Molecular analysis of transcriptional repression

描述（由申请人提供）：果蝇囊胚胚胎为真核生物转录调控机制提供了关键见解，特别是在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。我们的研究重点是了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间隙基因抑制因子队列。在上一个资助期，我们利用生化、遗传和数学建模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，并定量分析描述这些蛋白质调节的增强子结构的顺式调控代码。这些研究首次证明了短链和长链阻遏子在染色质水平上诱导的质性非常不同的效应，并且同一组协阻遏子可以根据环境引起非常不同的效应。我们对短程阻遏子的测试和建模揭示了其中的一些环境效应，为解开基因组调控信息的转录“语法”解剖提供了基础。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控代码的理解；2)在基因组水平上辨别由阻遏子募集的多个协阻遏子的活性和重要性，包括由机制上不同的短和远程阻遏子所产生的不同影响；3)表征新型协阻遏子——DEAD盒RNA解旋酶在抑制中的重要性。我们已经确定它是Knirps蛋白的辅助因子。这些综合目标的影响将是提供在后生动物发育过程中使用的保守转录调控过程的详细机制和系统范围的理解。从这些研究中获得的见解将为高等真核生物的发展和疾病的种群和进化研究提供必要的基础。

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.