Cse4 is part of an octameric nucleosome in budding yeast.
The budding yeast CenH3 histone variant Cse4 localizes to centromeric nucleosomes and is required for kinetochore assembly and chromosome segregation. The exact composition of centromeric Cse4-containing nucleosomes is a subject of debate. Using unbiased biochemical, cell-biological, and genetic approaches, we have tested the composition of Cse4-containing nucleosomes. Using micrococcal nuclease-treated chromatin, we find that Cse4 is associated with the histones H2A, H2B, and H4, but not H3 or the nonhistone protein Scm3. Overexpression of Cse4 rescues the lethality of a scm3 deletion, indicating that Scm3 is not essential for the formation of functional centromeric chromatin. We also find that octameric Cse4 nucleosomes can be reconstituted in vitro. Furthermore, Cse4-Cse4 dimerization occurs in vivo at the centromeric nucleosome, and this requires the predicted Cse4-Cse4 dimerization interface. Taken together, our experimental evidence supports the model that the Cse4 nucleosome is an octamer, containing two copies each of Cse4, H2A, H2B, and H4.
Stowers Institute for Medical Research, Kansas City, MO 64110, USA.
Raymond
R
Camahort
Manjunatha
M
Shivaraju
Mark
M
Mattingly
Bing
B
Li
Shima
S
Nakanishi
Dongxiao
D
Zhu
Ali
A
Shilatifard
Jerry L
JL
Workman
Jennifer L
JL
Gerton
D002503:Centromere / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015966:Gene Expression Regulation, Fungal; D006657:Histones / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes; D009154:Mutation; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D011487:Protein Conformation; D055503:Protein Multimerization; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
C092746:CSE4 protein, S cerevisiae; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D006657:Histones; D046912:Multiprotein Complexes; D009707:Nucleosomes; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins