Alternative titles; symbols
HGNC Approved Gene Symbol: DOT1L
Cytogenetic location: 19p13.3 Genomic coordinates (GRCh38) : 19:2,163,933-2,232,578 (from NCBI)
DOT1L is a methyltransferase that methylates lys79 of nucleosomal histone H3 (see 602810) (Feng et al., 2002). DOT1L functions within multiprotein complexes that provide specificity for gene targets to be activated by DOT1L histone H3 methyltransferase activity (Ogoh et al., 2017).
By sequencing clones obtained from a size-fractionated adult hippocampus cDNA library, Nagase et al. (2001) cloned a partial DOT1L cDNA, which they called KIAA1814. RT-PCR followed by ELISA revealed variable but ubiquitous expression. Highest levels were detected in lung, testis, and kidney. Moderate to high levels were found in whole brain and in most individual brain regions tested.
By searching an EST database for sequences homologous to yeast Dot1, Feng et al. (2002) obtained a full-length cDNA encoding DOT1L. The deduced 1,537-amino acid protein has a calculated molecular mass of 165 kD and contains a SAM (sterile alpha motif) domain for protein-protein interaction. DOT1L does not, however, have the SET domain found in most histone lysine methyltransferases.
By radiation hybrid analysis, Nagase et al. (2001) mapped the DOT1L gene to chromosome 19. By genomic sequence analysis, Feng et al. (2002) mapped the DOT1L gene to chromosome 19p13.3.
By in vitro assay of recombinant protein, Feng et al. (2002) determined that DOT1L is inactive against free core histones, but shows significant histone methyltransferase activity against nucleosomes. Analysis of truncated protein or protein mutated within the SAM domain demonstrated that DOT1L is a nucleosomal H3-specific methyltransferase that requires the SAM and sequences between amino acids 351 and 472 for activity. Overexpression of DOT1L in human embryonic kidney cells significantly increased methylation of lys79 of H3, but had no effect on lys4 or lys9. Methylation of lys79, attributed to DOT1L, was regulated during the cell cycle in synchronized HeLa cells. Levels decreased during S phase, reached their lowest point in G2, rose during M phase, and remained high throughout G1.
McGinty et al. (2008) demonstrated direct stimulation of DOT1L-mediated intranucleosomal methylation of H3 at lysine-79 using chemically ubiquitylated H2B (see 609904). Two traceless orthogonal expressed protein ligation (EPL) reactions were used for site-specific H2B ubiquitylation. Reconstitution of ubiquitylated H2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that ubiquitylated H2B directly activated methylation of H3 lysine-79 by DOT1L and was mediated by the DOT1L catalytic domain, most likely through allosteric mechanisms. Asymmetric incorporation of ubiquitylated H2B into dinucleosomes showed that enhancement of methylation was limited to nucleosomes bearing ubiquitylated H2B. McGinty et al. (2008) concluded that their work demonstrates a direct biochemical crosstalk between 2 modifications on separate histone proteins within a nucleosome.
Onder et al. (2012) used short hairpin RNAs to target genes in DNA and histone methylation pathways and identified positive and negative modulators of induced pluripotent stem cell (iPSC) generation. Inhibition of the H3K79 histone methyltransferase DOT1L by short hairpin RNA or a small molecule accelerated reprogramming, significantly increased the yield of iPSC colonies, and substituted for KLF4 (602253) and c-Myc (190080). Inhibition of DOT1L early in the reprogramming process is associated with a marked increase in 2 alternative factors, NANOG (607937) and LIN28 (611043), which play essential functional roles in the enhancement of reprogramming. Genomewide analysis of H3K79me2 distribution revealed that fibroblast-specific genes associated with the epithelial to mesenchymal transition lose H3K79me2 in the initial phases of reprogramming. DOT1L inhibition facilitates the loss of this mark from genes that are fated to be repressed in the pluripotent state. Onder et al. (2012) concluded that their findings implicated specific chromatin-modifying enzymes as barriers to or facilitators of reprogramming, and demonstrated how modulation of chromatin-modifying enzymes can be exploited to more efficiently generate iPSCs with fewer exogenous transcription factors.
Using mice lacking Mllt10 (602409), a cofactor in a subset of Dot1l complexes, Ogoh et al. (2017) found that these Dot1l complexes were required for H3K79 methylation and midfacial development. Mllt10 -/- mouse embryos developed midline facial clefting and ocular hypertelorism by embryonic day 12.5 (E12.5), with reduced H3K79 dimethylation in developing nasal processes, reduced expression of Ap2-alpha (TFAP2A; 107580) and Six2 (604994), reduced cell proliferation in midline, and death around E13.5 due to vascular defects. Pharmacologic inhibition of Dot1l in cultured E9.5 whole mouse embryos resulted in findings at E10.5 that were similar to, but more severe than, those of Mllt10 -/- mice.
Crystal Structure
Min et al. (2003) solved the 2.5-angstrom resolution structure of the catalytic domain of human DOT1L in complex with S-adenosyl-L-methionine (SAM). The structure revealed a unique organization of a mainly alpha-helical N-terminal domain and a central open alpha/beta structure, an active site consisting of a SAM-binding pocket, and a potential lysine-binding channel. The authors showed that a flexible, positively charged region at the C terminus of the catalytic domain is critical for nucleosome binding and enzymatic activity.
Feng, Q., Wang, H., Ng, H. H., Erdjument-Bromage, H., Tempst, P., Struhl, K., Zhang, Y. Methylation of H3-lysine 79 is mediated by a new family of HMTases without a SET domain. Curr. Biol. 12: 1052-1058, 2002. [PubMed: 12123582] [Full Text: https://doi.org/10.1016/s0960-9822(02)00901-6]
McGinty, R. K., Kim, J., Chatterjee, C., Roeder, R. G., Muir, T. W. Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation. Nature 453: 812-816, 2008. [PubMed: 18449190] [Full Text: https://doi.org/10.1038/nature06906]
Min, J., Feng, Q., Li, Z., Zhang, Y., Xu, R.-M. Structure of the catalytic domain of human DOT1L, a non-SET domain nucleosomal histone methyltransferase. Cell 112: 711-723, 2003. [PubMed: 12628190] [Full Text: https://doi.org/10.1016/s0092-8674(03)00114-4]
Nagase, T., Nakayama, M., Nakajima, D., Kikuno, R., Ohara, O. Prediction of the coding sequences of unidentified human genes. XX. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res. 8: 85-95, 2001. [PubMed: 11347906] [Full Text: https://doi.org/10.1093/dnares/8.2.85]
Ogoh, H., Yamagata, K., Nakao, T., Sandell, L. L., Yamamoto, A., Yamashita, A., Tanga, N., Suzuki, M., Abe, T., Kitabayashi, I., Watanabe, T., Sakai, D. Mllt10 knockout mouse model reveals critical role of Af10-dependent H3K79 methylation in midfacial development. Sci. Rep. 7: 11922, 2017. Note: Electronic Article. [PubMed: 28931923] [Full Text: https://doi.org/10.1038/s41598-017-11745-5]
Onder, T. T., Kara, N., Cherry, A., Sinha, A. U., Zhu, N., Bernt, K. M., Cahan, P., Mancarci, B. O., Unternaehrer, J., Gupta, P. B., Lander, E. S., Armstrong, S. A., Daley, G. Q. Chromatin-modifying enzymes as modulators of reprogramming. Nature 483: 598-602, 2012. [PubMed: 22388813] [Full Text: https://doi.org/10.1038/nature10953]