Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub

• Published in: Cell reports (Cambridge) Vol. 37; no. 5; p. 109924
• Main Authors: Salinas-Luypaert, Catalina, Allu, Praveen Kumar, Logsdon, Glennis A., Dawicki-McKenna, Jennine M., Gambogi, Craig W., Fachinetti, Daniele, Black, Ben E.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 02.11.2021; Elsevier
• Subjects: Cellular Biology; CENP-A; centromere; functional protein tags; genome editing; histone variant; Life Sciences; mitosis; posttranslational modification; ubiquitin; genome editing; functional protein tags; centromere; posttranslational modification; histone variant; mitosis; CENP-A; ubiquitin
• ISSN: 2211-1247; 2211-1247
• DOI: 10.1016/j.celrep.2021.109924

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-AK124 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. [Display omitted] •Preventing CENP-A ubiquitylation on K124 does not interfere with cell viability•Lysine-free tagged CENP-AK124R supports long-term centromere function•Common functional tags do not mask an essential function for K124 modification•General strategies presented for interrogating the function of specific PTM sites Using three gene replacement strategies, Salinas-Luypaert et al. demonstrate that CENP-AK124ub is not essential for CENP-A function at centromeres. Thus, functional tags do not mask the role of K124 when it is mutated. These strategies can be employed to interrogate posttranslational modifications at the centromere and other cellular locations.