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

摘要 在空间和时间上对基因表达进行严格调控是发育和动态平衡所必需的 多细胞生物。基因编码序列外的基因组区域(顺式调控模块，或 CRM/增强子)作为转录因子的组装平台，促进基因在 对细胞或环境提示的反应。尽管许多基因受到多个CRM的调控， 协调这些CRM的活动并调节其局部染色质动态的机制是 人们对此知之甚少。转录因子Brinker(BRK)在果蝇中的表达 在早期胚胎中被BRK基因的两个远端CRM激活，一个是BRK基因的5‘和一个是3’。最初被认为是 冗余的，这些CRM被发现驱动顺序的，部分重叠的表达模式沿着 胚胎背腹轴。此外，这些CRM依赖于启动子-近端元件(PPE)，该元件 似乎促进了每个CRM与启动者的顺序、远程互动。我们还有额外的 有证据表明BRK PPE是BRK在其他几个组织中表达所必需的。我们假设这是 位于果蝇BRK基因上游的PPE代表了一种普遍的协调机制 多个顺式调节模块(CRM‘)与启动子的相互作用，以及这种局部的协调 染色质动态对于适当的基因表达、发育和维持动态平衡是很重要的。 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BRK PPE管理BRK基因位点的染色质构象；Aim 2将识别分子效应器 支持BRK PPE活动；Aim 3将研究一种PPE结合蛋白Odd配对(OpA)在 除BRK外，还支持全球CRM-启动子在其他基因座的相互作用。不同类群中的许多基因 由多个CRM管理--包括所谓的超级、拉伸或阴影增强器--但我们非常了解 几乎没有关于这些不同的监管贡献在正常发展期间是如何协调的。真知灼见 将来自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以及通过染色质的全基因组分析来协调CRM 整合以揭示总体上调节CRM/增强子-启动子相互作用的机制。新的 实验方法，允许有针对性地操纵和直接观察活体中的染色质， 完整有机体的分化细胞，结合久经考验的遗传分析技术 果蝇中的发育现象可以在CRM-启动子相互作用和 转录因子结合对基因表达调控的贡献。因为很多基因， 果蝇和高等生物共有的途径和调节机制，经过改进 对果蝇基因调控如何在复杂的位点上协调的理解可能会为新的方法提供依据 在野生型和与疾病相关的人类环境中理解这些现象。

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.