# The role of EWSR1 at the centromere--

> **NIH NIH R01** · UNIVERSITY OF TEXAS HLTH SCIENCE CENTER · 2024 · $56,953

## Abstract

The centromere is a unique chromosomal region that is essential for high-fidelity chromosome transmission
during cell division. In mitosis, microtubules attached to the kinetochore help ensure the faithful segregation of
sister chromatids into daughter cells. Our goal is to understand the mechanisms that govern centromere function.
Centromere identity relies on the deposition of the centromere-specific histone H3 variant, CENP-A but not on
specific DNA sequences. After DNA replication, “old” centromeric nucleosomes are transferred onto the newly
replicated chromatids. In mammals, deposition of CENP-A occurs in the G1 phase of the cell cycle. The
mechanisms of CENP-A deposition and maintenance are crucial for proper centromere inheritance and function
but remain to be delineated. We recently found that EWSR1 (Ewing Sarcoma Breakpoint Region 1) is required
for CENP-A deposition and maintenance at the centromere. We show that EWSR1 and EWSR1-FLI1 (the
oncogenic fusion protein in Ewing sarcoma) bind to CENP-A through the SYGQ2 region within its prion-like
domain known to be important for phase separation. We found that EWSR1 is required for CENP-A deposition
at the centromere. CENP-A is associated with centromeric RNA, which is important for centromere function. We
demonstrate that EWSR1 plays a key role in promoting this association. We also have biochemical evidence
that EWSR1 binds R-loops, a three-stranded RNA-DNA hybrid, through its RNA-recognition motif. Based on
these findings, we hypothesize that EWSR1 plays crucial roles in maintaining centromere structure and function
by binding RNA and anchoring CENP-A in centromeric chromatin. We will test this hypothesis. Aim 1: To
determine the function of EWSR1 and RNA/R-loops in centromere maintenance. We generated EWSR1
conditional KO cells using an AID (auxin-inducible degron) system, which allows us to remove EWSR1 upon
auxin addition. We will test if auxin-induced degradation of EWSR1 abolishes CENP-A signals at the centromere
at different cell cycle stages. We will induce the expression of SETX, which clears RNA-DNA hybrids, at different
cell cycle stages to ask whether centromeric RNA/R-loops are required for CENP-A maintenance at the
centromere. Aim 2: To investigate the function of EWSR1-CENP-A interaction. We will generate EWSR1 mutants
defective in binding to CENP-A by introducing mutations into the SYGQ2 region of EWSR1. We will examine the
phenotypes of the EWSR1 KO cells expressing CENP-A binding deficient mutants. We will also identify the
EWSR1-binding domain of CENP-A. We will then isolate CENP-A mutants impaired for EWSR1 binding. We will
express Flag-tagged mutants in CENP-A KO cells to examine the localization of CENP-A and the phenotypes of
the mutant cells. We will investigate if EWSR1 association with CENP-A is unique for interphase. We will
investigate the mechanism that regulates cell cycle specificity of EWSR1-CENP-A interaction. Aim 3: To test if
EWSR1 anchors CENP-A at the c...

## Key facts

- **NIH application ID:** 11042908
- **Project number:** 3R01GM147636-02S2
- **Recipient organization:** UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
- **Principal Investigator:** KATSUMI KITAGAWA
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $56,953
- **Award type:** 3
- **Project period:** 2023-04-01 → 2025-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11042908

## Citation

> US National Institutes of Health, RePORTER application 11042908, The role of EWSR1 at the centromere-- (3R01GM147636-02S2). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/11042908. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*