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

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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.