Chromatin Function During Transcription and DNA Repair at Single Molecule Resolutionin Living Cells

总结 真核生物的基因组被包装在染色质中，与非组蛋白相关的核小体线性阵列 执行结构、酶和调节功能的蛋白质。本提案旨在阐明 染色质组织、重塑和修饰与两个关键的核功能：基因转录 和DNA修复，在活细胞中使用单分子成像，以获得实时的综合数据集。 转录和DNA修复蛋白质和染色质运动的动力学，以及它们与理论和 具有预测能力的建模。 我们将应用单分子跟踪（SMT）以高时空分辨率对组织进行成像， 果蝇中一个典型的先驱转录因子GAGA因子（GAF）的动态、调节和功能。 我们将对野生型和突变型GAF结合到GAF的全局和局部核组织以及动力学进行成像。 同源DNA元件全基因组，并在Hsp 70启动子在活血细胞。我们将在全球范围内 与GAF功能相关的八种突出的染色质和转录蛋白效应物的局部动力学。SMT 来自上述因素的数据集用于构建GAF相互作用的理论模型， 染色质靶标和测试模型。研究将扩展到人类NF-Y， 一种独特的先锋因子，使人类细胞中Hsp 70启动子处的染色质易于接近。 我们将研究染色质组织和动态与DNA修复之间的相互作用，使用非常 快速（vf）CRISPR，其可以在基因组中的任何地方产生双链断裂（DSB），具有高的 时空分辨率我们将通过时间来确定DSB修复动力学和染色质重组- 在产生DSB后，分辨染色质分析和修复因子的实时成像。康贝特人将以 拓扑相关结构域和环挤出对染色质修饰和松弛的影响， 伴随DNA修复，并整合染色质和DNA修复动力学数据集，构建理论 DSB修复过程中4D染色质重组的模型。我们将使用一系列染色质重塑剂， DNA损伤反应突变体，以记录因果关系，并将vfCRISPR的范围扩大到其他 DNA修复过程包括碱基切除修复和错配修复。 我们将合并上述方法，以探讨DNA修复介导的染色质改变如何影响 人类细胞中的转录，以及转录和相关的染色质变化如何影响 DNA修复动力学我们将对先锋和非先锋因子以及关键DNA修复酶的动态进行成像 在活的人类细胞中的活性Hsp 70基因，改变DSB和热休克的时间，以评估其影响。 在转录的不同阶段。标记位点和新生Hsp 70 mRNA的同时成像 将揭示转录如何影响受损基因座的动力学。

Summary Eukaryotic genomes are packaged in chromatin, linear arrays of nucleosomes in association with nonhistone proteins performing structural, enzymatic, and regulatory functions. This proposal aims to elucidate the interplay between chromatin organization, remodeling and modification and two key nuclear functions: gene transcription and DNA repair, using single molecule imaging in living cells to obtain comprehensive datasets on the real-time dynamics of transcription and DNA repair proteins and chromatin motions, and their integration with theory and modeling with predictive power. We will apply single molecule tracking (SMT) to image at high spatiotemporal resolution the organization, dynamics, regulation and function of a prototypical pioneer transcription factor, GAGA factor (GAF) in Drosophila. We will image the global and local nuclear organization and dynamics of wild-type and mutant GAF binding to cognate DNA elements genome-wide, and at Hsp70 promoters in live hemocytes. We will image the global and local dynamics of eight prominent chromatin and transcription protein effectors linked to GAF functions. SMT datasets from the factors imaged above are used to construct theoretical models for GAF interactions with chromatin targets and test models by experimental manipulation. Studies will be extended to human NF-Y, a distinct pioneer factor that makes accessible chromatin at the Hsp70 promoter in human cells. We will examine the interplay between chromatin organization and dynamics and DNA repair, using very fast (vf) CRISPR that can generate a double strand break (DSB) anywhere in the genome with high spatiotemporal resolution. We will determine DSB repair kinetics and chromatin reorganization through time- resolved chromatin analysis and real-time imaging of repair factors after generating DSB. We will determine the impact of topologically associated domains and loop extrusion on chromatin modifications and relaxations that accompany DNA repair, and integrate chromatin and DNA repair kinetics datasets to construct theoretical models for 4D chromatin reorganization during DSB repair. We will employ a series of chromatin remodeler and DNA damage response mutants to document causal relationships, and expand the reach of vfCRISPR to other DNA repair processes including base excision repair and mismatch repair. We will merge the above approaches to explore how DNA repair-mediated chromatin alterations affect transcription in human cells, and reciprocally, how transcription and associated chromatin changes influence DNA repair dynamics. We will image dynamics of pioneer and non-pioneer factors and key DNA repair enzymes at the active Hsp70 gene in living human cells, varying the timing of DSB and heat shock to evaluate the influence of DSB on different stages of transcription. Simultaneous imaging of labeled locus and nascent Hsp70 mRNA will reveal how transcription affects dynamics of the damaged locus.