A METHOD FOR POSITIVELY OR NEGATIVELY REGULATING THE ASSEMBLY OF NEWLY SYNTHESIZED CENP-A TO EXOGENOUS ALPHOID DNA CONTAINING CENP-B BOXES IN MAMMALIAN CELL LINES

摘要

We have discovered that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays is regulated by a histone H3K9 acetyl/methyl switch. Tethering of histone acetyltransferases (HATs) to alphoid DNA arrays breaks a cell type-specific barrier for de novo stable CENP-A assembly and induces assembly of other kinetochore proteins at the ectopic alphoid site similarly with the tethering of CENP-A deposition factors hMis18alpha or HJURP. HAT tethering bypasses the need for hMis18alpha, but requires HJURP for de novo kinetochore assembly. In contrast, tethering of H3K9 tri-methylase (Suv39h1) to the array causes methylation of H3K9, preventing de novo CENP-A assembly and kinetochore formation. CENP-A arrays assembled de novo by this mechanism can form kinetochores of human artificial chromosomes (HACs) that are propagated indefinitely in human cells.