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

描述(申请人提供)：这项建议的目标是阐明Groucho/GRG/TLE(GRG)辅阻遏子复合体的抑制机制，并确定其在延迟诱导多能内胚层前体细胞方面的作用。GRG蛋白结合并抑制几种参与发育的蛋白的靶标，包括Hes1和FoxA，导致细胞类型程序表达的延迟。然而，这种抑制的机制尚不完全清楚：Zaret实验室表明，虽然GRG同源异构体能够在不与其他因素相关的情况下覆盖染色质，但仍不清楚为什么GRG同源异构体除非被转录因子招募，否则不能进行抑制。Notch信号通路通过GRG募集使Hes1沉默，从而延迟胰腺细胞的分化。虽然有证据表明GRG在内胚层中扮演着类似的角色，但当GRG与先驱因子FoxA结合时，GRG在延迟肝脏程序中的作用尚未确定。因此，本研究提案旨在解决以下具体目标。特殊目的1将直接检验这样一个假设，即GRG蛋白的抑制活性需要在被招募到DNA时发生构象变化。将利用各种分析来表征GRG蛋白在Hes1或FoxA结合和募集到体外组装的核小体阵列时的构象变化。特定目标2将确定FoxA在内胚层招募Grg3的基因，并确定这些基因是否在GRG蛋白减少和/或失活时失去抑制标记而被激活。在FACS分选的小鼠内胚层细胞上进行的CHIP-SEQ实验将确定Grg3和FoxA1重叠的蛋白结合模式，通过抑制GRG基因或使其蛋白产物失活来操纵半小鼠胚胎模型中功能GRG蛋白的水平，将阐明GRG靶基因对功能Grg3蛋白水平下降的反应。总而言之，这些目标将有助于了解参与发育调控的许多方面的因素是如何发挥抑制作用的，包括Notch信号转导。此外，他们将阐明这一因素在多能祖细胞中延迟组织指定的参与。 公共卫生相关性：该提案的目的是阐明Groucho/GRG/TLE共抑制蛋白在延缓多能祖细胞分化中的作用。将分化推迟到适当的发育阶段，也就是在细胞群体扩大之后，对于适当的器官形成至关重要。从这项工作中获得的知识最终可以为干细胞疗法中健康组织的发展做出贡献。

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. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to elucidate the role of the Groucho/Grg/TLE corepressor protein in delaying the differentiation of multipotent progenitor cells. It is crucial for proper organ formation to defer differentiation until the appropriate developmental stage, after cell populations have expanded. Knowledge gained from this work can ultimately contribute to the development of healthy tissue in stem cell therapies.