This study investigates the role of histone H4-K20 trimethylation in the establishment of pericentric heterochromatin. The authors demonstrate that H4-K20 trimethylation is enriched at pericentric heterochromatin and is induced by H3-K9 trimethylation, which is mediated by the Suv39h HMTases. They identify two novel SET domain HMTases, Suv4-20h1 and Suv4-20h2, which specifically methylate H4-K20 in a nucleosomal context. These enzymes interact with HP1 isoforms, suggesting a sequential mechanism for the induction of H3-K9 and H4-K20 trimethylation. The evolutionary conservation of H4-K20 trimethylation is highlighted, with the Drosophila homolog, CG13563, acting as a dominant suppressor of position-effect variegation. The study suggests that H3-K9 and H4-K20 trimethylation form a repressive pathway that indexes pericentric heterochromatin.This study investigates the role of histone H4-K20 trimethylation in the establishment of pericentric heterochromatin. The authors demonstrate that H4-K20 trimethylation is enriched at pericentric heterochromatin and is induced by H3-K9 trimethylation, which is mediated by the Suv39h HMTases. They identify two novel SET domain HMTases, Suv4-20h1 and Suv4-20h2, which specifically methylate H4-K20 in a nucleosomal context. These enzymes interact with HP1 isoforms, suggesting a sequential mechanism for the induction of H3-K9 and H4-K20 trimethylation. The evolutionary conservation of H4-K20 trimethylation is highlighted, with the Drosophila homolog, CG13563, acting as a dominant suppressor of position-effect variegation. The study suggests that H3-K9 and H4-K20 trimethylation form a repressive pathway that indexes pericentric heterochromatin.