c Methylation of H3K79 is associated with chromatin at expressed genes, though it is unclear if this histone modification is required for transcription of all genes. Recent studies suggest that Wnt-responsive genes depend particularly on H3K79 methylation, which is catalyzed by the methyltransferase DOT1L. Human leukemias carrying MLL gene rearrangements show DOT1L-mediated H3K79 methylation and aberrant expression of leukemogenic genes. DOT1L inhibitors reverse these effects, but their clinical use is potentially limited by toxicity in Wnt-dependent tissues such as intestinal epithelium. Genome-wide positioning of the H3K79me2 mark in Lgr5 ؉ mouse intestinal stem cells and mature intestinal villus epithelium correlated with expression levels of all transcripts and not with Wnt-responsive genes per se. Selective Dot1l disruption in Lgr5؉ stem cells or in whole intestinal epithelium eliminated H3K79me2 from the respective compartments, allowing genetic evaluation of DOT1L requirements. The absence of methylated H3K79 did not impair health, intestinal homeostasis, or expression of Wnt target genes in crypt epithelium for up to 4 months, despite increased crypt cell apoptosis. Global transcript profiles in Dot1l-null cells were barely altered. Thus, H3K79 methylation is not essential for transcription of Wnt-responsive or other intestinal genes, and intestinal toxicity is not imperative when DOT1L is rendered inactive in vivo.C ovalent histone modifications influence chromatin structure and diverse nuclear functions, including gene regulation (1, 2). Expressed genes are associated with di-or trimethylated H3K4, H3K36, and H3K79 and monomethylated H3K9 and H4K20, whereas repressed genes are enriched for trimethylated H3K9, H3K27, and H4K20 (2-4); various lysine methyltransferases (KMTs) place these marks. H3K79me2 denotes active gene transcription in Saccharomyces cerevisiae, Drosophila, and mammalian cells (5-8). Unlike other modified histone N-terminal "tail" residues, H3K79 is exposed on the nucleosome surface, may be methylated at both heterochromatin and euchromatin (5, 9), and is aberrantly methylated in human leukemias that carry MLL1 gene rearrangements (10, 11).Disruption of Dot1 in yeast or its fly (grappa) and mammalian (Dot1l) homologs eliminates H3K79 methylation, revealing these as the only enzymes capable of H3K79 mono-, di-, and trimethylation (8, 12-15). Dot1 and DOT1L/KMT4 differ from other KMTs in possessing an arginine methyltransferase-like domain instead of a canonical SET domain (12), and H3K79 lacks known demethylases (9). Dot1-dependent H3K79 methylation is associated with telomere silencing and meiotic checkpoint controls (16), DNA repair (17), and modulation of constitutive heterochromatin (15), but its role in transcriptional control has drawn particular attention. Fruit fly grappa mutants dysregulate developmental genes and show embryonic defects (14). Dot1l-null mouse embryos are stunted and die in midgestation of restricted cardiovascular defects (15, 18) that seem incompatible with a ...