Abstract
Centromeres are defined by a unique single chromatin domain featuring the histone H3 variant, centromere protein A (CENP-A), and ensure proper chromosome segregation. Centromeric chromatin typically occupies a small subregion of low DNA methylation within multimegabase arrays of hypermethylated alpha-satellite repeats and constitutive pericentric heterochromatin. Here, we define the molecular basis of how heterochromatin serves as a 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 noncanonical 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 surrounding DNA methylation and nucleation of additional functional CENP-A domains across the same alpha-satellite sequences. Our study identifies the functional importance and specialization of different H3K9 methyltransferases across centromeric and pericentric domains, crucial for maintaining centromere domain size and suppressing ectopic centromere nucleation events.