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

总结 基因表达在空间和时间上的严格调节对于哺乳动物的发育和体内平衡是必要的。 多细胞生物基因编码序列以外的基因组区域（顺式调控模块，或 CRMs/增强子）作为转录因子的组装平台，促进基因在细胞中的表达。 对细胞或环境线索的反应。虽然许多基因受多种CRM的调控， 协调这些CRM的作用并调节其局部染色质动力学的机制是 不太了解。在果蝇中，转录因子Brinker（Brk） 在早期胚胎中被两个远端CRM激活，一个在brk基因的5'端，一个在3'端。最初被认为是 多余的，这些标准物质被发现驱动顺序，部分重叠的表达模式，沿沿着 胚背腹轴此外，这些CRM依赖于启动子近端元件（PPE），其 似乎促进了每个CRM与启动子的连续的、长距离的相互作用。我们有额外 这表明brk PPE是brk在其他几种组织中表达所必需的。我们假设这 位于果蝇brk基因上游的PPE代表了一种协调 多个顺式调节模块（CRMs）与启动子的相互作用，并且这种局部协调 染色质动力学对于适当的基因表达、发育和稳态的维持是重要的。 为了验证这一假设，我们提出了以下实验方向：目标1将测试的想法，brk PPE管理brk基因位点的染色质构象; Aim 2将识别分子效应子 支持brk PPE作用;目标3将研究PPE结合蛋白Odd配对（Opa）在 支持除brk之外的其它基因座处的全局CRM-启动子相互作用。许多基因跨越不同的分类群 是由多种标准物质-包括所谓的超级，拉伸或阴影增强剂-但我们知道非常 关于这些不同的调节作用在正常发育过程中是如何协调的，我们知之甚少。见解 将来自于对已经很好表征的基因的研究，如brk，其表达取决于 通过启动子近端元件的CRM协调;以及通过染色质的全基因组测定 构象，以揭示调控CRM/增强子-启动子相互作用的机制。新 实验方法，允许靶向操作和直接观察活体染色质， 一个完整的生物体的分化细胞，结合久经考验的基因分析技术， 果蝇中的发育现象可以提供CRM-启动子相互作用与 转录因子结合对基因表达调控的贡献。因为很多基因， 果蝇和高等生物之间共享的途径和调节机制， 了解果蝇复杂基因座的基因调控是如何协调的，可能会为新的研究方法提供信息。 以了解野生型以及与疾病相关的人类环境中的这些现象。

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.