Heterochromatin boundaries maintain centromere position, size and number
Centromeres are chromosomal loci that ensure proper chromosome segregation, defined by a unique single chromatin domain featuring the histone H3 variant, Centromere Protein A (CENP-A). Centromeric chromatin typically occupies a small subregion of low DNA methylation within multi-megabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. Here, we define the molecular basis for heterochromatin as the primary driver of centromere and neocentromere position, size and number. Using single-molecule epigenomics, we uncover roles for H3K9me3 methyltransferases SUV39H1/H2 and SETDB1, in addition to non-canonical roles for SUZ12 in maintaining H3K9me3 boundaries at centromeres. Loss of these heterochromatin boundaries leads to the progressive expansion and/or repositioning of the primary CENP-A domain, erosion of DNA methylation, and the nucleation of new functional centromeres across the same alpha satellite sequences. Our study identifies the functional specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size, and suppressing ectopic centromere nucleation events.