Figure 1
Quantitative H3K79 methylation analysis on a series of Dot1 alleles.
A) Schematic overview of yeast Dot1, trypanosome Dot1A and Dot1B and human Dot1L. The enzymes share a conserved catalytic domain (grey box, CD). In addition, yDot1 contains an N-terminal domain with a lysine-rich domain (light grey and white shaded box, respectively). Human Dot1L was expressed with part of its C-terminal domain that has weak similarity with the yDot1 N-terminal domain. B) Western-blot analysis of H3K79 methylation and yDot1 expression using specific antibodies. H3 and Pgk1 were used as loading controls. A wild-type yeast strain (NKI6061; WT) was used as a reference throughout the manuscript; its H3K79 methylation levels were determined by mass spectrometry. A dot1Δ (–) was included to determine antibody specificity. Dot1 enzymes were expressed from the following promoters at the endogenous yeast DOT1 locus: DOT1pr (D), TEFpr + yDOT1 5’UTR (T*), GPDpr + yDOT1 5’UTR (G*), ADHpr (A), TEFpr (T) or GPDpr (G). H3K79 methylation patterns confirmed by mass spectrometry (MS) are indicated with ♦. C) Western-blot analysis of a series of reference yeast strains (Y1-Y7) containing a range of known H3K79 methylation levels (see below) to determine the linearity of the H3K79me1, -me2 and -me3 home-made (hm) antibodies22 and the H3K79me2 antibody from Millipore (mp). Y1-Y7 refers to strains: NKI6081, NKI6077, NKI6084, NKI6099, NKI6100, NKI6085 and NKI6083. D) Samples described in (C) were quantified using an Odyssey scanner and by MS. To determine the linearity of the H3K79 methylation antibodies, MS data were plotted against the quantified western-blot data and a non-linear regression fit was performed (see Supplemental Methods). The linear function resulting from the fit was used to correct the quantified western-blot data described in this manuscript and to subsequently estimate the unmethylated H3 fraction. No linear fit was obtained for the H3K79me2 home-made antibody; the low correlation between MS and western-blot data of the H3K79me2 home made antibody was caused by cross-reactivity of this antiserum with H3K79me322. Unless stated otherwise, H3K79me2 was quantified using the Millipore antibody, since this showed very little cross-reactivity to H3K79me3.
