Figure 1.
Myc overexpression increases the number of γ-H2A.X positive cells.
(A, B) Wing imaginal disc of genotype ap-Gal4, UAS-GFP/+; UAS-p35/+. (C, D) Wing imaginal disc of genotype ap-Gal4, UAS-GFP/+; UAS-p35/UAS-Myc. GFP channel is shown in A and C, whereas γ-H2A.X staining is shown in B and D. (E) Quantitation of the number of γ-H2A.X positive cells from ap-Gal4, UAS-GFP/+; UAS-p35/+ wing discs (shown as ap>p35) and ap-Gal4, UAS-GFP/+; UAS-p35/UAS-Myc (shown as ap>Myc+p35). The number of γ-H2A.X positive cells within the ap>Gal4 expressing region (shown in GFP channel) from at least 10 imaginal discs was quantitated, and the error bars represent standard error. *indicates statistical significance of p<0.01 (student’s t-test). (F) Western blot analyses showing levels of Myc and the loading control γ-tubulin of wing imaginal discs of the genotypes ap-Gal4, UAS-GFP/+; UAS-p35/+ (labeled as p35) and ap-Gal4, UAS-GFP/+; UAS-p35/UAS-Myc (labeled as Myc+p35). Eight discs were loaded per lane. Myc is overexpressed 5.3-fold as quantitated using LiCOR software and normalized to the level of γ-tubulin (average of three Westerns).
Figure 2.
Myc overexpression increases mutation frequency.
(A) Schematic representation of the lacZ reporter we used to quantitate mutation frequency, and potential mutation events that can occur in this transgene. Adapted from Garcia et al. [29] (B) Western analyses showing 9-fold overexpression of Myc compared to the loading control of γ-tubulin. Samples are wing imaginal discs of the genotypes hs-FLP; lacZ #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; lacZ #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae were heat shocked (37°C) for 45 min during the 3rd larval instar and allowed to develop for 12 hours. Eight discs were loaded per lane. (C) lacZ mutation frequency of hs-FLP; lacZ #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; lacZ #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae are collected 3 days after a 45 minute heat shock at 37°C during the 1st instar larval stage and separated by sex. Experiments shown are from at least four biological repeats (D) lacZ mutation frequency of hs-FLP; lacZ #9/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; lacZ #9/+; act>CD2>Gal4, UAS-GFP/UAS-Myc (shown as “Myc”) larvae. (E) A selection (at least 48) plasmids were chosen to digest with AvaI to determine plasmid size per experiment for hs-FLP; lacZ #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; lacZ #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc (shown as “Myc”) larvae. Male and female data are shown separately for these analyses and do not show any differences in mutation spectra. White filled area of bar represents frequency of size change mutation (genome rearrangement), where as black solid area shows frequency of mutant plasmids that do not show a size change (point mutations). *indicates statistical significance of p<0.001 (student’s t-test).
Table 1.
Myc increases the severity of spontaneous DSB-mediated mutations.
Figure 3.
Overexpression of Myc increases mutation frequency and decreases lifespan at 29
°C. (A) Western blot from five adult heads at 29°C for three days from lacZ #2/Tub-Gal80TS; Actin-Gal4/+ (shown as ActTS>+) and lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc (shown as ActTS>Myc). Myc is induced ∼9-fold in this system (LiCOR quantitation). (B) lacZ mutation frequency of lacZ #2/Tub-Gal80TS; Actin-Gal4/+ (shown as “ActTS>+”) and lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc (shown as “ActTS>Myc”). Adults were placed at 29°C for 5 days before lacZ analyses to allow transgene expression. Black solid areas indicate frequency of point mutations and white areas indicate frequency of genome rearrangements. *indicates statistical significance of p<0.01 (student’s t-test). (C) Lifespan analyses of females of the genotype lacZ #2/Tub-Gal80TS; Actin-Gal4/+ (shown as “ActTS>+”) and of the genotype lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc (shown as “ActTS>Myc”). These lifespans are statistically significantly different from each other p<0.001 (Log-rank test).
Figure 4.
Myc overexpression in the adult increases mutation frequency and reduces lifespan at 25
°C. (A) Western analyses showing levels of Myc and the loading control γ-tubulin from four female adult heads of the genotypes lacZ #2/Tub-Gal80TS; Actin-Gal4/+ (shown as “ActTS>+”) and lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc (shown as “ActTS>Myc”). Adults were placed at 25°C for 10 days before Western analyses. (B) lacZ mutation frequency of females of genotype lacZ #2/Tub-Gal80TS; Actin-Gal4/+ (shown as “ActTS>+”) and lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc (shown as “ActTS>Myc”). Adults were placed at 25°C for 10 days before lacZ analyses. *indicates statistical significance of p<0.001 (student’s t-test). (C) Lifespan analyses at 25°C of females of the genotypes lacZ #2/Tub-Gal80TS; Actin-Gal4/+ (shown as “ActTS>+”) and lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc (shown as “ActTS>Myc”). These lifespans are statistically significantly different from each other p<0.001 (Log-rank test). (D) Lifespan analyses at 25°C of females of the genotypes lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-p35 (shown as “ActTS>p35”) and lacZ #2/Tub-Gal80TS; Actin-Gal4/UAS-Myc, UAS-p35 (shown as “ActTS>Myc+p35”). These are statistically significantly different (p<0.001, Log-rank test).
Figure 5.
myc heterozygotes have a reduced mutation frequency and increased lifespan.
(A) Western analyses showing levels of Myc and the loading control γ-tubulin from 20 female adult heads of the genotypes +/+; lacZ #2/+ (labeled as +), myc4/+; lacZ #2/+ (labeled as myc/+) and myc4/+; lacZ #2/+; myc:GFP/+ (labeled as myc/+, myc:GFP). Adults were 1–4 days old. (B) lacZ mutation frequency of +/+; lacZ #2/+ (labeled as +) and myc4/+; lacZ #2/+ (labeled as myc4/+) and myc4/+; lacZ #2/+; myc:GFP/+ (labeled myc4/+; myc:GFP/+). *indicates p<0.05 (student’s t-test). (C) Lifespan analyses of +/+; lacZ #2/+ (labeled as +), myc4/+; lacZ #2/+ (labeled as myc4/+) and myc4/+; lacZ #2/+; myc:GFP/+ (labeled as myc4/+, myc:GFP). The lifespan of myc4/+; lacZ #2/+ heterozygotes is significantly different to +/+; lacZ #2/+ and myc4/+; lacZ #2/+; myc:GFP/+ (which are indistinguishable) p = 0.002 (Log-rank test).