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09:02
Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A
Published on: June 10, 2020
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CENP-A Ubiquitylation Is Indispensable to Cell Viability.
Yohei Niikura1, Risa Kitagawa2, Lei Fang3
1Greehey Children's Cancer Research Institute, Department of Molecular Medicine, UT Health Science Center San Antonio, 8403 Floyd Curl Drive, San Antonio, TX 78229-3000, USA; MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, Nanjing, Jiangsu Province 210061, China.
Developmental Cell
|September 25, 2019
Summary
Centromere-specific histone CENP-A ubiquitylation is crucial for cell viability. Our study demonstrates that CENP-A K124 ubiquitylation is essential, as CENP-A K124R mutants exhibit lethality rescued only by ubiquitin fusion.
Area of Science:
- Epigenetics
- Molecular Biology
- Cell Biology
Background:
- Centromere identity is epigenetically determined by CENP-A, a histone H3 variant.
- The mechanism of CENP-A deposition at centromeres is not fully understood.
- Previous work implicated CENP-A K124 ubiquitylation in deposition, but conflicting data exists.
Purpose of the Study:
- To investigate the essentiality of CENP-A ubiquitylation for centromere function and cell viability.
- To resolve conflicting reports regarding CENP-A K124 ubiquitylation and mutant phenotypes.
- To elucidate the role of CENP-A ubiquitylation in centromere deposition.
Main Methods:
- CRISPR-Cas9 gene editing to create CENP-A K124R knockin mutants.
- Conditional CENP-A knockout system.
- Monoubiquitin fusion rescue experiments.
- Analysis of mutant protein localization and cell viability.
Main Results:
- EYFP tagging of CENP-A K124R induced compensatory ubiquitylation, allowing HJURP binding.
- Flag-tagged or untagged CENP-A K124R mutants exhibited lethality.
- Monoubiquitin fusion rescued the lethality of CENP-A K124R mutants.
- Ubiquitylation of CENP-A is demonstrated as essential for cell viability.
Conclusions:
- CENP-A ubiquitylation, independent of EYFP tagging, is essential for cell viability.
- The K124 residue's ubiquitylation status is critical for CENP-A function.
- This study clarifies the role of CENP-A ubiquitylation in maintaining centromere integrity and cell survival.
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