Hierarchical inactivation of a synthetic human kinetochore by a chromatin modifier.
We previously used a human artificial chromosome (HAC) with a synthetic kinetochore that could be targeted with chromatin modifiers fused to tetracycline repressor to show that targeting of the transcriptional repressor tTS within kinetochore chromatin disrupts kinetochore structure and function. Here we show that the transcriptional corepressor KAP1, a downstream effector of the tTS, can also inactivate the kinetochore. The disruption of kinetochore structure by KAP1 subdomains does not simply result from loss of centromeric CENP-A nucleosomes. Instead it reflects a hierarchical disruption of the outer kinetochore, with CENP-C levels falling before CENP-A levels and, in certain instances, CENP-H being lost more readily than CENP-C. These results suggest that this novel approach to kinetochore dissection may reveal new patterns of protein interactions within the kinetochore.
Cardinale
Stefano
S
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, United Kingdom.
Bergmann
Jan H
JH
Kelly
David
D
Nakano
Megumi
M
Valdivia
Manuel M
MM
Kimura
Hiroshi
H
Masumoto
Hiroshi
H
Larionov
Vladimir
V
Earnshaw
William C
WC
D001324:Autoantigens / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D002503:Centromere / Q000235:genetics / Q000378:metabolism; D000076247:Centromere Protein A; D002843:Chromatin / Q000235:genetics / Q000378:metabolism*; D047369:Chromatin Immunoprecipitation; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D022222:Chromosomes, Artificial, Human / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D006822:Hybrid Cells; D017404:In Situ Hybridization, Fluorescence; D018386:Kinetochores / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000076183:Tripartite Motif-Containing Protein 28
D001324:Autoantigens; C000619700:CENPA protein, human; D000076247:Centromere Protein A; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D009707:Nucleosomes; D012097:Repressor Proteins; C060905:centromere protein C; C101116:TRIM28 protein, human; D000076183:Tripartite Motif-Containing Protein 28