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

活细胞中单分子分辨率转录和 DNA 修复过程中的染色质功能

基本信息

批准号：
10687212
负责人：
Taekjip Ha
金额：
$ 4.92万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2023-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10687212
关键词：
Affect Architecture Base Excision Repairs Binding Biological Cell Nucleus Cells Chromatin Chromatin Fiber Chromatin Modeling Clustered Regularly Interspaced Short Palindromic Repeats Computer Models DNA DNA Damage DNA Repair DNA Repair Enzymes DNA Repair Gene Data Set Double Strand Break Repair Drosophila genus Elements Experimental Models Genes Genetic Transcription Genome Heat-Shock Response Hemocytes Human Image Kinetics Knowledge Label Light Link Mediating Messenger RNA Mismatch Repair Modeling Modification Motion Mus Nuclear Nucleosomes Outcome Pathway interactions Process Proteins Regulation Relaxation Resolution Series System Testing Theoretical model Time chromatin modification genome-wide genomic locus molecular imaging mutant non-histone protein promoter real-time images repaired response single molecule spatiotemporal submicron theories transcription factor

项目摘要

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.

总结