Functional Architecture of Developmental Cis-Regulatory Modules

7.项目总结/摘要 后生动物的发育、生理和进化都在很大程度上依赖于转录因子的活动。 顺式调节模块（CRM），也称为增强子。揭开了他们卓越的监管基础 因此，性质是生物科学的中心目标。虽然过去十年是一个非凡的时代， 尽管我们对发育增强子模块的研究取得了进展，但我们显然仍处于理解的初级阶段。 增强子的各种转录因子（TF）输入如何正确整合以产生新的转录因子。 基因表达输出。我们在这里提出的研究计划旨在解决这一基本问题 问题直接。 具体目标1。调查保留的CRM语法元素的功能基础。 进化上保守的“语法元素”--两个不同TF的相邻结合位点对-- 成为理解增强子模块非凡整合能力的下一个前沿。我们 我已经鉴定了两种这样的元件，它们介导Achaete/Scute前神经蛋白的协同激活 和无毛抑制因子，Notch信号通路的转导TF。我们将进行 一系列全面的体外和体内研究，旨在阐明生物化学和功能基础 他们的合作行动。 具体目标2。阐明复杂CRM活动模式的机制基础。我们以前的 研究揭示了果蝇基因组中存在大量增强子模块， 驱动着令人惊讶的时空复杂性的表达模式。这一发现引发了几个 有趣的问题这些复杂的模式是如何具体产生的？有没有一种逻辑 这种模式的不同组成部分？不同的子模式是否依赖于不同的 增强子的子元素我们将对选定的复合物进行详细的结构/功能研究， CRM，目标是测试不同的模型如何合成其输出模式。 具体目标3。研究增强子中新的保守CRM的性质和功能 分裂基因复合体我们最近发现了一个新的和高度保守的增强子模块， 分裂复合物增强子[E（spl）-C]。它位于一个不寻常的位置，并驱动一个异常广泛的模式， 报告基因的表达，这是不同于任何其他CRM的复杂。建议的实验 具体目标3将详细研究这个模块的功能，包括它服务的有趣的可能性 这可能有助于解释E（spl）-C的长期进化稳定性。 转录调控序列是人类基因组中广泛的基因表达的重要贡献者。 疾病状态。通过深入研究增强子特异性的机制基础，我们的工作将有助于 阐明调控序列变异如何影响增强子输出并因此影响人类表型。

7. Project Summary/Abstract Metazoan development, physiology, and evolution are all heavily founded on the activities of transcriptional cis-regulatory modules (CRMs), also known as enhancers. Unraveling the basis of their remarkable regulatory properties is thus a central goal of biological science. While the past decade has been an era of remarkable progress in the study of developmental enhancer modules, we are clearly still in our infancy in understanding exactly how an enhancer's various transcription factor (TF) inputs are properly integrated to generate a novel gene expression output. The research program we propose here is designed to address this fundamental problem directly. Specific Aim 1. Investigate the functional basis for conserved CRM grammar elements. Evolutionarily conserved "grammar elements" — pairs of adjacent binding sites for two different TFs — have emerged as the next frontier in understanding the extraordinary integrative capacity of enhancer modules. We have identified two such elements that mediate synergistic activation by the Achaete/Scute proneural proteins and Suppressor of Hairless, the transducing TF for the Notch signaling pathway. We will conduct a comprehensive series of in vitro and in vivo studies designed to eludicate the biochemical and functional basis of their cooperative action. Specific Aim 2. Elucidate the mechanistic basis of complex CRM activity patterns. Our previous studies have revealed the existence in the Drosophila genome of a large number of enhancer modules that drive expression patterns of surprising spatial and temporal complexity. This discovery raises several intriguing questions. How specifically are these complex patterns generated? Is there a logic that ties together the different components of such a pattern? Do the different subpatterns rely for their generation on distinct subelements of the enhancer? We will carry out detailed structure/function studies of selected sets of complex CRMs, with the goal of testing different models for how they synthesize their output patterns. Specific Aim 3. Investigate the nature and function of a novel conserved CRM in the Enhancer of split gene complex. We have recently identified a novel and deeply conserved enhancer module in the Enhancer of split complex [E(spl)-C]. It lies in an unusual location, and drives an exceptionally broad pattern of reporter gene expression that is unlike that of any other CRM in the Complex. The experiments proposed for Specific Aim 3 will investigate this module's function in detail, including the intriguing possibility that it serves more than one gene, and thus may help explain the long-term evolutionary stability of the E(spl)-C. Transcriptional regulatory sequences are critically important contributors to a broad spectrum of human disease states. By delving deeply into the mechanistic basis of enhancer specificity, our work will help illuminate how regulatory sequence variation affects enhancer output and hence human phenotype.