Loss of CHFR in human mammary epithelial cells causes genomic instability by disrupting the mitotic spindle assembly checkpoint.
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.
Privette
Lisa M
LM
Department of Human Genetics, University of Michigan, Ann Arbor, MI48109, USA.
Weier
Jingly Fung
JF
Nguyen
Ha Nam
HN
Yu
Xiaochun
X
Petty
Elizabeth M
EM
D000782:Aneuploidy; D064127:Aurora Kinases; D002135:Calcium-Binding Proteins / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics* / Q000378:metabolism; D002478:Cells, Cultured; D020090:Chromosome Segregation / Q000235:genetics; D004847:Epithelial Cells / Q000378:metabolism; D017353:Gene Deletion; D005786:Gene Expression Regulation; D018816:Genes, cdc; D042822:Genomic Instability / Q000235:genetics* / Q000502:physiology; D006801:Humans; D064246:Mad2 Proteins; D042361:Mammary Glands, Human / Q000378:metabolism*; D008954:Models, Biological; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism; D008941:Spindle Apparatus / Q000235:genetics*; D014404:Tubulin / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases
D002135:Calcium-Binding Proteins; D018797:Cell Cycle Proteins; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009363:Neoplasm Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; D012097:Repressor Proteins; D014404:Tubulin; C412002:CHFR protein, human; D044767:Ubiquitin-Protein Ligases; D064127:Aurora Kinases; D017346:Protein Serine-Threonine Kinases