# A high-throughput, comprehensive, and quantitative approach for measuring nucleosome-protein binding

> **NIH NIH R01** · STATE UNIVERSITY OF NEW YORK AT BUFFALO · 2021 · $354,127

## Abstract

DNA binding to chromosomal DNA is essential for many fundamental biological processes
including: transcriptional regulation, DNA replication and repair, recombination, and chromosome
segregation. There is a fundamental gap in understanding how transcription factors (TF) bind
regulatory regions located in compacted, high-order chromatin. Therefore, there is a fundamental
need to determine the mechanistic rules defining TF binding to chromatin. The long-term goal for
this project is to define the biological rules dictating TF binding to chromosomal DNA these rules
include transcription factor binding site orientation within a nucleosome, DNA and histone
modifications, cofactor and cooperative binding, and binding to subnucleosome particles. The
overall objective of this application is to develop a high throughput approach to measure the
principles of transcription factor binding to nucleosomal DNA. To accomplish this objective a new
high-throughput next-generation sequencing assay will be developed to allow the simultaneous
and quantitative examination of thousands of different nucleosomes in a single assay. The goal
of this application will be accomplished by three specific aims: 1) Formation of a nucleosome
library, 2) High-throughput protein-nucleosome binding assays with a library of nucleosomes, and
3) Define TF-nucleosome binding after histone modifications. Under the first aim, in vitro
nucleosomes will be generated from thousands of in silica designed and naturally occurring DNA
sequences in a single reaction, allowing a TF binding site to occur in all possible nucleosomal
orientations with various neighboring sequence context. In the second aim, a new methodology,
Pioneer-seq, will be developed where a transcription factor's binding affinity is determined to
thousands of nucleosomes within a nucleosome library containing differing sequences,
orientations, and variants. In the third aim, Pioneer-seq will be extended to examine how histone
tail modifications amend TF-nucleosome binding. Overall, this project will develop a high-
throughput quantitative method to determine binding principles for any protein to nucleosomal
DNA in the presence or absence of histone modifications. This contribution will be significant
because it can be applied to study many biological responses including: cell growth, regulation of
cell-division, embryonic development, differentiation, response to environmental stresses,
apoptosis and the development of a variety of disease states. In addition, biological principles
can be addressed involving cooperative binding, binding to subnucleosomes, TF-histone
interactions, and sequence content.

## Key facts

- **NIH application ID:** 10240593
- **Project number:** 5R01GM132199-03
- **Recipient organization:** STATE UNIVERSITY OF NEW YORK AT BUFFALO
- **Principal Investigator:** Michael Joseph Buck
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $354,127
- **Award type:** 5
- **Project period:** 2019-09-23 → 2023-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10240593

## Citation

> US National Institutes of Health, RePORTER application 10240593, A high-throughput, comprehensive, and quantitative approach for measuring nucleosome-protein binding (5R01GM132199-03). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/10240593. Licensed CC0.

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