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

> **NIH NIH U01** · JOHNS HOPKINS UNIVERSITY · 2020 · $765,851

## Abstract

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.

## Key facts

- **NIH application ID:** 10129627
- **Project number:** 1U01DK127432-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Taekjip Ha
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $765,851
- **Award type:** 1
- **Project period:** 2020-09-15 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10129627

## Citation

> US National Institutes of Health, RePORTER application 10129627, Chromatin Function During Transcription and DNA Repair at Single Molecule Resolutionin Living Cells (1U01DK127432-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10129627. Licensed CC0.

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