Deciphering the contribution of enhancer transcription to enhancer function

项目摘要/摘要 申请者要求获得Ruth L.Kirschstein国家研究服务奖两年的支持 个人博士前奖学金以促进健康相关研究的多样性(F31-多样性)，以审查 增强子转录与增强子功能的关系。增强剂已被证明能启动 短的非编码转录本在数量上与启动子相媲美，但它们在增强子功能上的作用仍然存在 有争议的。我的初步数据表明，增强剂和启动子同步他们的活动，形成联合- 受管制的领域。这一结果令人惊讶，因为之前使用总RNA测量的研究已经 表明增强子eRNAs在其相关的mRNAs之前倾斜，这表明增强子获得 首先激活，然后将该激活传送给相关联的发起人。共调控结构域 解释教科书中的增强子功能模型未能解释的几种机制。其中包括 增强子序列与启动子没有区别，因为它们被RNA结合并启动转录 聚合酶II，增强子和启动子在细胞核中形成枢纽，增强子以添加剂或 协同方式，以及在敲除多个增强子中的单个增强子时观察到的冗余 轨迹。这项研究的目标是检验增强剂可能通过以下方式发挥作用的假设 同时激活CRD内的转录起始点，并相互加强。这一假设 这意味着增强子转录对于增强子功能是必不可少的。我将在三个月内检验这一假设 具体目标：(1)确定跨信号的增强子-启动子共调节域的共性 反应，以揭示共调控结构域的形成是否是一种一般的调控机制 潜在的信号反应(2)决定了增强子转录和增强子功能之间的关系 在它们的天然基因组环境之外，来研究增强子转录和 增强子在其天然基因组环境之外的功能，以及(3)决定沉默增强子的影响 转录对体内基因激活的影响，以确定关闭增强子转录的影响 关于其天然基因组背景下的增强子功能。这次培训将使我能够(1)发展这些领域的技能 使用下一代测序和分子生物学进行研究设计、分析和解释 技术；(2)学习分子、染色质和增强子生物学的基础；(3)成功防御 论文；以及(4)获得竞争激烈的博士后奖学金，长期目标是成为一名 在一所研究密集的大学里，成功的、独立资助的科学家。

PROJECT SUMMARY/ABSTRACT The applicant is requesting two years of support from a Ruth L. Kirschstein National Research Service Award for Individual Predoctoral Fellowship to Promote Diversity in Health-Related Research (F31-Diversity) to examine the relationship between enhancer transcription and enhancer function. Enhancers have been shown to initiate short, non-coding transcripts in quantities rivaling promoters, yet their role on enhancer function remains controversial. My preliminary data suggests that enhancers and promoters synchronize their activity to form co- regulated domains. This result is surprising given that previous studies using total RNA measurements have indicated that enhancer eRNAs ramp up before their associated mRNAs, suggesting that enhancers get activated first and that this activation then gets transmitted to the associated promoters. Co-regulated domains explain several of the mechanisms that the textbook model of enhancer function fails to. These include that enhancer sequences are indistinguishable from promoters, that they are bound and initiate transcription by RNA polymerase II, that enhancers and promoters form hubs in the nucleus, that enhancers act in an additive or synergistic manner, and the redundancies observed when knocking out single enhancers in a multi-enhancer locus. The goal of this research is to test the hypothesis that enhancers may function by serving as simultaneously activated and mutually reinforcing transcription initiation sites within CRDs. This hypothesis implies that enhancer transcription would be essential for enhancer function. I will test this hypothesis in three specific aims: (1) determine the generality of enhancer-promoter co-regulatory domains across signaling responses, to uncover whether the formation of co-regulated domains are a general regulatory mechanism underlying signal response (2) determine the relationship between enhancer transcription and enhancer function outside of their native genomic environment, to examine the relationship between enhancer transcription and enhancer function outside of their native genomic context, and (3) determine the impact of silencing enhancer transcription on gene activation in vivo, to determine the influence that shutting down enhancer transcription ahs on enhancer function in its native genomic context. This training will allow me to (1) develop skills in the areas of research design, analysis and interpretation using next-generation sequencing and molecular biology techniques; (2) learn the fundamentals of molecular, chromatin, and enhancer biology; (3) successfully defend a dissertation; and (4) obtain a competitive post-doctoral fellowship with the long-term goal to become a successful, independently-funded scientist at a research-intensive university.