# Dissecting the Mechanisms of Pioneer Factor Facilitated Chromatin Opening

> **NIH NIH R01** · JOHNS HOPKINS UNIVERSITY · 2022 · $343,875

## Abstract

Project Summary
Nucleosomes cover most of the eukaryotic genome and present a barrier for the competent binding of many
regulatory proteins that control gene expression. A special set of transcription factors called pioneer factors can
bind their target sites on nucleosomal DNA, loosen chromatin, and promote the activity of other chromatin factors
and enzymes, leading to nucleosome repositioning and gene activation. Sox2 and Oct4 are pioneer factors that
orchestrate changes in cell fate during embryogenesis and adult neurogenesis and are major players in the
reprogramming of somatic cells into a pluripotent state. Mutation and deficiency in these proteins are linked to
neurological disorders, while their enrichment is tied to cancer. Despite ample studies, little is known about how
to Sox2, Oct4 and other pioneer factors exert their pioneering activity at the molecular level. Our goal is to
address key unresolved questions about how Sox2 and Oct4 engage with nucleosomes in distinct binding modes
and alter the structure and dynamics of nucleosomes, and how these changes affect the downstream action of
chromatin modifying and remodeling enzymes. Towards this goal, we propose to integrate chemical probing
assays with high-resolution nuclear magnetic resonance (NMR) spectroscopy to obtain site-specific structural
and dynamic information about pioneer factor interaction with nucleosomes and the perturbations they elicit in
DNA and histones to potentially regulate other factors. In Aim 1, we will utilize in vitro binding assays to
characterize distinct binding modes of Sox2 and Oct4 to artificial and natural nucleosomes and their dependence
on DNA sequence, co-factor cooperativity, and histone composition. We will also adapt methyl TROSY NMR,
suitable for studies of super-large biomolecules, to interrogate the site-specific binding conformation and
dynamics of these proteins to select nucleosomes with atomic detail. In Aim 2, we will examine the effect of Sox2
and Oct4 binding in various nucleosome positions on the conformation and accessibility of nucleosomal DNA
and the disordered histone tails by using NMR, chemical and enzymatic probing methods sensitive to structural
and dynamic perturbations. We will further assess whether Sox2 and Oct4, through direct contact and
nucleosome perturbations, stimulate the activity of histone methyltransferase enzymes that are found to
collaborate in vivo with these pioneer factors. Finally, in Aim 3, we will adapt our approach to characterize how
a methyl-DNA binding protein and proposed pioneer factor Kaiso binds to and alters nucleosomes, providing
insight into the recognition and function of epigenetic DNA marks in chromatin regulation. The proposed work
will lay the basis for future investigations of other pioneer factors and the role of DNA context, synergistic action,
histone variants and epigenetic modifications into pioneer factor-mediated chromatin opening. This will improve
our ability to modify pioneer f...

## Key facts

- **NIH application ID:** 10501478
- **Project number:** 1R01GM147642-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Evgenia Nikolaevna Nikolova
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $343,875
- **Award type:** 1
- **Project period:** 2022-08-01 → 2027-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10501478

## Citation

> US National Institutes of Health, RePORTER application 10501478, Dissecting the Mechanisms of Pioneer Factor Facilitated Chromatin Opening (1R01GM147642-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10501478. Licensed CC0.

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