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Loss of CHFR in human mammary epithelial cells causes genomic instability by disrupting the mitotic spindle assembly
Lisa M Privette1, Jingly Fung Weier, Ha Nam Nguyen
1Department of Human Genetics, University of Michigan, Ann Arbor, MI48109, USA.
Abstract:
CHFR is an E3 ubiquitin ligase and an early mitotic checkpoint protein implicated in many cancers and in the maintenance of genomic stability. To analyze the role of CHFR in genomic stability, by siRNA, we decreased its expression in genomically stable MCF10A cells. Lowered CHFR expression quickly led to increased aneuploidy due to many mitotic defects. First, we confirmed that CHFR interacts with the mitotic kinase Aurora A to regulate its expression. Furthermore, we found that decreased CHFR led to disorganized multipolar mitotic spindles. This was supported by the finding that CHFR interacts with alpha-tubulin and can regulate its ubiquitination in response to nocodazole and the amount of acetylated alpha-tubulin, a component of the mitotic spindle. Finally, we found a novel CHFR interacting protein, the spindle checkpoint protein MAD2. Decreased CHFR expression resulted in the mislocalization of both MAD2 and BUBR1 during mitosis and impaired MAD2/CDC20 complex formation. Further evidence of a compromised spindle checkpoint was the presence of misaligned metaphase chromosomes, lagging anaphase chromosomes, and defective cytokinesis in CHFR knockdown cells. Importantly, our results suggest a novel role for CHFR regulating chromosome segregation where decreased expression, as seen in cancer cells, contributes to genomic instability by impairing the spindle assembly checkpoint.
Insights
The study reveals that decreased expression of the CHFR protein, an E3 ubiquitin ligase, leads to genomic instability. This occurs through mitotic defects, impaired spindle assembly, and compromised chromosome segregation, particularly relevant in cancer cells.
Area of Science:
- Cell Biology
- Genetics
- Cancer Research
Background:
- CHFR (Checkpoint with FHA and RING domains) is an E3 ubiquitin ligase and mitotic checkpoint protein crucial for genomic stability.
- Its role in maintaining genomic integrity and its implication in various cancers highlight the need for further investigation.
Purpose of the Study:
- To elucidate the specific functions of CHFR in maintaining genomic stability.
- To investigate the molecular mechanisms by which CHFR regulates mitosis and the spindle assembly checkpoint.
Main Methods:
- Utilized siRNA to decrease CHFR expression in MCF10A cells.
- Confirmed protein interactions using co-immunoprecipitation.
- Assessed mitotic spindle organization, chromosome segregation, and cytokinesis through microscopy and molecular assays.
Main Results:
- Reduced CHFR expression caused aneuploidy and mitotic defects, including disorganized multipolar spindles.
- CHFR was found to interact with Aurora A and alpha-tubulin, regulating their expression and ubiquitination.
- CHFR knockdown led to mislocalization of MAD2 and BUBR1, impaired MAD2/CDC20 complex formation, and defects in chromosome segregation and cytokinesis.
Conclusions:
- CHFR plays a critical role in regulating chromosome segregation and the spindle assembly checkpoint.
- Decreased CHFR expression, observed in cancers, contributes to genomic instability by compromising these essential cellular processes.
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