# Assessing the basis of chromatin opening by hematopoietic pioneer factors

> **NIH NIH F31** · UNIVERSITY OF PENNSYLVANIA · 2022 · $34,179

## Abstract

Project Summary/Abstract
The goal of this proposal is to investigate the intrinsic ability of hematopoietic transcription factors (TFs) to
engage closed chromatin and initiate chromatin opening during cell fate changes. Cell fate control is a
fundamental process in biology and de novo generation of clinically relevant cell types has enormous
implications for research and therapeutics; however, the general principles by which closed and unmarked
chromatin is initially accessed by these factors are not fully understood. In eukaryotic cells, chromatin
compaction serves as a mechanism for gene regulation by modulating the accessibility of TFs to target DNA
sequences. Development of macrophages from hematopoietic stem cells requires coordinated expression of
PU.1 with myeloid TFs C/EBPα and C/EBPβ, and ectopic expression of PU.1 and C/EBPα/β converts
fibroblasts to the macrophage lineage. I hypothesized that PU.1, C/EBPα, and C/EBPβ act as “pioneer” factors
and that initiating development requires these factors to directly bind compacted chromatin, initiate chromatin
opening, and allow subsequent binding of additional TFs that activate gene expression. Our lab identified
nucleosomes targeted by PU.1, C/EBPα, and C/EBPβ in vivo, and reconstituted these nucleosomes in vitro. To
assess the interactions of these factors with higher-order chromatin substrates, I reconstituted fluorescently
end-labeled synthetic nucleosome arrays and inserted a central nucleosome capable of binding by PU.1,
C/EBPα, and C/EBPβ. I have found that PU.1, C/EBPα, and C/EBPβ bind and initiate accessibility of H1-
compacted nucleosome arrays with different efficiencies. Furthermore, I have found that the DNA binding
domains of PU.1 and C/EBPα open chromatin with less efficiency compared to the full-length proteins. I am
now investigating the ability of these pioneer factors to open chromatin and how their mechanisms of action
compare to another established pioneer factor, FoxA. I hypothesize that PU.1, C/EBPα, and C/EBPβ possess
protein domains that enable chromatin interactions, and that initiating chromatin accessibility is fundamental to
the ability of these factors to direct cell fate in vivo. To address this hypothesis I will 1) use nanopore
sequencing to define TF-chromatin interactions and determine if PU.1, C/EBPα, and C/EBPβ interact with
histones using crosslinking mass spectrometry 2) perform a deletion analysis of PU.1, C/EBPα, and C/EBPβ
to map domains required for chromatin opening in vitro and determine if chromatin opening by pioneer factors
is required for TF directed macrophage transdifferentiaiton. My project will provide insights into the
mechanisms of chromatin structure regulation by hematopoietic TFs during cellular development and
reprogramming. Furthermore, biochemical analysis will provide details of the molecular features that endow
TFs with pioneering activity.

## Key facts

- **NIH application ID:** 10368949
- **Project number:** 5F31DK123886-03
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Megan A. Frederick
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $34,179
- **Award type:** 5
- **Project period:** 2020-03-01 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10368949

## Citation

> US National Institutes of Health, RePORTER application 10368949, Assessing the basis of chromatin opening by hematopoietic pioneer factors (5F31DK123886-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10368949. Licensed CC0.

---

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