# Spreading of Xist RNA and Polycomb complexes along the inactive X-chromosome.

> **NIH NIH R01** · MASSACHUSETTS GENERAL HOSPITAL · 2022 · $690,348

## Abstract

PROJECT SUMMARY
Our research program is unified by an effort to understand the mechanisms underlying X-chromosome
inactivation (XCI) and how functional interactions between Polycomb repressive complex 2 (PRC2) and long
noncoding RNA (lncRNA) spread the silencing process through the X-chromosome. XCI is the dosage
compensation process in mammals that leads to silencing of one X-chromosome in the peri-implantation
embryo. The X-inactivation center (Xic) — the X-linked region that controls the initiation, spread, and
maintenance of silencing — harbors a large number of genes encoding functional lncRNAs, including the
prototype, Xist, a 17-kb RNA that “coats” the inactive X (Xi) to initiate silencing. Towards understanding
mechanisms, in 2008 we identified Polycomb repressive complex 2 (PRC2) as an interacting protein partner
for Xist RNA. PRC2 is the epigenetic complex responsible for trimethylating H3 lysine27 (H3K27me3). Under
this NIH award, we have identified of a “nucleation center” required for loading of Xist-Polycomb complexes
onto the Xi prior to spreading, defined YY1 as the tether, and demonstrated that Xist RNA is but one of
thousands of RNA partners for EZH2, the catalytic subunit of PRC2. We have hypothesized that regulatory
interactions between PRC2 and RNA would become a recurrent theme in epigenetic regulation. Two major
unsolved problems in the field are how the interaction specificity between PRC2 and RNA is achieved and how
they regulate gene expression in a locus-specific manner. In the past funding cycle, we have made significant
progress towards addressing these problems. We have: (i) mapped Xi-binding sites for Xist RNA and PRC2,
(ii) performed single-molecule imaging of the Xist-PRC2 relationship on the Xi, (iii) defined the interaction
specificity and regulatory interactions between Xist's Repeat A motif and various subunits of PRC2, (iv)
identified an in vivo chaperone that helps determine the interaction specificity between Xist and PRC2, and (v)
revealed a role for SMCHD1 (a chromosome architectural protein) in the regional spreading of Xist-PRC2
complexes. We have furthermore discovered a surprising non-canonical activity of EZH2 — an activity
independent of its histone methyltransferase activity and that triggers a site-specific cleavage of a SINE repeat
RNA (B2) during the stress response. In the next five years, we will address key follow-up questions. First, we
aim to define specific motifs for the Polycomb-RNA interaction and understand how ATRX influences the
interaction. Second, we will investigate underlying mechanisms for the Xist-PRC2 "spreading" function on the
Xi through mutagenesis of Xist and perturbation of factors required for higher order Xi structures. Finally, we
will examine how EZH2 triggers site-specific cleavage of RNA and determine whether this activity impacts XCI.

## Key facts

- **NIH application ID:** 10428516
- **Project number:** 5R01HD097665-12
- **Recipient organization:** MASSACHUSETTS GENERAL HOSPITAL
- **Principal Investigator:** JEANNIE T LEE
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $690,348
- **Award type:** 5
- **Project period:** 2019-09-01 → 2024-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10428516

## Citation

> US National Institutes of Health, RePORTER application 10428516, Spreading of Xist RNA and Polycomb complexes along the inactive X-chromosome. (5R01HD097665-12). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10428516. Licensed CC0.

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