Developmental gene expression through coordinate action of cis-regulatory modules

描述（由申请人提供）：使用果蝇胚胎作为模型系统，我们有特殊的机会来研究多个顺式调控模块（CRM）如何合作，以支持时空调控的基因表达在发展过程中。在由父母资助的研究中，我们发现，背侧转录因子，这是有助于图案的背腹（DV）轴，表现出动态，因为它的靶基因。在每个核周期之间以及每个核周期内，都观察到Dorsal及其靶基因水平的显着变化，大约为分钟。染色质免疫沉淀实验也进行了检查在体内DNA占用的转录因子，重要的胚胎图案。这些实验表明，胚胎中的许多基因受到成对的同时活跃的顺式调控模块（CRM）的调控，这些模块以相似的时空模式驱动表达。这里提出的实验旨在了解为什么以及如何多个CRM协调控制果蝇胚胎中的时空基因表达，特别是，提供深入了解如何转移活动从一个CRM到下一个被调节，以支持发展进程。我们将利用丰富的背景信息和我们的知识DV模式，以帮助指导选择特别相关的顺式调控系统的研究。许多分子和遗传学工具可用于支持果蝇中的这些研究，包括遗传方法的简便性以及通过重组工程操纵大型转基因的能力，这有助于在天然环境中对CRM和其他调控序列进行功能测定。此外，我们将使用标准技术（3C）以及我们正在开发的一种新的成像方法来研究染色质构象对基因表达的影响，以深入了解特定CRM何时以及如何与时间和空间分辨率的启动子相互作用。在这个更新申请中，我们提出了三个具体的目标，这将大大促进我们对CRM如何协调支持发育基因表达的理解：目标1 -分析从一个CRM到下一个CRM的过渡如何支持连续的基因表达。目的2 -研究基因表达动态、边界和水平之间的关系。目的3 -以细胞为基础在体内测定染色质构象。基因的表达在时间和空间上都受到严格的控制，这是正常发育所必需的。由于大约三分之一的所有标准物质控制背腹图案可能作为“坐标对”，我们认为，多个标准物质的整合功能的基因表达的共同调节可能是顺式调节控制的一般机制，这是刚刚开始曝光。分裂 联合影响基因表达的跨多个模块的顺式调节信息可以提供输出的灵活性，这可能是有利的，特别是对于存在遗传扰动和/或变化的环境条件的发育中的胚胎。所有动物的基因调控机制的保守性保证了这些研究将具有深远的意义。

DESCRIPTION (provided by applicant): Using the Drosophila embryo as a model system, we have the exceptional opportunity to investigate how multiple cis-regulatory modules (CRMs) cooperate to support spatiotemporally-regulated gene expression during the course of development. In studies supported by the parent grant, we found that the Dorsal transcription factor, which is instrumental for patterning the dorsal-ventral (DV) axis, exhibits dynamics as do its target genes. Significant changes in levels of Dorsal and its target genes were observed both between as well as within each nuclear cycle, on the order of minutes. Chromatin immunoprecipitation experiments were also conducted to examine in vivo DNA occupancy by transcription factors, important for embryonic patterning. These experiments showed that many genes in the embryo are regulated by pairs of concurrently active cis-regulatory modules (CRMs) that drive expression in similar spatiotemporal patterns. The experiments proposed here aim to understand why and how multiple CRMs coordinate to control spatiotemporal gene expression in the Drosophila embryo, and, in particular, to provide insight into how transfer of activity from one CRM to the next is regulated to support developmental progression. We will capitalize on ample background information and our knowledge of DV patterning to help guide choice of particularly relevant cis-regulatory systems for study. Many molecular and genetic tools are available to support these studies in Drosophila, including ease of genetic approaches as well as the ability to manipulate large transgenes through recombineering, that facilitate functional assays of CRMs and other regulatory sequences in native context. Also, we will investigate the impact of chromatin conformation on gene expression using standard techniques (3C) as well as a novel imaging approach we are developing, to provide insight into when and how particular CRMs interact with the promoter with temporal and spatial resolution. Here in this renewal application we propose three specific aims that will significantly advance our understanding of how CRM coordinate to support developmental gene expression: Aim 1 - To analyze how transition from one CRM to the next supports continuous gene expression. Aim 2 - To investigate the relationship between gene expression dynamics, boundaries, and levels. Aim 3 - To assay chromatin conformation in vivo on a cell-by-cell basis. Carefully timed and spatially controlled expression of genes is required for normal development to proceed. As roughly a third of all CRMs controlling dorsal-ventral patterning may function as 'coordinate pairs', we argue that co-regulation of gene expression by integrative function of multiple CRMs is likely a general mechanism of cis-regulatory control; one that is just beginning to come to light. Splitting cis-regulatory information across multiple modules that jointly influence gene expression may provide flexibility of output that can be advantageous, especially for the developing embryo that is presented with genetic perturbation and/or changing environmental conditions. The conservation of gene regulatory mechanisms across all animals promises that these studies will have far reaching implications.