Visualizing mechanisms at the intersection of chromatin, transcription, and epigenetics

染色质、转录和表观遗传学交叉点的可视化机制

基本信息

批准号：
10685736
负责人：
Lucas Farnung
金额：
$ 152.55万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-09-19 至 2026-08-31
项目状态：
未结题

项目摘要

PROJECT SUMMARY Transcription of protein-coding genes governs cell identity and fate through cell specific gene expression programs. During transcription, RNA polymerase II must traverse through its native chromatin template. Chromatin and its fundamental unit, the nucleosome, impose a significant hurdle to nuclear processes such as transcription. Importantly, the transcription machinery must not only traverse the genome without disrupting chromatin organization, but transcriptional activity is also epigenetically regulated through post-translational modifications of histone proteins. Often these processes are embedded in elaborate feedback loops where chromatin architecture, transcription, and epigenetics regulate each other. These regulatory mechanisms play a pivotal for gene expression control and dysregulation of gene expression often results in the emergence of cancers. The biochemical and structural foundation for the crosstalk between the transcription machinery, chromatin, and epigenetics, however, remains poorly understood, partly because of the complexity of cellular systems and limitations in experimental approaches. The goal of this proposal is to overcome our lack of understanding of gene expression regulation by using an integrated reconstitution strategy to study chromatin organization, transcription, and epigenetics in parallel. Here, I propose the development of a combinatorial biochemical and structural approach termed visual biochemistry. Visual biochemistry combines a fully reconstituted chromatin transcription system and time-resolved single-particle cryogenic electron microscopy. Our work has revealed that visual biochemistry can be successfully employed to understand how nucleosomes and epigenetic information are retained during transcription. Our proposed research will identify the structural basis of how the transcription machinery transcribes higher-order chromatin in a native-like environement (Aim 1). Coupled with a broad biochemical screen, our experiments will identify novel factors that regulate gene expression in chromatin (Aim 2). Next, we will reveal how transcription affects and is affected by epigenetic crosstalk and feedback loops (Aim 3). Our research will define the molecular rules that govern transcription through chromatin in human biology and explain fundamental mechanisms that drive differential gene expression in health and disease.

项目概要蛋白质编码基因的转录通过细胞特异性基因控制细胞身份和命运表达程序。在转录过程中，RNA 聚合酶 II 必须穿过其天然结构染色质模板。染色质及其基本单位核小体对转录等核过程的障碍。重要的是，转录机器必须不仅在不破坏染色质组织的情况下遍历基因组，而且转录活性还通过组蛋白的翻译后修饰进行表观遗传调节蛋白质。通常这些过程嵌入在复杂的反馈循环中，其中染色质结构、转录和表观遗传学相互调节。这些监管机制在基因表达控制中发挥着关键作用，基因表达失调通常会导致癌症的出现。之间串扰的生化和结构基础然而，转录机制、染色质和表观遗传学仍然知之甚少，部分原因是细胞系统的复杂性和实验方法的限制。该提案的目标是克服我们对基因表达缺乏了解的情况通过使用综合重构策略来研究染色质组织进行调节，转录和表观遗传学并行。在这里，我建议开发一个组合生物化学和结构方法称为视觉生物化学。视觉生物化学结合完全重建的染色质转录系统和时间分辨单粒子低温电子显微镜。我们的工作表明视觉生物化学可以成功地用于了解核小体和表观遗传信息在过程中如何保留转录。我们提出的研究将确定转录的结构基础机器在类似天然的环境中转录高阶染色质（目标 1）。耦合通过广泛的生化筛选，我们的实验将识别调节基因的新因子染色质中的表达（目标 2）。接下来，我们将揭示转录如何影响以及被影响表观遗传串扰和反馈回路（目标 3）。我们的研究将定义分子规则在人类生物学中通过染色质控制转录并解释基本原理驱动健康和疾病中差异基因表达的机制。