Molecular analysis of transcriptional repression

描述（由申请人提供）：果蝇胚盘胚胎提供了对真核转录调控机制的关键见解，特别是发育环境中基本生化过程的整合，其中差异基因表达用于驱动特定细胞和组织的发育命运。我们的研究集中在理解转录抑制因子在这种情况下的作用，因为模式生成的主要部分依赖于合子表达的gap基因抑制因子的队列。在过去的补助金期间，我们已经利用生物化学，遗传学和数学建模的方法来了解转录抑制因子的机制，识别和表征与这些蛋白质相互作用的辅抑制因子，并定量分析顺式调控代码，描述了这些蛋白质调控的增强子的结构。这些研究首次证明，短距离和长距离阻遏物在染色质水平上诱导了性质上非常不同的效应，并且同一组辅阻遏物可以根据上下文引起非常不同的效应。我们的测试和建模的短程阻遏物揭示了一些这些上下文的影响，提供了基础的解剖转录“语法”，解锁基因组调控信息。在我们的下一个资助期，我们提出了三个目标：1）通过内源性增强子的分析和建模来扩展我们对顺式调节密码的理解，2）在基因组水平上识别由阻遏物招募的多个辅阻遏物的活性和重要性，包括由机械上不同的短程和长程阻遏物造成的不同效应，和3）表征一种新型的辅阻遏物，一个死亡盒RNA解旋酶，我们已经确定为Knirps蛋白的辅因子的阻遏的重要性。这些综合目标的影响将是提供一个详细的机制和系统范围内的理解保守的转录调控过程，用于整个后生动物的发展。从这些研究中获得的见解将为高等真核生物的发育和疾病的种群和进化研究提供必要的基础。 公共卫生相关性：这项研究计划旨在了解编码在DNA中的基因调控的“语言”。通过确定作为“关闭开关”的蛋白质如何中断基因表达，我们将能够识别动物发育的基础过程，并辨别它们在疾病背景下如何被错误调节。此外，通过使用建模方法，我们将能够更好地解释破坏这些调节蛋白结合的DNA开关完整性的基因突变。

DESCRIPTION (provided by applicant): The Drosophila blastoderm embryo has provided key insights into eukaryotic transcriptional regulatory mechanisms, in particular the integration of basic biochemical processes in a developmental setting, where differential gene expression is used to drive the developmental fate of particular cells and tissues. Our research has focused on understanding the action of transcriptional repressors in this setting, because a major portion of the pattern generation relies on a cohort of zygotically expressed gap gene repressors. In the last grant period, we have utilized biochemical, genetic, and mathematical modeling approaches to understand the mechanisms of transcriptional repressors, to identify and characterize the corepressors that interact with these proteins, and quantitatively analyze the cis-regulatory code that describes the architecture of enhancers regulated by these proteins. These studies have demonstrated for the first time that short- and long-range repressors induce qualitatively very different effects at the chromatin level, and that the same set of corepressors can elicit very different effects depending on context. Our testing and modeling of short-range repressors revealed some of these contextual effects, providing the basis for a dissection of transcriptional "grammar" that unlocks genomic regulatory information. In our next funding period, we propose three aims to 1) extend our understanding of the cis-regulatory code through analysis and modeling of endogenous enhancers, 2) discern at a genomic level the activity and importance of multiple corepressors recruited by repressors, including distinct effects wrought by the mechanistically divergent short- and long-range repressors, and 3) characterize the importance in repression of a new type of corepressor, a DEAD box RNA helicase, which we have identified as a cofactor of the Knirps protein. The impact of these integrated aims will be to provide a detailed mechanistic and systems-wide understanding of conserved transcriptional regulatory processes that are used throughout metazoan development. The insights gained from these studies will provide essential underpinnings for population and evolutionary studies of development and disease in higher eukaryotes. PUBLIC HEALTH RELEVANCE: This research program seeks to understand the "language" of gene regulation that is encoded in the DNA. By determining how the proteins that serve as "off switches" act to interrupt gene expression, we will be able to identify processes that underlie animal development, and discern how they are misregulated in the context of disease. Furthermore, by use of modeling approaches, we will be able to better interpret genetic mutations that disrupt the integrity of the DNA switches to which these regulatory proteins bind.