Functional Architecture of Developmental Cis-Regulatory Modules

7.项目摘要/摘要 后生动物的发育、生理和进化都在很大程度上建立在转录活动的基础上。 顺式调节模块(CRM)，也称为增强剂。揭开了他们卓越的监管基础 因此，属性是生物科学的中心目标。虽然过去十年是一个值得注意的时代 发育增强子模块的研究进展，我们显然仍处于理解的初级阶段 到底增强子的各种转录因子(TF)输入是如何正确整合的，以产生一个新的 基因表达输出。我们在这里提出的研究计划就是为了解决这一根本问题。 直接出问题了。 具体目标1。研究保守的CRM语法成分的功能基础。 进化上保守的“语法元素”--两个不同的TF的相邻结合位点对--有 成为了解增强器模块非凡的综合能力的下一个前沿。我们 我已经确定了两个这样的元件，它们介导了Achaete/Scute神经蛋白的协同激活 和无毛的抑制因子，Notch信号通路的转导因子。我们将进行一项 旨在揭示生化和功能基础的一系列全面的体外和体内研究 他们的合作行动。 具体目标2.阐明复杂客户关系管理活动模式的机理基础。我们以前的 研究表明，果蝇基因组中存在大量的增强子模块，这些模块 驱动令人惊讶的空间和时间复杂性的表达模式。这一发现引发了几个问题 引人入胜的问题。这些复杂的模式是如何具体生成的？有没有一种逻辑联系在一起 这种模式的不同组成部分是什么？不同的子模式的生成依赖于不同的 增强子的亚基？我们将对选定的复合体进行详细的结构/功能研究 CRM，目的是测试不同的模型如何合成其输出模式。 具体目的3.研究增强子中一种新的保守的CRM的性质和功能 分裂基因复合体。我们最近发现了一种新的、高度保守的增强子模块 裂解复合体增强剂[E(SPL)-C]。它位于一个不同寻常的位置，并驱动着一个异常广泛的模式 报告基因的表达不同于复合体中任何其他CRM的表达。为以下目的提出的实验 具体目标3将详细研究该模块的功能，包括它所服务的耐人寻味的可能性 因此，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广谱基因的重要组成部分 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的输出，从而影响人类的表型。

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.