# Dynamics of chromosome organization and chromatin states in single cells

> **NIH NIH U01** · CALIFORNIA INSTITUTE OF TECHNOLOGY · 2022 · $1,136,227

## Abstract

SUMMARY
Cellular differentiation involves tightly coupled changes in gene expression, chromatin state, and
sub-nuclear arrangements of chromosomes. Understanding and controlling differentiation
requires understanding how each of these processes occurs dynamically within the same cell and
how they influence one another. Existing techniques can provide genome scale analysis of
interactions or spatial organization of a few chromosomal positions. However, we have lacked a
generalizable framework for simultaneous reconstruction of the overall dynamics of the nucleus
across all three levels. Recent work from our labs has opened up the possibility of achieving such
coupled analysis. Our track first, identify later approach allows many DNA to be simultaneously
tracked in living cells. RNA and DNA seqFISH allows a large number of transcripts and DNA loci
to be imaged in single fixed cells and MEMOIR allows lineage information to be recovered from
endpoint measurements. In this project, we propose to combine live imaging, multiplexed RNA,
DNA, and immunofluorescence measurements, and MEMOIR lineage tracking to capture whole-
genome dynamics of chromosomal loci and chromatin states. Using mouse embryonic stem cells
(mESCs) as a model system, we will study the transition from the pluripotent state to an earlier 2-
cell (2C) like state which shows drastic chromosome re-arrangement and changes in nascent
gene expression patterns. In addition, we will study the chromosomal dynamics of X-inactivation
based on the initial observations that sister X chromosomes are in contact with each other during
early phases of the inactivation process. Both of these biological questions require tracking
chromosomal dynamics and chromatin state simultaneously in single cells. The “Track First and
ID later” approach allows a large number of loci to be tracked in living cells. The combined
MEMOIR approach with multiplex immunofluorescence allows us to infer the kinetics of chromatin
states transitions. Bringing these tools to study X inactivation and 2C state transition will
demonstrate the capability of this approach for addressing a broad range of cell fate decision
questions. We will also develop analysis and visualization tools to integrate genomics (SPRITE)
and imaging data. The technology developed in this project can be readily implemented in human
cell lines and adopted by other labs in the 4DN consortium.

## Key facts

- **NIH application ID:** 10456124
- **Project number:** 5U01DK127420-03
- **Recipient organization:** CALIFORNIA INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Long Cai
- **Activity code:** U01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,136,227
- **Award type:** 5
- **Project period:** 2020-09-19 → 2025-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10456124

## Citation

> US National Institutes of Health, RePORTER application 10456124, Dynamics of chromosome organization and chromatin states in single cells (5U01DK127420-03). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10456124. Licensed CC0.

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