Imaging the fate of histone Cse4 reveals de novo replacement in S phase and subsequent stable residence at centromeres.
The budding yeast centromere contains Cse4, a specialized histone H3 variant. Fluorescence pulse-chase analysis of an internally tagged Cse4 reveals that it is replaced with newly synthesized molecules in S phase, remaining stably associated with centromeres thereafter. In contrast, C-terminally-tagged Cse4 is functionally impaired, showing slow cell growth, cell lethality at elevated temperatures, and extra-centromeric nuclear accumulation. Recent studies using such strains gave conflicting findings regarding the centromeric abundance and cell cycle dynamics of Cse4. Our findings indicate that internally tagged Cse4 is a better reporter of the biology of this histone variant. Furthermore, the size of centromeric Cse4 clusters was precisely mapped with a new 3D-PALM method, revealing substantial compaction during anaphase. Cse4-specific chaperone Scm3 displays steady-state, stoichiometric co-localization with Cse4 at centromeres throughout the cell cycle, while undergoing exchange with a nuclear pool. These findings suggest that a stable Cse4 nucleosome is maintained by dynamic chaperone-in-residence Scm3.DOI: http://dx.doi.org/10.7554/eLife.02203.001.
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, United States wisniewskij@janelia.hhmi.org.
Jan
J
Wisniewski
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
Bassam
B
Hajj
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
Jiji
J
Chen
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, United States.
Gaku
G
Mizuguchi
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, United States.
Hua
H
Xiao
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, United States.
Debbie
D
Wei
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States.
Maxime
M
Dahan
Janelia Farm Research Campus, Howard Hughes Medical Institute, Ashburn, United States Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, United States wuc@janelia.hhmi.org.
Carl
C
Wu
D000705:Anaphase; D002503:Centromere; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D004261:DNA Replication; D004271:DNA, Fungal / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005453:Fluorescence; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
C092746:CSE4 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D029701:Saccharomyces cerevisiae Proteins
3D-PALM; Cse4 histone variant; Scm3 chaperone; centromeric nucleosome; fluorescence pulse-chase; multifocus microscopy