Role of the Groucho/Grg/TLE corepressor protein in delaying tissue specification

描述（由申请人提供）：本提案的目的是阐明Groucho/Grg/TLE （Grg）协同抑制复合物的抑制机制，并确定其在延迟多能内胚层祖细胞诱导中的作用。Grg蛋白结合并抑制几种参与发育的蛋白，包括Hes1和FoxA，导致细胞类型程序的表达延迟。然而，这种抑制的机制尚不完全清楚：Zaret实验室表明，虽然Grg四聚体能够在不与其他因子相关的情况下覆盖染色质，但仍不清楚为什么Grg四聚体除非被转录因子募集而不能抑制。Notch信号通路，包括通过Grg募集Hes1沉默，延迟胰腺细胞分化。虽然有证据表明，当与先锋因子FoxA结合时，Grg在内胚层中起着类似的作用，但Grg在延迟肝脏程序中的作用尚未明确。因此，本研究计划旨在解决以下具体目标。特异性目标1将直接测试一个假设，即在募集到DNA时蛋白质构象的改变是抑制Grg蛋白活性所必需的。在体外组装的核小体阵列中，各种检测方法将用于表征Grg蛋白的构象变化，以响应Hes1或FoxA的结合和募集。特异性目标2将确定FoxA在内胚层中招募Grg3的基因，并确定这些基因是否会随着Grg蛋白的减少和/或失活而失去其抑制标记并被激活。在FACs分类的小鼠内胚层细胞上进行ChIP-seq实验，将确定Grg3和FoxA1重叠蛋白结合模式，并通过抑制Grg基因或使其蛋白产物失活，在半小鼠胚胎模型中操纵功能Grg蛋白水平，将阐明Grg靶基因对功能Grg3蛋白水平降低的反应。总之，这些目标将有助于了解参与发育调节的许多方面的因子，包括Notch信号，如何作为抑制因子发挥作用。此外，他们将阐明该因子参与延迟多能祖细胞的组织规范。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the repression mechanism of the Groucho/Grg/TLE (Grg) corepressor complex, and establish its role in delaying the induction of multipotent endoderm progenitors. Grg proteins bind and repress the targets of several proteins involved in development, including Hes1 and FoxA, causing the delay in expression of cell type programs. However, the mechanism of this repression is yet to be fully understood: the Zaret lab showed that while Grg homotetramers are capable of blanketing chromatin without associating with other factors, it remains unknown why Grg homotetramers are not capable of repression unless recruited by a transcription factor. The Notch signaling pathway, involving Hes1 silencing through Grg recruitment, delays pancreatic cell differentiation. While evidence suggests that Grg plays a similar role in the endoderm, when bound to the pioneer factor, FoxA, the role of Grg in delaying the liver program has yet to be defined. Thus, this research proposal is designed to address the following specific aims. Specific Aim 1 will directly test the hypothesis that a protein conformation change upon recruitment to the DNA is required for the repressive activity of Grg proteins. Various assays will be utilized to characterize conformational changes of the Grg protein in response to Hes1 or FoxA binding and recruitment to nucleosome arrays assembled in vitro. Specific Aim 2 will identify the genes to which FoxA recruits Grg3 in the endoderm, and ascertain whether these genes lose their repressive mark and become activated as Grg proteins diminish and/or are inactivated. ChIP-seq experiments on FACs sorted mouse endoderm cells will determine Grg3 and FoxA1 overlapping protein binding patterns, and manipulation of functional Grg protein levels in the half mouse embryo model, by repression of the Grg gene or inactivation of its protein product, will elucidate the response of Grg target genes to the diminishing levels of functional Grg3 protein. Together, these aims will contribute to the knowledge of how a factor involved in many aspects of developmental regulation, including Notch signaling, functions as a repressor. Furthermore, they will shed light upon this factor's involvement in delaying tissue specification in multipotent progenitor cells.