# Dissecting the mechanism of epigenetic spreading by targeted degradation of architectural proteins

> **NIH NIH F31** · HARVARD MEDICAL SCHOOL · 2021 · $27,331

## Abstract

Dissecting the mechanism of epigenetic spreading by targeted degradation of architectural proteins
In female mammals, one X chromosome is silenced in order to balance dosage of X-linked genes with males,
a process known as X chromosome inactivation (XCI). XCI is mediated by a long noncoding RNA Xist, which
spreads across the inactive X, triggering recruitment of repressive factors such as Polycomb Repressive
Complexes 1 and 2 (PRC1/2), heterochromatin formation and gene silencing. In this way, XCI serves as a
model for the spreading of heterochromatin marks, a process which is essential for gene silencing across the
mammalian genome, but which is not well understood. XCI also dramatically alters the three-dimensional (3D)
chromatin structure of the X chromosome. In particular, chromosome conformation studies revealed that the X
chromosome, along with the rest of the mammalian genome is organized by architectural proteins CTCF and
cohesin into Topologically Associating Domains (TADs), megabase-sized regions within which chromatin
interactions form more frequently than across domain borders. Mammalian chromosomes are also organized
independently of CTCF and cohesin into longer range ‘compartmental’ interactions between active and inactive
regions (A/B compartments). While TADs and compartments generally remain stable during differentiation,
both structures are dramatically altered during XCI. Specifically, TADs and compartments are greatly
attenuated during XCI, with the inactive X instead being structured into megadomains. As such dramatic
restructuring of chromosome conformation is not seen during any other cellular process, a pivotal question is
whether these changes in X chromosome conformation are required for XCI or are merely a byproduct of
silencing the entire chromosome. To address this question, I generated female mouse embryonic stem cells
(mESCs) in which architectural proteins CTCF, RAD21, and WAPL can be rapidly degraded using the dTAG
degron system during XCI. Using my CTCF and RAD21 degron cell lines, I first propose to weaken TADs and
strengthen compartments in early XCI and study the impact of these changes on Xist spreading and
subsequent steps of XCI. In contrast, I propose to use my WAPL degron cell lines to strengthen TADs and
weaken compartments in early XCI and study the impact of these opposing changes on Xist spreading. By
elucidating which, if any, 3D chromatin structures are needed for Xist spreading, I expect to shed light not only
on XCI, but also on the mechanism of heterochromatin spreading outside of XCI—and the role of 3D genome
organization in gene regulation in general as well.

## Key facts

- **NIH application ID:** 10049187
- **Project number:** 5F31HD100109-02
- **Recipient organization:** HARVARD MEDICAL SCHOOL
- **Principal Investigator:** Andrea J Kriz
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $27,331
- **Award type:** 5
- **Project period:** 2019-12-01 → 2021-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10049187

## Citation

> US National Institutes of Health, RePORTER application 10049187, Dissecting the mechanism of epigenetic spreading by targeted degradation of architectural proteins (5F31HD100109-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/10049187. Licensed CC0.

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