Towards fully reconstituting mammalian transcription in a test tube

项目摘要/摘要 人类基因组的长度接近2米，必须被压缩以适应细胞核，这 直径只有几微米。除了紧凑外，DNA必须被组织起来以保持 基因组的完整性，但也保持了对读取基因组的分子机器的访问。怎么了？ 基因组的组织直接影响特定细胞中哪些基因的表达。基因组脱钩 组织和基因表达对细胞有深远的影响，导致癌症，智力 残疾和发育迟缓。目前尚不清楚基因组的组织和第一步是如何进行的 基因表达和转录在物理上是相互耦合的。确定这两个流程的链接方式对于 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水平 核小体是通过将DNA包裹在组蛋白上而形成的，其紧凑和组织是由核小体介导的 蛋白质。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和转录的过程 基因组紧凑是通过在体外重组染色质上的转录反应而交织在一起的 使用(1)一种新的高通量生化分析和(2)时间分辨低温电子技术来观察它们 显微镜(EM)。 在本提案的第一部分，我们将开发一种高通量的单分子转录检测方法。 在数千个DNA序列上使用重组的转录复合体。我们将评估DNA如何 序列、染色质状态和转录因子在早期直接调节转录活性 转录延伸。这项测试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 上下文。 在这个提案的第二部分，我们将定义染色质和DNA序列如何影响 通过捕获转录复合体的结构快照来实现转录动力学 核小体。我们将使用时间分辨低温电子显微镜直接评估转录复合体采用哪些状态 在早期转录期间，并识别稳定的在线和不稳定的离线状态。总而言之，这项雄心勃勃的提案 将解决有关核小体和DNA序列如何用于直接影响 转录输出。这份建议书中获得的方法和数据将有助于实现我的长期愿景 实验室将完全重建哺乳动物转录，以确定细胞命运如何受偶联蛋白的调节 基因组组织和基因表达，以及疾病突变如何扰乱这种耦合。

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.