# Probing the dynamics of chromosome organization in single cells

> **NIH NIH R35** · UNIVERSITY OF WASHINGTON · 2024 · $31,901

## Abstract

Project Summary
Mammalian cells package two meters of linear DNA into a nucleus whose diameter is on average only a few
microns. This packaging must be done in such a way that DNA transactions such as transcription, replication,
and repair can faithfully occur. The development of the `Hi-C' technique has enabled the mapping of
chromosome-chromosome interaction frequency on the genome-wide scale in large populations of cells. Hi-
C has generated detailed datasets that have revealed key organizational features, including `domains' of
elevated interaction frequency and the broad partitioning of the genome into active and inactive
`compartments.' Disruption of these features is associated with cancer and developmental disease. However,
in part due to technical limitations, pressing open questions remain about the causes and consequences of
3D genome organization, particularly in single cells. For instance, it is not fully clear whether features such as
domains actually form in individual cells, or instead represent the averaging of many individual states that are
present in the population of cells profiled because single-cell Hi-C methods provide very sparse datasets in
which most features of genome organization remain invisible. Furthermore, the physical properties of these
genomic features have yet to be examined in single cells, and it is not currently known if these regions create
distinct diffusive environments that can impact the activity of trans-acting factors. Finally, the discovery of
additional proteins that influence nuclear architecture has been limited by the high financial and resource cost
of Hi-C. Our previous work established a powerful new platform for investigating 3D genome organization in
single cells using microscopy. We introduced a programmable fluorescent in situ hybridization (FISH)
approach that uses sets of bioinformatically designed oligonucleotide probes to create complex hybridization
patterns in fixed samples. We have harnessed this technology to pioneer the use of single-molecule super-
resolution microscopy to study chromosome structure on the nanoscale, introduced a technique capable of
visually distinguishing homologous chromosomes, and also have developed an approach that facilitates the
multiplexed amplification of fluorescent signals in fixed cells and tissues. We will build upon this foundational
work by developing and applying single-cell imaging technologies and leveraging advanced optical
approaches to directly address these unsolved questions. Specifically, we will introduce a broadly enabling
single-cell imaging strategy capable of resolving features of 3D genome organization with unprecedented
detail using multiplexed FISH (Objective 1) and investigate the biophysical properties of 3D organizational
features with live-cell imaging (Objective 2). We will augment these efforts with high-throughput, unbiased
screening approaches to identify new regulators of 3D genome structure (Objective 3). Our detailed
c...

## Key facts

- **NIH application ID:** 10840704
- **Project number:** 3R35GM137916-04S1
- **Recipient organization:** UNIVERSITY OF WASHINGTON
- **Principal Investigator:** Brian Joseph Beliveau
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $31,901
- **Award type:** 3
- **Project period:** 2020-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10840704

## Citation

> US National Institutes of Health, RePORTER application 10840704, Probing the dynamics of chromosome organization in single cells (3R35GM137916-04S1). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10840704. Licensed CC0.

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