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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.

项目总结蛋白质编码基因的转录通过细胞特异性基因来控制细胞的身份和命运表情程序。在转录过程中，RNA聚合酶II必须遍历其固有的染色质模板。染色质及其基本单位--核小体--对核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，而且转录活性也是通过组蛋白的翻译后修饰来表观遗传调节的蛋白质。通常，这些过程被嵌入到精细的反馈环中，其中染色质建筑、转录和表观遗传学相互调节。这些监管机制在基因表达调控中起着关键作用，基因表达的失调往往会导致癌症的出现。串扰产生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。部分原因是蜂窝系统的复杂性和实验方法的局限性。这项提议的目标是克服我们对基因表达缺乏理解的问题通过使用整合的重组策略来研究染色质组织来进行调控，转录，和表观遗传学并行。在这里，我建议开发一种组合生物化学和结构方法称为视觉生物化学。视觉生物化学组合一种完全重组的染色质转录系统和时间分辨的单粒子低温电子显微镜。我们的工作揭示了视觉生物化学可以成功地用来理解核小体和表观遗传信息是如何在抄写。我们提出的研究将确定转录如何进行的结构基础机器在类似自然的环境中转录高阶染色质(目标1)。耦合有了广阔的生化屏幕，我们的实验将识别出调控基因的新因素。染色质表达(目标2)。接下来，我们将揭示转录是如何影响和影响的表观遗传串扰和反馈环路(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调控转录并解释基本原理在健康和疾病中驱动差异基因表达的机制。