Towards fully reconstituting mammalian transcription in a test tube

在试管中完全重建哺乳动物转录

• 批准号: 10242352
• 负责人: Seychelle Monqiue Vos
• 金额: $ 130.36万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-21 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10242352
• 关键词: Address; Adopted; Affect; Behavior; Biochemical; Biological Assay; Caliber; Cell Nucleus; Cells; Chromatin; Complex; Coupled; Coupling; Cryoelectron Microscopy; DNA; DNA Sequence; Data; Developmental Delay Disorders; Disease; Enzymes; Gene Expression; Genes; Genetic Transcription; Genome; Goals; Histones; Human; Human Genome; In Vitro; Intellectual functioning disability; Length; Link; Malignant Neoplasms; Measures; Mediating; Methods; Molecular Machines; Mutation; Nucleosomes; Output; Process; Proteins; Reaction; Specificity; Structure; Testing; Time; Transcription Elongation; Transcription Process; Tube; Vision; cell type; developmental disease; genome integrity; in vitro Assay; meter; novel; promoter; reconstitution; single molecule; time use; transcription factor

Project Summary/Abstract The human genome measures nearly 2 meters in length and must be compacted to fit in the nucleus, which measures only a few microns in diameter. In addition to compaction, the DNA must be organized to maintain genome integrity but also remain accessible to the molecular machines that read the genome. How the genome is organized directly influences which genes are expressed in a particular cell. Decoupling of genome organization and gene expression has profound consequences for cells, leading to cancer, intellectual disability, and developmental delay. It is mechanistically unclear how genome organization and the first step of gene expression, transcription, are physically coupled. Determining how both processes are linked is critical for understanding how cell type function and specificity are achieved. The first level of eukaryotic genome compaction and organization is mediated by nucleosomes that are formed by wrapping DNA around histone proteins. Here I will investigate how DNA sequence and nucleosomes impact gene expression in promoter proximal regions of human genes. I will mechanistically decipher how the processes of transcription and genome compaction are intertwined by reconstituting transcription reactions on chromatin in vitro and observing them using (1) a novel high throughput biochemical assay and (2) time-resolved cryo-electron microscopy (EM). In the first part of this proposal, we will develop a high throughput, single molecule transcription assay using reconstituted transcription complexes on thousands of DNA sequences. We will assess how DNA sequence, chromatin state, and transcription factors directly regulate transcriptional activity during early transcription elongation. This assay will overcome major hurdles in the field by revealing directly how nucleosomes and protein factors affect transcription behavior on an unprecedented number of DNA sequence contexts. In the second part of this proposal, we will define how chromatin and DNA sequence influence transcription dynamics by capturing structural snapshots of transcription complexes as they transcribe through nucleosomes. We will use time-resolved cryo-EM to directly assess which states transcription complexes adopt during early transcription and identify stable online and labile offline states. Together, this ambitious proposal will address important questions regarding how nucleosomes and DNA sequence are used to directly influence transcriptional output. The methods and data acquired in this proposal will help fulfill the long-term vision of my lab to fully reconstitute mammalian transcription to determine how cell fate is regulated by the coupling of genome organization and gene expression and how disease mutations can perturb this coupling.

项目总结/摘要 人类基因组长度接近2米，必须压缩以适应细胞核， 直径只有几微米除了压实，DNA必须组织起来， 基因组的完整性，但也保持可访问的分子机器读取基因组。如何 基因组的组织直接影响哪些基因在特定细胞中表达。基因组解耦 组织和基因表达对细胞有深远的影响，导致癌症，智力低下， 残疾和发育迟缓。目前还不清楚基因组的组织结构和基因表达的第一步是如何进行的。 基因的表达和转录都是物理耦合的。确定这两个过程是如何联系在一起的， 了解细胞类型的功能和特异性是如何实现的。真核生物基因组的第一层 致密化和组织化是由核小体介导的，核小体是通过将DNA包裹在组蛋白周围而形成的 proteins.在这里，我将研究DNA序列和核小体如何影响启动子中的基因表达 人类基因的近端区域。我将机械地破译转录过程和 基因组压缩通过体外染色质上的重建转录反应而交织在一起， 使用（1）新的高通量生物化学测定和（2）时间分辨冷冻电子 显微镜（EM）。 在这个建议的第一部分，我们将开发一个高通量，单分子转录检测 利用数千个DNA序列上的重组转录复合物。我们将评估DNA 序列，染色质状态和转录因子直接调节早期转录活性， 转录延伸这项试验将克服该领域的主要障碍，直接揭示如何 核小体和蛋白质因子影响前所未有数量的DNA序列的转录行为 contexts. 在这个建议的第二部分，我们将定义染色质和DNA序列如何影响 转录动力学通过捕获转录复合物的结构快照，因为它们转录通过 核小体我们将使用时间分辨cryo-EM直接评估转录复合体采用的状态 并识别稳定的在线和不稳定的离线状态。这个雄心勃勃的计划 将解决有关核小体和DNA序列如何直接影响 转录产物本提案中所采用的方法和数据将有助于实现我的长期愿景。 实验室完全重建哺乳动物的转录，以确定细胞的命运是如何调节耦合 基因组组织和基因表达以及疾病突变如何干扰这种耦合。