# Developmental constraints shaping human sex chromosomes and escape from X inactivation

> **NIH NIH R35** · UNIVERSITY OF CONNECTICUT SCH OF MED/DNT · 2024 · $449,550

## Abstract

PROJECT SUMMARY
 Biological sex in placental mammals is determined by their X and Y chromosomes, which, though once
a pair, diverged after the Y was confined to the male germline, and progressively lost most of its ancestral
genes. To maintain dosage parity of X-linked genes with XY males, XX females evolved a mechanism to
randomly silence one X in each cell during early development, a process termed X chromosome inactivation
(XCI). However, some genes evolved to escape XCI and resist attrition on the Y. These “escapee” genes
are expressed from two copies in males and females alike, because development is sensitive to their
dosage. Indeed, lack of the second sex chromosome (monosomy-X) causes miscarriage in many placental
mammals. Another group of genes is specific to the X, but nonetheless escapes XCI, and thereby
contributes to sex-divergent gene expression.
 The relevance of genes escaping XCI to human health and development is evidenced by sex
differences in disease, monosomy-X leading all causes of miscarriage, and live-born Turner syndrome (TS),
which predisposes TS females to fatal cardiac events due to altered metabolism and vascular
malformations. Yet, it remains unknown how and which escapees contribute to this outsized health burden,
because far fewer genes escape XCI in the pre-eminent mouse model, which therefore tolerates
monosomy-X without miscarriage and little developmental impact overall.
 To address this gap, we generated monosomy-X alongside isogenic euploid human induced pluripotent
stem cells (hiPSCs) from male and female samples mosaic for the second sex chromosome. We exploited
this unique resource to demonstrate impaired placental gene expression in a trophoblast model of
monosomy-X, and herein propose to extend these studies to link specific X/Y gene pairs and downstream
pathways to this miscarriage-relevant phenotype. Our deep characterization of euploid XX hPSCs also
revealed that escapee genes likely seed X reactivation when XCI master regulator XIST is repressed. Here,
we propose to use novel hPSC lines we since established to elucidate the mechanisms of XCI, escape and
reactivation, all of which have remained largely enigmatic in humans. Our sophisticated epigenetic tools
enable de novo establishment and maintenance of XCI in female hPSCs, which also will benefit the
generation of validated and stable hiPSC lines for in vitro modeling of X-linked disease and sex differences.
We anticipate this research program to ultimately yield mechanistic insights that may inform new means for
reactivation of functional alleles in heterozygous females manifesting X-linked recessive and dominant
disorders, and provide molecular-genetic approaches towards dissecting escapee gene contributions to TS
and human sex differences.

## Key facts

- **NIH application ID:** 10842881
- **Project number:** 1R35GM153326-01
- **Recipient organization:** UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
- **Principal Investigator:** Stefan F. Pinter
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $449,550
- **Award type:** 1
- **Project period:** 2024-08-01 → 2029-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10842881

## Citation

> US National Institutes of Health, RePORTER application 10842881, Developmental constraints shaping human sex chromosomes and escape from X inactivation (1R35GM153326-01). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10842881. Licensed CC0.

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