# Cross-regulation between loop extrusion, chromatin fiber structure and chromatin-associated RNAs

> **NIH NIH DP2** · ROCKEFELLER UNIVERSITY · 2022 · $1,485,041

## Abstract

The cohesin complex is a major factor driving the 3-D organization of mammalian genomes at the scale of tens
to kilobases to megabases. Recent single-molecule experiments have shown that it can extrude loops of DNA,
which are an organizing principle of genome architecture. Together with CTCF, a DNA-binding protein that stalls
cohesin’s translocation and defines loop boundaries, and several regulators of cohesin that promote loading,
such as NIPBL, or release from chromatin, such as WAPL, cohesin defines interaction domains in chromosomes
that affect patterns of gene expression during development and can lead to developmental diseases or cancer
when disrupted. Although loop extrusion on naked DNA has been studied, cohesin in cells must navigate
nucleosome-packed chromatin fibers that restrict access to binding sites on DNA, self-organize into
compartments of similar epigenetic state that are independent of and compete with loop domains, potentially
regulate DNA supercoiling, and are decorated with chromatin-associated RNAs. How cohesin and CTCF interact
with chromatin in cells is the next frontier in understanding 3-D genome organization. Progress in this arena will
require a multi-scale approach, with methods that probe both nucleosome-scale and megabase-scale features.
I propose experiments to probe (1) how loop extrusion by cohesin perturbs the local structure of the chromatin
fiber; (2) how the local structure of the chromatin fiber, modulated by depletion of linker histones and
destabilization of nucleosomes, regulates cohesin’s ability to load and extrude loops; (3) how changes in the
balance of supercoiling due to excess cohesin looping affect local nucleosome-nucleosome interactions; and (4)
the chromatin-associated RNA interactome of CTCF at RNA-dependent and RNA-independent loop boundaries.
To dissect the specific effects of cohesin, its regulators and the chromatin fiber, we will use a combination of
stable protein depletion, acute degradation, and pharmacological inhibition in human and mouse cell lines. We
will read out changes in chromatin fiber structure and chromatin-associated RNAs using RICC-seq, a method I
recently developed for measuring DNA-DNA contacts at sub-nucleosome resolution in intact cells, and using
novel technology development to probe the chromatin-associated RNA interactome of specific proteins. These
methods will be combined with more established epigenome and transcription profiling tools and with coarse-
grained simulations to develop and test multi-scale models for the interaction of loop extrusion machinery with
the chromatin fiber. I anticipate that the results of these experiments will shed new light on how loop extrusion
and chromatin’s self-association interact in specific contexts, which models for cohesin’s engagement with DNA
are relevant to loop extrusion, how supercoiling is disseminated across chromosomes, and how the local
molecular context defines loop boundaries. This knowledge may reveal new stra...

## Key facts

- **NIH application ID:** 10472889
- **Project number:** 1DP2GM150021-01
- **Recipient organization:** ROCKEFELLER UNIVERSITY
- **Principal Investigator:** Viviana I Risca
- **Activity code:** DP2 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $1,485,041
- **Award type:** 1
- **Project period:** 2022-09-01 → 2025-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10472889

## Citation

> US National Institutes of Health, RePORTER application 10472889, Cross-regulation between loop extrusion, chromatin fiber structure and chromatin-associated RNAs (1DP2GM150021-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10472889. Licensed CC0.

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