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Updated: Oct 26, 2025
Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells
Published on: June 14, 2014
Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes
Yuka Tamura1,2, Tatsuya Ohhata1, Hiroyuki Niida1
1Department of Molecular Biology, Hamamatsu University School of Medicine, Hamamatsu, Japan.
Abstract:
The reactivation of X-linked genes is observed in some primary breast tumors. Two active X chromosomes are also observed in female embryonic stem cells (ESCs), but whether double doses of X-linked genes affect DNA repair efficiency remains unclear. Here, we establish isogenic female/male ESCs and show that the female ESCs are more sensitive to camptothecin and have lower gene targeting efficiency than male ESCs, suggesting that homologous recombination (HR) efficiency is reduced in female ESCs. We also generate Xist-inducible female ESCs and show that the lower HR efficiency is restored when X chromosome inactivation is induced. Finally, we assess the X-linked genes with a role in DNA repair and find that Brcc3 is one of the genes involved in a network promoting proper HR. Our findings link the double doses of X-linked genes with lower DNA repair activity, and this may have relevance for common diseases in female patients, such as breast cancer.
Insights
Female embryonic stem cells with two active X chromosomes show reduced DNA repair efficiency compared to male cells, potentially impacting disease risk in women.
Area of Science:
- Genetics
- Cell Biology
- Genomic Stability
Background:
- X-linked gene reactivation occurs in some breast tumors.
- Female embryonic stem cells (ESCs) possess two active X chromosomes, unlike male ESCs with one.
Purpose of the Study:
- To investigate the impact of double X-chromosome doses on DNA repair efficiency in female ESCs.
- To explore the role of X-linked genes in homologous recombination (HR) and its implications for diseases like breast cancer.
Main Methods:
- Established isogenic female and male ESC lines for direct comparison.
- Utilized camptothecin sensitivity assays and gene targeting efficiency measurements.
- Generated Xist-inducible female ESCs to study X chromosome inactivation (XCI).
- Assessed the involvement of X-linked genes in DNA repair pathways.
Main Results:
- Female ESCs exhibited increased sensitivity to camptothecin and reduced gene targeting efficiency, indicating lower homologous recombination (HR) rates.
- Induction of X chromosome inactivation (XCI) in female ESCs restored HR efficiency to levels comparable to male ESCs.
- Identified Brcc3 as an X-linked gene crucial for a network that promotes efficient HR.
Conclusions:
- The presence of two active X chromosomes in female cells is associated with diminished DNA repair capacity, specifically reduced HR efficiency.
- This finding suggests a potential link between X-linked gene dosage and increased susceptibility to diseases like breast cancer in females.
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