1. Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-A K124ub .
- Author
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Salinas-Luypaert C, Allu PK, Logsdon GA, Dawicki-McKenna JM, Gambogi CW, Fachinetti D, and Black BE
- Subjects
- Cell Line, Tumor, Cell Survival, Centromere genetics, Centromere Protein A genetics, Chromatin genetics, Chromatin Assembly and Disassembly, Colonic Neoplasms genetics, Gene Editing, Humans, Lysine, Mutation, Ubiquitination, Centromere metabolism, Centromere Protein A metabolism, Chromatin metabolism, Colonic Neoplasms metabolism, Genetic Techniques, Retinal Pigment Epithelium metabolism
- Abstract
Functional tags are ubiquitous in cell biology, and for studies of one chromosomal locus, the centromere, tags have been remarkably useful. The centromere directs chromosome inheritance at cell division. The location of the centromere is defined by a histone H3 variant, CENP-A. The regulation of the chromatin assembly pathway essential for centromere inheritance and function includes posttranslational modification (PTM) of key components, including CENP-A itself. Others have recently called into question the use of functional tags, with the claim that at least two widely used tags obscured the essentiality of one particular PTM, CENP-A
K124 ubiquitination (ub). Here, we employ three independent gene replacement strategies that eliminate large, lysine-containing tags to interrogate these claims. Using these approaches, we find no evidence to support an essential function of CENP-AK124ub . Our general methodology will be useful to validate discoveries permitted by powerful functional tagging schemes at the centromere and other cellular locations., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2021
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