Regulation of long distance enhancer-promoter interactions by promoter-proximal elements

启动子-近端元件对长距离增强子-启动子相互作用的调节

• 批准号: 10688129
• 负责人: Angelike Stathopoulos
• 金额: $ 36.21万
• 依托单位: CALIFORNIA INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-19 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10688129
• 关键词: 3-Dimensional; Affect; Architecture; Binding; Binding Proteins; Biological Assay; Cell Differentiation process; Cells; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Code; Complex; Coupled; Cues; DNA; DNA Binding; Data; Development; Disease; Distal; Drosophila genus; Drosophila melanogaster; Dysplasia; Elements; Embryo; Enhancers; Eukaryotic Cell; Event; Gene Expression; Gene Expression Regulation; Genes; Genome; Genomics; Homeostasis; Human; Hybrids; Image; Link; Maintenance; Molecular; Molecular Conformation; Mutagenesis; Mutation; Organism; Ovary; Pathway interactions; Pattern; Phenotype; RNA Interference; Regulation; Reporter; Research; Role; Stretching; System; Techniques; Testing; Time; Tissues; Untranslated RNA; chromatin immunoprecipitation; combinatorial; fly; genetic analysis; genomic locus; imaging approach; improved; in vivo; insight; mutant; novel strategies; promoter; response; transcription factor; whole genome

SUMMARY Tight regulation of gene expression in space and time is necessary for development and homeostasis in multicellular organisms. Regions of the genome outside gene coding sequences (cis-regulatory modules, or CRMs/enhancers) serve as assembly platforms for transcription factors that facilitate gene expression in response to cellular or environmental cues. Although many genes are regulated by multiple CRMs, mechanisms that coordinate the action of these CRMs and that regulate their local chromatin dynamics are poorly understood. In the fruit fly Drosophila melanogaster, expression of the transcription factor Brinker (Brk) is activated in the early embryo by two distal CRMs, one 5’ and one 3’ to the brk gene. Initially thought to be redundant, these CRMs were found to drive sequential, partially overlapping patterns of expression along the embryonic dorso-ventral axis. Further, these CRMs depend on a promoter-proximal element (PPE) that appears to facilitate the sequential, long-range interaction of each CRM with the promoter. We have additional evidence that the brk PPE is required for brk expression in several other tissues. We hypothesize that this PPE located upstream of the brk gene in Drosophila represents a general mechanism for coordinating multiple cis-regulatory modules’ (CRMs’) interaction with the promoter, and that this coordination of local chromatin dynamics is important for proper gene expression, development and maintenance of homeostasis. To test this hypothesis, we propose the following experimental directions: Aim 1 will test the idea that the brk PPE manages chromatin conformation at the brk gene locus; Aim 2 will identify molecular effectors supporting brk PPE action; and Aim 3 will investigate a role for one PPE-binding protein Odd paired (Opa) in supporting global CRM-promoter interactions at other loci in addition to brk. Many genes across diverse taxa are regulated by multiple CRMs – including so-called super, stretch or shadow enhancers – yet we know very little about how these various regulatory contributions are coordinated during normal development. Insights will come from the study of already well-characterized genes such as brk, whose expression depends on CRM coordination by a promoter proximal element; as well as through whole genome assays of chromatin conformation to uncover the mechanisms regulating CRM/enhancer-promoter interactions, in general. New experimental approaches, which permit targeted manipulation and direct observation of chromatin in live, differentiated cells of an intact organism, combined with tried-and-true techniques for the analysis of genetic and developmental phenomena in Drosophila can provide a link between CRM-promoter interaction and the contributions of transcription factor binding to the regulation of gene expression. Because many genes, pathways and regulatory mechanisms are shared between Drosophila and higher organisms, improved understanding of how gene regulation is coordinated at complex loci in flies is likely to inform new approaches to understand these phenomena in wild-type as well as disease-relevant human contexts.

概括 基因表达在空间和时间上的严格调控对于发育和体内平衡是必要的。 多细胞生物。基因编码序列之外的基因组区域（顺式调控模块，或 CRM/增强子）作为转录因子的组装平台，促进基因表达 对细胞或环境线索的反应。尽管许多基因受到多种 CRM 的调控， 协调这些 CRM 的作用并调节其局部染色质动态的机制是 不太了解。在果蝇果蝇中，转录因子 Brinker (Brk) 的表达 在早期胚胎中由两个远端 CRM 激活，一个位于 brk 基因的 5' 端，一个位于 3' 端。最初以为是 冗余，这些 CRM 被发现可以驱动顺序的、部分重叠的表达模式 胚胎背腹轴。此外，这些 CRM 依赖于启动子近端元件 (PPE)， 似乎促进了每个 CRM 与启动子的顺序、远程相互作用。我们还有额外的 有证据表明，brk PPE 是其他几种组织中 brk 表达所必需的。我们假设这 果蝇中位于 brk 基因上游的 PPE 代表了协调的一般机制 多个顺式调节模块（CRM）与启动子的相互作用，并且本地的这种协调 染色质动力学对于基因的正确表达、发育和体内平衡的维持非常重要。 为了检验这一假设，我们提出以下实验方向：目标 1 将检验 brk PPE 管理 brk 基因位点的染色质构象；目标 2 将识别分子效应器 支持brk个人防护装备行动；目标 3 将研究一种 PPE 结合蛋白奇数配对 (Opa) 在 支持除 brk 之外的其他位点的全球 CRM 启动子互动。许多基因跨越不同的分类群 受到多种 CRM 的监管——包括所谓的超级、拉伸或阴影增强剂——但我们非常了解 关于在正常开发过程中如何协调这些不同的监管贡献却知之甚少。见解 将来自对已经充分表征的基因（例如 brk）的研究，其表达取决于 由启动子近端元件进行的 CRM 协调；以及通过染色质的全基因组分析 一般来说，揭示调节 CRM/增强子-启动子相互作用的机制。新的 实验方法，允许有针对性地操作和直接观察活体中的染色质， 完整生物体的分化细胞，结合经过验证的遗传分析技术 果蝇的发育现象可以提供 CRM 启动子相互作用和 转录因子结合对基因表达调节的贡献。因为很多基因， 果蝇和高等生物之间共享途径和调节机制，改进 了解果蝇复杂基因座的基因调控如何协调可能会为新方法提供信息 了解野生型以及与疾病相关的人类环境中的这些现象。