biostudies-literatureCarty BLEuropean Research CouncilWellcome Trust220-234https://www.ebi.ac.uk/biostudies/studies/S-EPMC7618434biostudies-literatureUnknown33(2)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 reposNature structural & molecular biologyHeterochromatin boundaries maintain centromere position, size and number.PMC7618434101078838304643210645Murillo-Pineda MVolpe EWhittingham OGiunta SFachinetti DDubocanin DMikulski PAltemose NCarty BLJansen LETDumont MHumes JfalseHeterochromatin boundaries maintain centromere position, size and number.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 repos2026-01-01T00:00:00Z2026 Feb2026-07-16T09:39:55.41Z2026-07-09T10:47:04.031ZS-EPMC76184344129133410.1038/s41594-025-01706-2