Inactivation of a human kinetochore by specific targeting of chromatin modifiers.
We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric alpha-satellite repeat containing one natural monomer with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity.
Nakano
Megumi
M
Laboratory of Molecular Pharmacology, National Cancer Institute, National Institutes of Health, Building 37, Room 5040, 9000 Rockville Pike, Bethesda, MD 20892, USA.
Cardinale
Stefano
S
Noskov
Vladimir N
VN
Gassmann
Reto
R
Vagnarelli
Paola
P
Kandels-Lewis
Stefanie
S
Larionov
Vladimir
V
Earnshaw
William C
WC
Masumoto
Hiroshi
H
D000818:Animals; D001483:Base Sequence; D002503:Centromere / Q000235:genetics* / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D022222:Chromosomes, Artificial, Human / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D044127:Epigenesis, Genetic; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D018386:Kinetochores / Q000378:metabolism*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D037941:Silencer Elements, Transcriptional
D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D006657:Histones; D011993:Recombinant Fusion Proteins; D004247:DNA