The Retinoblastoma corepressor family: precision targeting for discerning gene regulatory mechanisms in the Drosophila eye

项目摘要/摘要 视网膜母细胞瘤是一种罕见的儿科癌症，由视网膜母细胞瘤(RB)肿瘤的两个拷贝丢失引起 抑制子基因。Rb是一种高度保守的转录因子，对基因的正确调控具有重要意义。 在真核细胞中表达。在包括乳腺癌在内的许多人类癌症中也观察到Rb基因突变 和肺癌。在人类中，Rb家族由Rb、p107和p130组成，它们显示出重叠和 在基因调控中的非冗余作用。类似地，果蝇谱系经历了基因复制事件 导致Rbf1和Rbf2对偶数的表达。RB家族多样性的意义和 他们在不同背景下调节基因表达的分工尚不清楚。他们的活动一直是 在特定启动子的背景下进行研究；然而，我们对这些因素缺乏更深层次的机制理解 在正常组织的背景下，在发育中。这项提案将研究组织特异性基因调控 用苍蝇模型系统研究视网膜母细胞瘤家族蛋白的活性。我们假设RB家族有 通过对启动子的不同靶向和通过不同的 镇压活动。在目标1中，我们将研究RB近亲是如何表现出机械差异转录的 在目标2中，我们将确定Rbf1蛋白本身的调控如何控制特定于启动子的 压抑。我们将使用我们新开发的方法来高度精确地将RB类似基因定位于不同的基因 启动子通过RB融合到核酸酶死亡的Cas9。这一强大的工具允许直接比较 Rb家族成员在发育系统中研究他们的基因和组织特异性活动。使用这个工具， 我们将确定Rbf1和Rbf2对基因表达的影响的差异，以及Rbf1和Rbf2在 染色质环境的调节。该项目将在密歇根州立大学的实验室中进行 作为全面学术和专业培训计划的一部分，为申请者做好准备 作为一名独立研究科学家，研究疾病背景下的基因调控。使用 随着工作的完成，结果将揭示Rb家族的基因调控机制 蛋白质，这将提高我们对为什么某些组织对失活特别敏感的理解 RB。了解Rb介导的基因调控机制及其作为转录抑制因子的活性 将允许开发基因疗法来治疗视网膜母细胞瘤和其他类型的癌症。

PROJECT SUMMARY/ABSTRACT Retinoblastoma is a rare pediatric cancer that is caused by loss of both copies of the retinoblastoma (Rb) tumor suppressor gene. Rb is a highly conserved and critical transcription factor for the proper regulation of gene expression in eukaryotes. Mutations in Rb have also been observed in many human cancers including breast and lung cancer. In humans, the Rb family comprises Rb, p107, and p130, which exhibit both overlapping and non-redundant roles in gene regulation. Similarly, the Drosophila lineage experienced a gene duplication event leading to the expression of the Rbf1 and Rbf2 paralogs. The significance of the multiplicity of the Rb family and their division of labor in regulating gene expression in different contexts is not clear. Their activity has been studied in the context of specific promoters; however, we lack deeper mechanistic understanding of these factors in the context of normal tissues, in development. This proposal will investigate the tissue-specific gene regulatory activities of retinoblastoma family proteins using the fly model system. We hypothesize that the Rb family has diversified to have gene-specific activities through differential targeting to promoters and through different repression activities. In Aim 1, we will investigate how Rb paralogs exhibit mechanistically different transcriptional control while in Aim 2 we will determine how regulation of the Rbf1 protein itself controls promoter-specific repression. We will use our newly developed method for highly precise targeting of Rb paralogs to diverse gene promoters through Rb fusions to a nuclease dead Cas9. This powerful tool allows for direct comparison of the Rb family members in a developmental system to study their gene- and tissue-specific activities. Using this tool, we will determine the differences between Rbf1 and Rbf2 impact on gene expression, and differences in modulation of the chromatin environment. This project will be carried out at Michigan State University in the lab of Dr. David Arnosti as part of a comprehensive academic and professional training plan to prepare the applicant for a career as an independent research scientist investigating gene regulation in the context of disease. With the completion of the proposed work, the results will uncover the mechanisms of gene regulation by Rb family proteins, which will improve our understanding of why certain tissues are especially sensitive to inactivation of Rb. Understanding the mechanisms of Rb-mediated gene regulation and its activity as a transcriptional repressor will allow for the development of gene therapies to treat Retinoblastoma and other cancer types.