biostudies-literatureTatarakis ANIH National Institute of General Medical SciencesHoward Hughes Medical InstituteHelen Hay Whitney FoundationNIGMS NIH HHS3388-3406.e12https://www.ebi.ac.uk/biostudies/studies/S-EPMC12478525biostudies-literatureUnknown85(18)Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing transposons and genes, but the mechanisms that establish and maintain these domains are not fully understood. Here, we use a CRISPR-based genetic screen to investigate the requirements for establishment and maintenance of histone H3 lysine 9 trimethylation (H3K9me3) heterochromatin. In mouse embryonic stem cells (mESCs), we show that transiently induced H3K9me3 heterochromatin is inherited for a limited number of cell divisions, independently of sequence-dependent recruitment, but becomes stable upon differentiation, concomitant with downregulation of enzymes erasing H3K9me and DNA methylation. In addition, ordered and non-redundant activities of multiple H3K9 and DNA methyMolecular cellRequirements for establishment and epigenetic stability of mammalian heterochromatin.PMC12478525R01 GM072805Moazed DSaini HPinzon-Arteaga CATatarakis AFeng WYu JfalseRequirements for establishment and epigenetic stability of mammalian heterochromatin.Heterochromatic domains of DNA account for a large fraction of mammalian genomes and play critical roles in silencing transposons and genes, but the mechanisms that establish and maintain these domains are not fully understood. Here, we use a CRISPR-based genetic screen to investigate the requirements for establishment and maintenance of histone H3 lysine 9 trimethylation (H3K9me3) heterochromatin. In mouse embryonic stem cells (mESCs), we show that transiently induced H3K9me3 heterochromatin is inherited for a limited number of cell divisions, independently of sequence-dependent recruitment, but becomes stable upon differentiation, concomitant with downregulation of enzymes erasing H3K9me and DNA methylation. In addition, ordered and non-redundant activities of multiple H3K9 and DNA methy2025-01-01T00:00:00Z2025 Sep2026-06-04T00:10:08.579Z2026-05-03T03:12:32.012ZS-EPMC124785254097252510.1016/j.molcel.2025.08.025