Histone H3 lysine methyltransferase activities control compartmentalization of human centromeres

Centromeres are essential chromosomal regions that ensure accurate genome segregation during cell division. They are organized into epigenetically discrete compartments: a Centromere Protein A (CENP-A)-rich core for microtubule attachment and surrounding heterochromatic pericentromeres that promote cohesion. Despite their importance, the mechanisms that define, enforce and partition these chromatin domains remain poorly understood. To address this, we disrupted key H3K9 methyltransferases- SUV39H1, SUV39H2, and SETDB1- that establish heterochromatin in humans. We find that SETDB1 is required for H3K9 dimethylation at core centromeres, while SUV39H1/2 complete trimethylation. Unexpectedly, depleting all three enzymes results in aberrantly high H3K9me3, driving CENP-A expansion into pericentromeres. This promiscuous deposition is mediated by G9a/GLP methyltransferases, which selectively reestablish H3K9me3 within the centromere core. SETDB1, regardless of its enzymatic activity, blocks G9a/GLP-mediated heterochromatin deposition and CENP-A expansion, revealing a novel, catalytic-independent function in safeguarding centromeres. Overall, our work defines the molecular logic governing centromeric repression, and uncovers foundational principles of epigenetic compartmentalization.