Histone lysine methylation encodes genomic functions into the chemical state of nucleosomes (38
). The collective actions of lysine methyltransferase and lysine demethylase enzymes maintain a landscape of steady-state methylation of histones around which eukaryotic DNA is packaged. Histone methylation can facilitate or abrogate a variety of protein-protein interactions occurring along the chromatin fiber, thus permitting stable regulation over localized regions of the genome. Several recent high-throughput descriptions of histone lysine methylation across mammalian genomes have documented the pervasiveness of this form of epigenetic organization (2
). However, the full biological significance of most histone lysine methylation pathways in mammals has yet to be revealed.
Methylation of histone H3 at lysine 79 (H3K79) is conserved among most eukaryotic species. In budding yeast, nearly 90% of histone H3 bears monomethylation (H3K79me1), dimethylation (H3K79me2), or trimethylation (H3K79me3) at lysine 79, all catalyzed exclusively by the histone methyltransferase Dot1 (27
). H3K79 methylation is widely distributed across the euchromatic yeast genome but markedly depleted at heterochromatic mating-type, ribosomal DNA, and telomeric loci (26
). Genes in these regions are controlled by silent information regulator (SIR) proteins, which can bind nucleosomes and silence transcription (reviewed in reference 33
). Genetic, as well as biochemical, evidence suggests a mutual antagonism between H3K79 methylation by Dot1 and the association of SIR proteins with chromatin (1
). Therefore, the action of Dot1 in yeast serves to impose a boundary that confines SIR proteins to silent telomeric heterochromatin. In Drosophila
, H3K79 methylation is also a prominent modification within euchromatin, with levels in transcribed regions correlating with gene activity (35
). Mutations in the Drosophila
Dot1 ortholog grappa
lead to Polycomb
), suggesting that H3K79 methylation may influence developmentally regulated gene expression in multicellular eukaryotes.
DOT1L, the mammalian ortholog, displays enzymatic properties similar to those of its counterpart in yeast (9
). Accordingly, H3K79 methylation can be detected on mammalian histones by mass spectrometry, with monomethylation being the most abundant species and correlating with the fraction of histone H3 modified by acetylation, suggesting enrichment at active genes (53
). Several reports of individual genes in mammalian cells have correlated H3K79 methylation with transcriptional activation but also with gene repression (15
). All three degrees of H3K79 methylation were recently examined across the human genome by Solexa sequencing of DNA obtained by chromatin immunoprecipitation (ChIP) (2
). It was reported that H3K79me3 is enriched at both silent and active genes, with silent regions having overall higher levels of this modification. H3K79me1 and H3K79me2 were not found to show a significant preference for either active or silent genes. These seemingly contradictory descriptions of H3K79 methylation within mammalian chromatin highlight the need to better understand the mechanisms that recruit DOT1L to genomic sites in vivo and the relationship of H3K79 methylation with gene transcription.
The MLL1 gene, which encodes a histone H3K4 methyltransferase, frequently undergoes chromosomal translocations in acute leukemia. The resulting oncogenic fusion protein encodes the N terminus of MLL1 (which lacks methyltransferase activity) fused to the C terminus of a heterogeneous group of partner molecules (19
). Interestingly, several fusion partners of MLL1 encode proteins that bind directly or indirectly to DOT1L (5
). The inappropriate recruitment of DOT1L to MLL1-regulated genes in the Hox cluster leads to H3K79 hypermethylation, increased transcription, and a block in hematopoietic cell differentiation (28
). Thus, DOT1L has the potential to be an important regulator of gene expression in mammalian cells and represents a potential therapeutic target in this disease.
In this study, we examined the recruitment of DOT1L and the patterning of mono-, di-, and trimethylation of H3K79 under dynamic conditions in several mammalian cell lineages. Our findings revealed the ubiquitous nature of DOT1L recruitment and H3K79 methylation at actively transcribed chromatin. We also observed strong similarities between the patterning of H3K79 methylation and that of H3K4 methylation within mammalian chromatin that may reflect parallel pathways specifying gene activity or antagonizing gene silencing. Our study also revealed a novel pattern of H3K79 methylation that correlates with binding of DNA-binding activators at regulatory elements.