A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor.
A regulatory role for the unstructured C-terminal domain of the CtBP transcriptional corepressor.
复制标题
CtBP 转录辅阻遏物非结构化 C 端结构域的调节作用。
DOI：
10.1101/2023.05.19.541472
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Arnosti,DavidN
中科院分区：
文献类型：
--
作者：
Raicu,Ana-Maria;Suresh,Megha;Arnosti,DavidN
C 端结合蛋白 (CtBP) 是一种转录辅阻遏物，在发育、肿瘤发生和细胞命运中发挥着关键作用。 CtBP 蛋白在结构上与 α 羟酸脱氢酶相似，并且在 C 末端具有显着的本质无序区域。在哺乳动物系统中，缺乏 C 端结构域 (CTD) 的 CtBP 蛋白能够充当转录调节因子并寡聚化，从而使人们对这种非结构化结构域对于基因调控的重要性产生疑问。然而，约 100 个残基的非结构化 CTD（包括一些短基序）的存在在双边对称动物中是保守的，表明在进化过程中维持该结构域的重要性。为了揭示 CtBP CTD 的重要性，我们对天然存在的具有或缺乏 CTD 的 CtBP 果蝇亚型（即 CtBP(L) 和 CtBP(S)）进行了功能测试。我们使用 CRISPRi 系统将 dCas9-CtBP(L) 和 dCas9-CtBP(S) 募集至内源启动子，以直接比较它们在体内的转录影响。有趣的是，CtBP(S)能够显着抑制Mpp6启动子的转录，而CtBP(L)则弱得多，这表明长CTD可能调节CtBP的抑制活性。相反，在细胞培养中，亚型在转染的 Mpp6 报告基因上表现相似。这两种发育调节同工型的活性的上下文特异性差异表明，CTD 可能有助于提供一系列适合发育程序的抑制活性。
The C-terminal binding protein (CtBP) is a transcriptional corepressor that plays critical roles in development, tumorigenesis, and cell fate. CtBP proteins are structurally similar to alpha hydroxyacid dehydrogenases and feature a prominent intrinsically disordered region in the C terminus. In the mammalian system, CtBP proteins lacking the C-terminal domain (CTD) are able to function as transcriptional regulators and oligomerize, putting into question the significance of this unstructured domain for gene regulation. Yet, the presence of an unstructured CTD of ∼100 residues, including some short motifs, is conserved across Bilateria, indicating the importance of maintaining this domain over evolutionary time. To uncover the significance of the CtBP CTD, we functionally tested naturally occurringDrosophilaisoforms of CtBP that possess or lack the CTD, namely CtBP(L) and CtBP(S). We used the CRISPRi system to recruit dCas9-CtBP(L) and dCas9-CtBP(S) to endogenous promoters to directly compare their transcriptional impactsin vivo. Interestingly, CtBP(S) was able to significantly repress transcription of theMpp6promoter, while CtBP(L) was much weaker, suggesting that the long CTD may modulate CtBP’s repression activity. In contrast, in cell culture, the isoforms behaved similarly on a transfectedMpp6reporter gene. The context-specific differences in activity of these two developmentally regulated isoforms suggests that the CTD may help provide a spectrum of repression activity suitable for developmental programs.