# Capturing the dynamic epigenome using single molecule and single cell approaches

> **NIH NIH R35** · BRANDEIS UNIVERSITY · 2024 · $398,920

## Abstract

ABSTRACT
 Retaining pre-existing patterns of histone modifications during early development and multicellular
differentiation is essential for the maintenance of cellular identity. Histone H3 lysine 9 methylation (H3K9me) is
associated with the formation of transcriptionally silent, specialized chromatin domains also referred to as
heterochromatin. Heterochromatin establishment is essential for normal centromere and telomere function,
silencing of transposons and repetitive DNA elements and preserving lineage-specific patterns of gene
expression. The loss of H3K9 methylation is associated with genome instability and aneuploidy which are
widely recognized as the most common abnormalities associated with cancer. Heterochromatin establishment
is a dynamic process which involves weak and transient interactions between histone modifiers and their
cognate nucleosome substrates. Counterintuitively, these weak protein-protein interactions produce epigenetic
states that remain heritable across generational timescales. These dynamic properties associated with the
epigenome exposes significant conceptual and methodological gaps that this proposal will principally address:
1) How do dynamic epigenetic complexes consisting of histone modification readers, writers and erasers
assemble in vitro and in living cells? 2) How do histone modifiers traverse a complex chromatin landscape to
locate their sites of action? 3) How is epigenetic memory stored and transmitted across subsequent
generations? The current belief is that histone modifications function as inert scaffolds which passively
promote the localization of histone modifiers to distinct sites in the genome. Based on our recent studies, we
propose an inversion of this paradigm. Our results reveal that H3K9 methylation has an active role in
catalyzing the cooperative assembly of a heterochromatin regulatory complex. Our studies underscore how the
assembly of epigenetic complexes in an H3K9 methylation dependent manner restricts protein-protein
interactions to specific chromatin contexts. In this proposal, we will use in vitro biochemistry to reconstitute
heterochromatin regulatory complexes, single molecule imaging to define the order and timing of assembly of
these complexes in vitro and within a native chromatin context and a high-throughput microfluidic platform to
define how epigenetic memory is propagated within individual lineages. The real-time visualization of
heterochromatin assembly at high spatial and temporal resolution will illuminate how transient molecular
interactions can synergize to establish stable and heritable patterns of gene expression.

## Key facts

- **NIH application ID:** 10895445
- **Project number:** 5R35GM137832-05
- **Recipient organization:** BRANDEIS UNIVERSITY
- **Principal Investigator:** Kaushik Ragunathan
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $398,920
- **Award type:** 5
- **Project period:** 2020-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10895445

## Citation

> US National Institutes of Health, RePORTER application 10895445, Capturing the dynamic epigenome using single molecule and single cell approaches (5R35GM137832-05). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10895445. Licensed CC0.

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