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.
Eukaryotic genomes are packaged in chromatin. 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 and light-based manipulation of genome.
