Characterization of silencer element-associated chromatin

沉默元件相关染色质的表征

基本信息

批准号：
10561612
负责人：
Julian Andrew Segert
金额：
$ 3.55万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-03-01 至 2025-02-28
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10561612
关键词：
3-Dimensional Address Affinity Architecture Attention Binding Binding Proteins Biological Assay Biology Cell Separation Cellular Assay ChIP-seq Characteristics Chemicals Chromatin Collection Complex DNA DNA Binding DNA-Binding Proteins Data Development Developmental Process Disease Drosophila genus Elements Embryo Enhancers Ensure Epigenetic Process Experimental Designs Fluorescent in Situ Hybridization Future Gene Expression Gene Expression Regulation Genes Genetic Genetic Transcription Genome Genomics Histone Code Histones Investigation Label Maps Mass Spectrum Analysis Mesoderm Methods Modeling Modification Molecular Conformation Nature Pathway interactions Positioning Attribute Post-Translational Protein Processing Process Proteomics Regulation Regulatory Element Reporter Repressor Proteins Research Research Personnel Research Project Grants Role Snails Statistical Models Surveys System Techniques Testing Transcriptional Silencer Elements Validation Variant Visualization Western Blotting career cell type chromatin modification cofactor combinatorial design experience experimental analysis falls genome wide association study high throughput screening histone modification human disease improved insight morphogens mutant prevent promoter protein protein interaction recruit spatiotemporal tool transcription factor

项目摘要

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.

抽象的基因表达的时空控制是生物学的基本过程。虽然增强剂的作用正向调节基因表达几十年来一直受到重视，但沉默子却明显缺失大多数型号。最近在多个系统中进行的一系列高通量筛选已经识别出数千个消音器。引人注目的是，这些消音器中的大量属于双功能顺式调节元件，这些元件也在其他细胞类型或发育时期充当增强子。沉默子也针对 eQTL 和 GWAS 进行了丰富表明它们对于理解复杂疾病的遗传学至关重要。消音器各有特色来自绝缘体，它阻止增强子-启动子相互作用。消音器主动抑制他们的表达靶基因，与增强子一样，其作用独立于位置和方向。尽管大量消音器已在报告基因检测中进行了功能鉴定，但仍很难预测新的消音器元件。虽然可以通过 ChIP-seq 预测特定组蛋白后的增强子、启动子和绝缘子翻译修饰，已知没有染色质修饰可以预测沉默子。此外，一组 DNA 结合蛋白及其沉默子的辅助因子在很大程度上是未知的。 ChIP-seq 靶标的鉴定高度预测消音器将极大地提高研究人员研究这些消音器的能力新环境中的基本要素，可以深入了解沉默机制。为了解决关于消音器的基本挥之不去的问题，我将进行蛋白质组学调查，以确定影响消音器的因素区分果蝇中胚层双功能顺式调节元件的沉默子和增强子状态。这将为消音器元件的机制提供重要见解。我也会研究一下三维消音器与其目标之间形成接触。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，这一过程称为抗- 循环播放。该项目的完成将解决有关消音器的许多悬而未决的问题元素，包括导致沉默的因素及其对靶基因的影响。这项目将发现消音器的特定标记，使未来的研究人员能够通过 ChIP 识别它们 seq 类似于其他功能元素的注释方式。该研究项目将提供宝贵的我希望将实验设计和分析方面的经验带入我的学术生涯研究者。