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

摘要 真核基因组包装在染色质中，染色质是核小体与非组蛋白结合的线性阵列。 蛋白质执行结构、酶和调节功能的蛋白质这项提议旨在阐明这种相互作用 在染色质组织、重塑和修饰与两个关键的核功能之间：基因转录 以及DNA修复，使用活细胞中的单分子成像来实时获得全面的数据集 转录和DNA修复蛋白和染色质运动的动力学及其与理论和 具有预测能力的建模。 我们将应用单分子跟踪(SMT)来以高时空分辨率对组织进行成像， 典型先锋转录因子GAF在果蝇中的动态、调节和功能 我们将描绘全球和局部的核组织和野生型和突变型GAF结合到 基因组范围内的同源DNA元件，以及活血细胞中的Hsp70启动子。我们将把全球和 与GAF功能相关的八个重要染色质和转录蛋白效应器的局部动力学。SMT 以上因素的数据集被用来构建GAF与 染色质靶标和实验操作测试模型。研究将扩展到人类核因子-Y，a 独特的先驱因子，使人类细胞中Hsp70启动子上的染色质变得容易获得。 我们将研究染色质组织和动力学与DNA修复之间的相互作用，使用非常 快速(VF)CRISPR，可在基因组中任何位置产生双链断裂(DSB) 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- 产生DSB后，可分辨染色质分析和修复因子的实时成像。我们将确定 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修复动力学数据集以构建理论 DSB修复过程中4D染色质重组模型。我们将使用一系列染色质重建器和 DNA损伤反应突变体记录因果关系，并将vfCRISPR的覆盖范围扩大到其他 DNA修复过程包括碱基切除修复和错配修复。 我们将结合上述方法来探索DNA修复介导的染色质改变如何影响 人类细胞中的转录，反过来，转录和相关染色质的变化如何影响 DNA修复动力学。我们将描绘先锋和非先锋因子的动态以及关键的DNA修复酶 在活性Hsp70基因的活细胞中，改变DSB和热休克的时间来评估其影响 DSB在转录的不同阶段。标记基因座和新生Hsp70基因的同时成像 将揭示转录如何影响受损基因的动态。

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.