtBP2与RIP140的相互作用，并描绘了体外相互作用差异所需的两个基序（PIDLS和PINLS）。使用不同方法（内源性CtBPs的滴定、诱变以及在CtBP基因敲除细胞中的转染），我们发现CtBPs的募集仅部分解释了RIP140施加的负调控。然后我们证明RIP140在体外不仅与I类HDACs相关，还与II类酶如HDAC5相关。这种相互作用主要涉及RIP140的N末端（第27 - 199位氨基酸残基）和HDAC5的两个结构域。此外，这两种蛋白在转染实验中功能上相互干扰，共聚焦显微镜显示它们在细胞核中共定位。有趣的是，使用特异性HDAC抑制剂曲古抑菌素A，我们表明HDAC活性对于RIP140的活性反式抑制是不必要的。最后，我们证明RIP140的C末端区域包含另外两个沉默结构域，并且独立于HDAC活性和CtBPs赋予强大的活性反式抑制作用。总之，这些数据表明辅因子RIP140的转录抑制涉及依赖多个结构域和伙伴的复杂机制。
