# Identifying the Xist interactome during dynamic X inactivation in healthy and lupus-derived B cells

> **NIH NIH F32** · UNIVERSITY OF PENNSYLVANIA · 2020 · $64,926

## Abstract

PROJECT SUMMARY
I am presently a postdoctoral researcher at the University of Pennsylvania (Penn); under the supervision of Dr.
Montserrat Anguera I am training to become an independent scientist with an eye toward starting my own
laboratory and pursuing questions relevant to human immune health and disease. In Dr. Anguera’s lab, we are
interested in X inactivation mechanisms in lymphocytes and their implications for human autoimmune disease.
Inactivation of one X chromosome usually happens very early in development and remains constant throughout
the life of the cell in order to keep gene expression equal between XX and XY individuals. X Chromosome
Inactivation (XCI) is initiated and maintained by expression of an RNA transcript known as Xist, which is
transcribed from the future inactive X (Xi) then spreads up and down the chromosome to cover it in a “cloud” of
Xist RNA, which remains on the Xi for the life of the cell and any daughter cells. Unlike nearly all other XX cells
in a woman’s body, B- and T- immune cells, also known as lymphocytes, have a dynamic X chromosome
inactivation mechanism. The Xist cloud on the Xi dissipates in naïve lymphocytes and returns to coat the Xi once
the cells are activated. In patients with lupus, Xist successfully evacuates the Xi in naïve lymphocytes but fails
to fully return after activation. My project takes a closer look at the mechanisms by which Xist leaves and returns
to the Xi during B lymphocyte activation. Between 80 and 200 proteins have been shown to bind to Xist and
additional experiments have elucidated their role in XCI, however, none of the previous high-throughput analyses
have been performed in cells that exhibit this novel approach to X inactivation, which so far appears to be specific
to lymphocytes. Based on previous findings from our laboratory and publicly available gene expression data from
other labs, I hypothesize that two proteins, cohesin (SMC3) and Lamin B Receptor (LBR) play critical roles in
this process in healthy and lupus B cells. In order to test this hypothesis, I propose to perform a biotin-probe
based Xist RNA pull-down called Comprehensive identification of RNA-binding proteins by mass spectrometry
(ChIRP-MS) in B cells from mice and humans. The ChIRP-MS approach collects all the Xist RNA in the cell and
brings with it any proteins bound to the Xist RNA, those proteins are then identified using a mass spectrometer
for simultaneous identification of every protein. I’ll perform ChIRP-MS in healthy naïve and activated B cells as
well as activated B cells from mouse models of lupus and human lupus patients. After mass spectrometry
identification of proteins, I will compare the proteins associated with Xist in naïve to activated B cells and healthy
to lupus B cells and look for those proteins enriched in either subset. Results from these experiments will not
only confirm or modify my predictions about the role of LBR and SMC3 in X inactivation but will allow for
comparisons bet...

## Key facts

- **NIH application ID:** 10068735
- **Project number:** 1F32AI154797-01
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Sarah Catherine Pyfrom
- **Activity code:** F32 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $64,926
- **Award type:** 1
- **Project period:** 2020-07-01 → 2023-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10068735

## Citation

> US National Institutes of Health, RePORTER application 10068735, Identifying the Xist interactome during dynamic X inactivation in healthy and lupus-derived B cells (1F32AI154797-01). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/10068735. Licensed CC0.

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