Dot1 forms a 2-MDa complex with AF10, AF9, ENL, Skp1, and TRRAP. (A) Schematic showing the steps involved in the purification of the DotCom. Flag-tagged Dot1, expressed under an inducible promoter in HEK293T cells, was isolated by Flag affinity chromatography. Resulting protein complexes were then separated by size exclusion on a Superose 6 column. (B) Expression and affinity purification of Dot1. Flag affinity-purified protein complexes were analyzed by silver staining and SDS-PAGE. The arrowhead indicates the position of Flag-Dot1. Flag affinity-purified preparations from nuclear extracts from non-Flag-expressing HEK293T cells were used as a control (293 Control). (C) A HMTase assay with Dot1 and 293 Flag eluates on recombinant yeast nucleosomes was performed overnight in the presence or absence of S-adenosyl methionine (SAM) and analyzed by Western blotting using antibodies specific toward H3K79me2 and H3K79me3. Signals for H3K79me2 and H3K79me3 demonstrate the expected specificity of DotCom in carrying out H3K79 methylation. Total H3 levels were assayed as a loading control. (D) Human Dot1 is in a 2-MDa complex. Silver staining of size exclusion chromatography for Flag-Dot1 preparations from A is shown. From fractions 8 to 16, a peak of Dot1 is seen at 2 MDa (fraction 10), and MudPIT analysis revealed the presence of Dot1, TRRAP, AF10, AF17, AF9, ENL, and Skp1 as indicated. Western blotting with anti-Flag and anti-Dot1 antibodies shows a similar peak at fraction 10. ENL comigrates with the Dot1 peak in these fractions. Control Flag eluates are prepared as in B. (E) Human Dot1 has HMT activity. HMT assay was performed as in C with Dot1-Flag eluate as input, Control 293 Flag eluate, and fractions 8 and 10 from the size exclusion chromatography from D. The HMT activity of fraction 10 demonstrates that the ∼2-MDa DotCom is enzymatically active. Coomassie staining shows the integrity of the four core histones in these nucleosomes.

Purification of AF10, ENL, and AF9 complexes identify Dot1 as a common component. (A) Clonal cell lines expressing Flag-tagged AF10, AF9, and ENL under a tetracycline-inducible promoter were generated in HEK293T cells, from which Dignam nuclear extracts were prepared for Flag purification and silver staining. Arrowheads show the positions of Flag-tagged proteins. A 293 Control Flag purification from untransfected 293 cells is shown as control. (B) Flag affinity purifications of Flag-AF10, Flag-AF9, Flag-ENL, and 293 Control nuclear extracts were probed with Dot1 antibody, revealing the presence of Dot1 in all purifications except the control 293 extracts. (C) Radioactive HMTase assay was performed using immunocomplexes purified from Flag-Dot1, Flag-AF9, and 293 Control extracts on yeast recombinant nucleosomes. The fluorograph shows the activity of Flag-Dot1 and Flag-AF9, but not 293 Control preparations, toward H3. Purified recombinant yeast Dot1 was used as a positive control in the reaction. (D) MudPIT analysis of two independent Flag-Dot1 purifications (Dot1-dNSAF-A and Dot1-dNSAF-B) and of the Superose 6 size exclusion peak fraction 10 from a Flag-Dot1 purification (Dot1-SE Fract. 10). dNSAF values indicate an estimate of the relative abundance of AF9, AF10, ENL, AF17, and Skp1. The presence of similar amounts of these proteins in the 2-MDa fraction 10 indicates that these proteins exist in DotCom. (E,F) MudPIT analysis of Flag eluates of Flag-AF10, Flag-ENL, and Flag-AF9. (E) dNSAF values of proteins are plotted on the Y-axis, and are given in the table shown in F. Numbers in bold indicate the values for the tagged protein.

dDot1 (gpp), dAF10 (Alhambra), ENL/AF9-related, and dSkp1 (skpB) specifically regulate the expression of a wingless target gene, senseless. (A′–F′) Staining of wing imaginal discs from Drosophila third instar larvae with antibodies to the Wingless target gene senseless. Using the UAS-GAL4 system, en-GAL4 was used to drive the expression of UAS-hairpin constructs targeting Drosophila Dot1 and its interactors. (A–H) The domain of knockdown is marked by GFP, which is also expressed from a UAS. (A′–F′) Arrows point to the location where senseless is normally expressed in two stripes. (A–A″) yw crossed to the en-GAL4 driver was used as a control and shows the wild-type expression of senseless in the wing pouch. (A″–G″) Merge of GFP and Senseless signals. Knockdowns of dDot1 (B–B″), dAF10 (C–C″), and dSkp1 (E–E″) lead to major reductions in Senseless, with lesser reductions in Senseless levels observed for knockdown of dENL/AF9 (D–D″). (F–F″) No change in Senseless levels after TRRAP RNAi were observed in this genetic background, but reductions could be seen in an enhanced RNAi background overexpressing Dicer-2 (see Supplemental Fig. 2). (G″–H″′) Immunofluorescence analysis of Distalless (Dll) and Vestigial (Vg), the products of two low-threshold wingless target genes; distalless (G–G″) and vestigial (H–H″) are not affected by the knockdown of dDot1 (gpp-v16001), as seen by similar broad stripes outside and inside the domain of RNAi knockdown as indicated by GFP staining in the domain of en-Gal4-driven expression.

Human Dot1 trimethylates H3K79 in the presence of monoubiquitination of H2B. (A) HMTase assays with Dot1 Flag immunocomplex and nucleosomal substrates extracted as crude yeast nuclear extracts from wild-type (DOT1), ΔDot1, and H2BK123A DOT1 strains. ΔDot1 nucleosomes serve as H2B monoubiquitinated substrates (Ubq Nuc), as they have wild-type levels of H2B monoubiquitination and are devoid of any H3K79 methylation, and H2BK123A DOT1 nucleosomes serve as nonmonoubiquitinated substrates, as they lack monoubiquitination on H2B with Lys123 mutated to alanine. Comparison of the HMTase activity of hDotCom toward H3K79me, H3K79me2, and H3K79me3 on monoubiquitinated H2B and nonmonoubiquitinated H2B nucleosomal substrates shows a preferential activity of hDotCom toward trimethylation of H3K79 and, to a lesser extent, toward dimethylation in the presence of monoubiquitinated H2B, but has no difference in the ability to mono- or dimethylate H3K79. An identical titration of hDotCom was used for both monoubiquitinated H2B and nonmonoubiquitinated H2B nucleosomal substrates. H3 was used as a loading control. (B) Fifty HMT reactions (25 μL each, with 0.88 pmol of nucleosome) were performed in the presence or absence of SAM overnight using the catalytic domain of recombinant human Dot1 (1–416), pooled, and then analyzed by Western blotting. (C–E) Detection, identification, and quantitation of histone H3 Arg-C-digested “R.LVREIAQDFKTDLR.F” peptides bearing no modification, or mono-, di-, or trimethylation on Lys79 from the reaction mixture in B. Endoproteinase Arg-C-digested peptides were analyzed with a 12-step MudPIT on an Eksigent NanoLC two-dimensional system coupled to a LTQ-Orbitrap hydrib mass spectrometer. (C) Full MS scans were acquired in FT mode at 60,000 resolution. The doubly charged ions at 852.48, 859.48, 866.49, and 873.50 m/z were fragmented in the ion trap and mapped by SEQUEST to histone H3 “R.LVREIAQDFKTDLR.F” peptides with unmodified (me0), monomethylated (me1), dimethylated (me2), and trimethylated (me3) Lys79, respectively. (D) All four differentially modified peptides coeluted within a 1- to 2-min retention time window. (E) A representative tandem mass spectrum matched to “R.LVREIAQDFK(me3)TDLR.F” was annotated. The doubly charged precursor ion with a protonated mass of 1745.99 Da was marked by the green tick along the m/z-axis. Fragment ions bearing trimethylated Lys79 (circled) were matched in both singly charged b and y ion series, which were labeled in red and blue, respectively. Doubly charged b and y ions were labeled in magenta and dark cyan, respectively. If the fragment ions lost ammonia, the resulting peaks were labeled with the same color as the source series with an asterisk (*) to indicate that they are 17 Da away from the fragment ion. Trimethylated histone H3 peptides matched by multiple spectra were observed only in the presence of SAM.

SiRNA-mediated knockdown of Dot1, AF10, AF9, and ENL affects global H3K79 methylation levels. (A) HeLa cells were treated with scrambled, ENL, AF9, AF10, and Dot1 siRNAs as indicated. Total cell lysates were subjected to immunoblot analyses with the indicated antibodies. Knockdown demonstrates that AF10 is the major regulator of H3K79 methylation by Dot1, whereas AF9 and ENL effects on H3K79 methylation are affected to a lesser extent, with AF9 having a stronger effect than ENL. Tubulin and H3 were used as loading controls. (B) qRT–PCR of the siRNA-treated HeLa cells shows the extent of knockdown after 72 h of incubation. Relative expression to Gapdh is plotted. (C) Flag affinity purification from Flag-Dot1 and Flag-AF10 293 cell extracts coimmunoprecipitate β-catenin, a key mediator of the Wnt signaling pathway. The presence of β-catenin in both the Flag-Dot1 and Flag-AF10, but not in Flag purification from HEK 293T extracts, indicates that β-catenin interacts with DotCom.

H2B monoubiquitination by Bre1 specifically links H3K79 trimethylation to Wingless signaling. (A) Bre1 mutant embryos show reduced levels of bulk monoubiquitinated H2B and H3K79me3. H3K79me and H3K79me2 are not affected. Histones were analyzed from 16 ± 1-h embryos of homozygous Bre1 mutants [l(3)01640] and wild-type controls for the presence of H3K79 methylation as indicated. (B) RNAi-mediated knockdown of Bre1 in wing imaginal discs leads to reduced expression of senseless. Expression of Wingless protein itself is not affected, and there is no change in the low-threshold Wg target distalless (dll). en-Gal4 was used to drive the expression of UAS-Bre1-RNAi, and the domain of knockdown is marked by UAS-GFP. The arrow shows the loss of senseless expression. (C) Bre1 mutant embryos show reduced expression of Notum and CG6234. RNA was extracted from 16 ± 1-h embryos of homozygous Bre1 mutants [l(3)01640] and wild-type controls, and expression levels were determined by qRT–PCR. Relative expression to Tubulin is plotted.