Characterization of silencer element-associated chromatin

摘要 基因表达的时空调控是生物学中的一个基本过程。虽然增强剂的作用是 正向调节基因表达几十年来一直受到重视，明显缺乏消音器 从大多数模特那里。最近在多个系统中进行的一系列高通量筛查已经识别出数千个 消音器。引人注目的是，大量这样的消音器属于双功能顺式调节元件，也 在其他细胞类型或发育时期起增强剂的作用。消音器还富含eQTL和GWAs 这表明它们对于理解复杂疾病的遗传学是必不可少的。消声器是独一无二的 绝缘体，防止增强子-启动子相互作用。消音器主动抑制他们的表达 靶基因和增强子一样，不受位置和方向的影响。即使有大量的 消音器已经在功能上被鉴定，但仍然很难预测新的消音器元件。 而组蛋白后的增强子、启动子和绝缘体可以通过CHIP-SEQ进行预测。 翻译修饰，没有染色质修饰是已知的预测消音器。此外， 一组DNA结合蛋白及其在沉默时的辅助因子在很大程度上是未知的。CHIP-SEQ目标识别 消声器的高度预测性将极大地提高研究人员研究这些消声器的能力 在新的背景下的基本要素，并可以提供对沉默机制的洞察。为了解决这个问题 关于消音器的基本问题，我将进行一项蛋白质组调查，以确定 区分果蝇中胚层双功能顺式调节元件的抑制子和增强子状态。这 将给出对消音器元件的机制的关键见解。我还将调查三维 消音器与其目标之间形成的接触。已经观察到消音器受 Snail不接触tss，而是破坏正常的增强子-启动子接触，这一过程被称为反- 循环。该项目的完成将填补有关消声器的许多悬而未决的问题 因素，包括负责沉默的因素和它们对目标基因的影响。这 该项目将发现消音器的特定标记，使未来的调查人员能够通过芯片识别它们- SEQ类似于其他功能元素的注释方式。这项研究项目将给我们带来有价值的 在实验设计和分析方面的经验，我希望把这些经验带到我的学术生涯中去 调查员。

Abstract Spatio-temporal control of gene expression is a fundamental process in biology. While the role of enhancers to positively regulate gene expression has been appreciated for decades, silencers are conspicuously absent from most models. A recent string of high-throughput screens in multiple systems has identified thousands of silencers. Strikingly, a large number of these silencers fall within bifunctional cis-regulatory elements that also act as enhancers in other cell types or developmental times. Silencers are also enriched for eQTLs and GWAS hits, suggesting they are essential for understanding the genetics of complex disease. Silencers are distinct from insulators, which prevent enhancer-promoter interactions. Silencers actively dampen expression of their target genes and, like enhancers, act independently of position and orientation. Even though a large number of silencers have been functionally identified in reporter assays, it is still difficult to predict new silencer elements. While enhancers, promoters, and insulators can be predicted by ChIP-seq for specific histone post- translational modifications, no chromatin modifications are known to be predictive of silencers. Additionally, the set of DNA binding proteins and their cofactors at silencers is largely unknown. Identification of ChIP-seq target that are highly predictive of silencers would vastly improve the ability of researchers to study these fundamental elements in new contexts and can give insights into silencing mechanisms. In order to address fundamental lingering questions regarding silencers, I will perform a proteomic survey to identify factors that distinguish silencer and enhancer states of bifunctional cis-regulatory elements in Drosophila mesoderm. This will give key insights into the mechanisms of silencer elements. I will also investigate the three-dimensional contacts formed between silencers and their targets. It has already been observed that silencers bound by Snail do not contact TSSs, but instead disrupt normal enhancer-promoter contacts, a process known as anti- looping. Completion of this project will fill address many of the outstanding questions regarding silencer elements, including the factors responsible for silencing and the effect they have on their target genes. This project will uncover specific markers of silencers that will allow future investigators to identify them via ChIP- seq similar to how other functional elements can be annotated. This research project will give valuable experience in experimental design and analysis that I hope to carry with me into a career as an academic investigator.