# Visualizing transcription factor binding and chromatin assembly on newly synthesized DNA

> **NIH NIH K99** · FRED HUTCHINSON CANCER CENTER · 2024 · $125,000

## Abstract

PROJECT SUMMARY
Newly synthesized DNA represents an ideal system to understand the molecule mechanisms governing
chromatin structure and gene expression. Chromatin structure controls transcription by modulating DNA
accessibility. During DNA replication, chromatin structure must be disassembled to allow for passage of the
replication fork, which generates an intermediate chromatin structure distinct from mature chromatin. Failure to
reestablish proper chromatin structure leads to spurious gene expression and is associated with a variety of
human diseases. Transcription factors (TFs) are a critical component of chromatin structure that regulate
transcription by binding to targeted sequences on DNA and altering chromatin structure. Following replication
fork passage, TF binding sites become occluded by nucleosomes. As nucleosomes are refractory to TF
binding, it is unclear when or how TFs rebind to targeted DNA sequences to reestablish mature chromatin
structure. Furthermore, in-spite of the established role for TFs in regulating gene expression, it is unclear what
effect TF binding has on chromatin structure. To address these gaps in knowledge, I will use novel techniques
termed Nascent CUT&Tag and Nascent Fiber-seq to profile TF binding on nascent DNA and during the
subsequent steps of chromatin maturation. In Aim 1, I will use Nascent CUT&Tag to determine the kinetics
and mechanisms of TF binding to nascent chromatin. This aim will test whether TFs can associate with
nucleosomal DNA during the process of rebinding, or whether accessible DNA is necessary for TFs to bind
following replication fork passage. In Aim 2, I will visualize TF binding and nascent chromatin maturation on
the single molecule level. By simultaneously visualizing TF binding and surrounding chromatin structure, I will
be able to assess the direct impacts of TF binding on critical chromatin features such as nucleosome
positioning and RNA polymerase II occupancy. In Aim 3, I will characterize the impacts of nucleosome
turnover on TF binding and chromatin maturation. Using the anti-cancer drug aclarubicin, I will drive elevated
nucleosome turnover and observe the effects on TF binding and nascent chromatin maturation. These studies
will provide critical insights into the features regulating TF binding and chromatin assembly, which will inform
our understanding of the underlying mechanisms regulating gene expression and cell fate specification. The
training outlined in this proposal will provide a strong foundation to develop as an independent investigator
studying the critical relationship between chromatin structure and gene expression.

## Key facts

- **NIH application ID:** 10785886
- **Project number:** 1K99GM152821-01
- **Recipient organization:** FRED HUTCHINSON CANCER CENTER
- **Principal Investigator:** Matthew Wooten
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $125,000
- **Award type:** 1
- **Project period:** 2023-12-01 → 2025-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10785886

## Citation

> US National Institutes of Health, RePORTER application 10785886, Visualizing transcription factor binding and chromatin assembly on newly synthesized DNA (1K99GM152821-01). Retrieved via AI Analytics 2026-06-01 from https://api.ai-analytics.org/grant/nih/10785886. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*