# Time-resolved chromatin accessibility by Single-Molecule FRET of +1 Nucleosome Dynamics

> **NIH NIH F31** · JOHNS HOPKINS UNIVERSITY · 2024 · $48,974

## Abstract

All eukaryotic organisms share the complex need for intricate control of gene expression according to
the needs of the cell. This is achieved largely in part by dynamic, precise positioning of nucleosomes at gene
promoters, which share a defined chromatin architecture characterized by a nucleosome depleted region
(NDR) surrounded by modified nucleosomes. The boundary of the NDR is defined by an upstream -1
nucleosome and a downstream +1 nucleosome. The gene promoter contains necessary sequence elements
that have been well established to control transcription initiation such as transcription factor binding sites, the
TATA box, and the necessary transcription start sites. It has been reported that the nucleosome can obstruct
these sequence motifs serving as a barrier to transcription machinery and its positioning provides an important
role in regulating gene expression. Nucleosomes are positioned by chromatin remodeling enzymes that use
energy from ATP hydrolysis for nucleosome translocation. Many gene promoters recruit multiple remodelers
from the sub-classes (SWI/SNF, INO80, ISWI, and CHD) and transcription factors that direct the necessary
transcription machinery. However, the kinetics of how these multiple factors that can all slide nucleosomes
work at the same gene promoter remains elusive with existing methods. This proposal is based on my
preliminary data designing a real-time reporter of +1 nucleosome position to elucidate how multiple remodelers
coordinate transcription initiation using the model organism Saccharomyces cerevisiae. Here, I propose a time-
resolved system to observe the kinetically separable steps of reconstituted +1 nucleosome movement on
native sequences in the context of sequence specific and general transcription factor binding. In Aim 1, I will
use DNA from the promoter of the inducible gene HIS3 to reconstitute a +1 nucleosome FRET construct that
will report +1 nucleosome positioning over time. In Aim 2, I will add purified chromatin remodeling enzymes to
my nucleosome construct and measure the temporal window of promoter accessibility caused by multiple,
opposing remodelers, testing the hypothesis that the push and pull by different remodelers are responsible for
promoter accessibility. Additionally, I will image the same construct in the presence of whole cell extracts that
contain all the native cell components for comparison with purified remodelers. In Aim 3, to measure the
functional impact of promoter accessibility, I will test the hypothesis that nucleosome dynamics control the
binding of sequence specific transcription factor GCN4 and the general transcription factor TFIID. These
experiments provide a direct reporter of +1 nucleosome positioning kinetics at single molecule resolution.
Overall, this work will provide unprecedented insight into the multifaceted kinetic interplay of major chromatin
and transcription components at a eukaryotic gene promoter for the transition from repressed to active
chromatin.

## Key facts

- **NIH application ID:** 10998135
- **Project number:** 1F31GM156098-01
- **Recipient organization:** JOHNS HOPKINS UNIVERSITY
- **Principal Investigator:** Maryam Yamadi
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 1
- **Project period:** 2024-09-01 → 2027-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10998135

## Citation

> US National Institutes of Health, RePORTER application 10998135, Time-resolved chromatin accessibility by Single-Molecule FRET of +1 Nucleosome Dynamics (1F31GM156098-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10998135. Licensed CC0.

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