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. 2023 Oct 11;110(11):1919–1937. doi: 10.1016/j.ajhg.2023.09.009

Figure 4.

Figure 4

Expression human DOT1L disrupts wing morphology and different variants display different amounts of toxicity and H3K79 methylation

(A) Heterozygous mutant flies, gppTG4/+, expressing reference or variant DOT1L cDNA have reduced viability compared to control flies (gppTG4/+> UAS-Empty) as shown by lower-than-expected genotypic ratios of survival into adulthood. The variants p.Glu123Lys and p.Glu129Lys show significant reduction in viability compared to flies expressing reference DOT1L. All the crosses were performed at 25°C. Percent viabilities (o/e ratios) from three independent experiments were plotted as mean ± SEM, and statistical significance was determined by one-way ANOVA for multiple groups (∗∗∗∗p < 0.0001).

(B) Relative DOT1L mRNA expression levels in gppTG4/+ flies expressing reference or variant DOT1L. Normalized DOT1L levels from three independent experiments were plotted as mean ± SEM, and statistical significance was determined by one-way ANOVA for multiple groups.

(C) Representative images of different wing phenotypes observed in in survivors of heterozygous mutant flies, gppTG4/+, expressing each DOT1L cDNA. Blue arrow, loss of cross-vein; black arrow, extra vein branching; orange arrow, blistered areas; green arrow, necrotic areas.

(D) Penetrance of strong wing phenotypes in survivors of heterozygous mutant flies, gppTG4/+, expressing each DOT1L cDNA. Percent penetrance for strong phenotype from three independent experiments were plotted as mean ± SEM, and statistical significance was determined by one-way ANOVA for multiple groups (∗p < 0.05, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

(E) All the DOT1L variants, except p.Arg292Cys, behave as gain-of-function (GoF) mutations. Survivors of heterozygous mutant flies, gppTG4/+, expressing reference or variant DOT1L cDNA show increased H3K79 methylation compared to control flies (gppTG4/+> UAS-Empty). Flies expressing variant DOT1L cDNAs, corresponding to p.Cys45Gly, p.Thr100Met p.Glu123Lys, p.Glu129Lys, p.Leu626Val, and p.Lys1025Met, show higher H3K79 methylation levels when compared to reference (Ei). Flies expressing variant DOT1L cDNAs p.Glu123Lys and p.Glu129Lys show higher DOT1L levels when compared to reference (Eii). Protein lysate from 10 adult flies were prepared for each sample. H3K79 methylation levels were normalized with loading control, H3, and fold change for each sample were calculated by comparing normalized H3K79 methylation levels to reference DOT1L-expressing flies. DOT1L levels were normalized with loading control, α-tubulin, and fold change for each sample were calculated by comparing normalized DOT1L levels to reference DOT1L-expressing flies. All the crosses were performed at 25°C. Blue numbers indicate the fold change of H3K79 methylation level in reference when compared to control (UAS-Empty).

(Fi) Normalized H3K79 methylation band intensities for each group from three independent experiments were plotted as mean ± SEM, and statistical significance was determined by one-way ANOVA for multiple groups (∗p < 0.05, ∗∗∗∗p < 0.0001).

(Fii) Normalized DOT1L band intensities for each group from three independent experiments were plotted as mean ± SEM, and statistical significance was determined by one-way ANOVA for multiple groups (∗∗∗p < 0.001).