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Updated: Jun 24, 2025
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Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A
Published on: June 10, 2020
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Native and tagged CENP-A histones are functionally inequivalent.
Minh Bui1, Songjoon Baek2, Reda S Bentahar2
1Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, 41 Medlars Drive, Bldg 41/Rm B1300, Bethesda, MD, 20892, USA. buim@mail.nih.gov.
Epigenetics & Chromatin
|June 2, 2024
Summary
Adding tags to the CENP-A protein, a histone variant, alters its function. Tagged CENP-A shows different binding, modifications, and chaperone use, impacting its biological role.
Area of Science:
- Cell Biology
- Molecular Biology
- Epigenetics
Background:
- Biochemical and fluorescent tags are crucial for studying cellular processes.
- Nuclear proteins, like CENP-A, present challenges due to low abundance and poor antibody recognition.
- Protein tagging is a common method to overcome these challenges for protein enrichment, purification, and visualization.
Purpose of the Study:
- To investigate whether C-terminally TAP-tagged CENP-A recapitulates the native function of the histone variant.
- To identify potential alterations in kinetochore binding, post-translational modifications, and chaperone utilization of tagged CENP-A.
Main Methods:
- Introduction of C-terminally TAP-tagged CENP-A in human cells.
- Analysis of kinetochore protein binding and post-translational modifications (PTMs) of tagged CENP-A.
- Assessment of histone chaperone utilization and centromeric incorporation of tagged CENP-A nucleosomes.
Main Results:
- C-terminally TAP-tagged CENP-A exhibited altered kinetochore protein binding and distinct post-translational modifications (PTMs).
- Tagged CENP-A utilized different histone chaperones compared to native CENP-A and could partially displace it.
- CENP-A-containing nucleosomes with tags showed reduced centromeric incorporation and poor association with linker histone H1.5.
Conclusions:
- Expressing tagged versions of the histone variant CENP-A may lead to the use of non-native pathways.
- Tagging CENP-A can unexpectedly alter its biological function and interactions within the centromere.
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