Gene replacement strategies validate the use of functional tags on centromeric chromatin and invalidate an essential role for CENP-AK124ub
Catalina Salinas-Luypaert,
Praveen Kumar Allu,
Glennis A. Logsdon,
Jennine M. Dawicki-McKenna,
Craig W. Gambogi,
Daniele Fachinetti,
Ben E. Black
Affiliations
Catalina Salinas-Luypaert
Institut Curie, PSL University, CNRS, UMR 144, 26 rue d’Ulm, 75005, Paris, France
Praveen Kumar Allu
Department of Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Glennis A. Logsdon
Department of Genome Sciences, University of Washington School of Medicine, Seattle, WA 98195, USA
Jennine M. Dawicki-McKenna
Department of Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA
Craig W. Gambogi
Department of Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Program in Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA
Daniele Fachinetti
Institut Curie, PSL University, CNRS, UMR 144, 26 rue d’Ulm, 75005, Paris, France; Corresponding author
Ben E. Black
Department of Biochemistry and Biophysics, Penn Center for Genome Integrity, Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Graduate Program in Biochemistry and Molecular Biophysics, University of Pennsylvania, Philadelphia, PA 19104, USA; Corresponding author
Summary: 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.
