Developmental gene expression through coordinate action of cis-regulatory modules

通过顺式调控模块的协调作用发育基因表达

• 批准号: 8854095
• 负责人: Angelike Stathopoulos
• 金额: $ 31.64万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-03-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8854095
• 关键词: Animals; Biological Assay; Biological Models; Cells; ChIP-seq; Chromatin; DNA; Data; Development; Developmental Gene; Disease; Dorsal; Drosophila genus; Ectoderm; Embryo; Embryonic Development; Enhancers; Exhibits; Gene Expression; Gene Expression Regulation; Gene Targeting; Genes; Genetic; Genetic Transcription; Genome; Genomics; Goals; Health; Heart; Image; In Vitro; Knowledge; Lead; Mesoderm; Molecular Conformation; Molecular Genetics; Nuclear; Organogenesis; Output; Pattern; Regulation; Regulator Genes; Research; Resolution; Sampling; Site; Snails; System; Techniques; Testing; Time; Transgenes; Untranslated RNA; base; chromatin immunoprecipitation; dorsal proteins; environmental change; flexibility; gene conservation; genetic approach; in vivo; insight; novel; parent grant; promoter; research study; spatiotemporal; tool; transcription factor

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.

描述(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，我们发现帮助形成背腹(DV)轴模式的背部转录因子和它的目标基因一样显示出动态。在每一个核循环之间以及在每个核周期内，背部及其靶基因的水平都观察到了在几分钟的量级上的显著变化。染色质免疫沉淀实验也被用来检测体内DNA被转录因子占据的情况，转录因子对胚胎构型很重要。这些实验表明，胚胎中的许多基因受到一对同时活动的顺式调控模块(CRM)的调控，这些模块以类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，特别是提供关于如何调节从一个CRM到下一个CRM的活动以支持发育进展的见解。我们将利用丰富的背景信息和我们对DV模式的知识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在果蝇中进行这些研究，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，这有助于在自然环境中对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，以深入了解特定的CRM何时以及如何以时间和空间分辨率与启动子相互作用。在这份续订申请中，我们提出了三个具体目标，它们将显著促进我们对CRM如何协调以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的过渡如何支持持续的基因表达。目的2-研究基因表达动力学、边界和水平之间的关系。目的3-在活体细胞的基础上分析染色质构象。正常发育需要经过仔细的时间和空间控制的基因表达。由于控制背腹模式的CRM大约有三分之一可能起到‘坐标对’的作用，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控的一种一般机制；这一机制才刚刚开始被发现。拆分 跨多个模块的顺式调控信息共同影响基因表达，可能提供输出的灵活性，这可能是有利的，特别是对于面临遗传扰动和/或不断变化的环境条件的发育中的胚胎。保护所有动物的基因调控机制保证了这些研究将具有深远的影响。