Nakano, M., Cardinale, S., Noskov, V. N., Gassmann, R., Vagnarelli, P., Kandels-Lewis, S., Larionov, V., Earnshaw, W. C., Masumoto, H.
Inactivation of a Human Kinetochore by Specific Targeting of Chromatin Modifiers
Dev Cell. 2008 Apr 01; 14(4): 507-22.
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.
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