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，我们有机会研究多个顺式调控模块（crm）如何在发育过程中协同支持时空调控的基因表达。在父母资助的研究中，我们发现背侧转录因子，这是对背腹轴（DV）模式的工具，与其靶基因一样表现出动态。在每个核周期之间以及在每个核周期内观察到Dorsal及其靶基因水平的显著变化，以分钟为单位。染色质免疫沉淀实验也用于检测转录因子在体内的DNA占用，这对胚胎模式的形成很重要。这些实验表明，胚胎中的许多基因受到一对同时活跃的顺式调控模块（CRMs）的调控，这些模块以相似的时空模式驱动表达。本文提出的实验旨在了解多个CRM协调控制果蝇胚胎中时空基因表达的原因和方式，特别是提供如何调节从一个CRM到下一个CRM的活动转移以支持发育进程的见解。我们将利用丰富的背景信息和我们对DV模式的了解来帮助指导选择特别相关的顺式调控系统进行研究。许多分子和遗传工具可用于支持果蝇的这些研究，包括易于使用的遗传方法以及通过重组操作大型转基因的能力，这有助于在天然环境下对crm和其他调控序列进行功能分析。此外，我们将使用标准技术（3C）以及我们正在开发的新型成像方法来研究染色质构象对基因表达的影响，以提供特定crm何时以及如何与时间和空间分辨率的启动子相互作用的见解。在此更新申请中，我们提出了三个具体目标，这些目标将显著推进我们对CRM如何协调支持发育性基因表达的理解：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 -在细胞的基础上测定体内染色质构象。正常发育的进行需要精心控制基因的时间和空间表达。由于大约三分之一的控制背-腹侧模式的crm可以作为“坐标对”，我们认为通过多个crm的整合功能共同调节基因表达可能是顺式调节控制的一般机制；一个刚刚开始浮出水面的问题。分裂

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.