Myc overexpression increases mutation frequency.

<p>(A) Schematic representation of the <i>lacZ</i> reporter we used to quantitate mutation frequency, and potential mutation events that can occur in this transgene. Adapted from Garcia et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074641#pone.0074641-Garcia3" target="_blank">[29]</a> (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; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/UAS-Myc larvae (shown as “Myc”). Larvae were heat shocked (37°C) for 45 min during the 3^rd larval instar and allowed to develop for 12 hours. Eight discs were loaded per lane. (C) <i>lacZ</i> mutation frequency of hs-FLP; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #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 1^st instar larval stage and separated by sex. Experiments shown are from at least four biological repeats (D) <i>lacZ</i> mutation frequency of hs-FLP; <i>lacZ</i> #9/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #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; <i>lacZ</i> #2/+; act>CD2>Gal4, UAS-GFP/+ (shown as “+”) and hs-FLP; <i>lacZ</i> #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).</p

Myc-Dependent Genome Instability and Lifespan in <i>Drosophila</i>

<div><p>The Myc family of transcription factors are key regulators of cell growth and proliferation that are dysregulated in a large number of human cancers. When overexpressed, Myc family proteins also cause genomic instability, a hallmark of both transformed and aging cells. Using an <i>in vivo lacZ</i> mutation reporter, we show that overexpression of Myc in <i>Drosophila</i> increases the frequency of large genome rearrangements associated with erroneous repair of DNA double-strand breaks (DSBs). In addition, we find that overexpression of Myc shortens adult lifespan and, conversely, that Myc haploinsufficiency reduces mutation load and extends lifespan. Our data provide the first evidence that Myc may act as a pro-aging factor, possibly through its ability to greatly increase genome instability.</p></div

Myc overexpression in the adult increases mutation frequency and reduces lifespan at 25

<p>°<b>C.</b> (A) Western analyses showing levels of Myc and the loading control γ-tubulin from four female adult heads of the genotypes <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/+ (shown as “Act^TS>+”) and <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/UAS-Myc (shown as “Act^TS>Myc”). Adults were placed at 25°C for 10 days before Western analyses. (B) <i>lacZ</i> mutation frequency of females of genotype <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/+ (shown as “Act^TS>+”) and <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/UAS-Myc (shown as “Act^TS>Myc”). Adults were placed at 25°C for 10 days before <i>lacZ</i> analyses. *indicates statistical significance of p<0.001 (student’s t-test). (C) Lifespan analyses at 25°C of females of the genotypes <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/+ (shown as “Act^TS>+”) and <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/UAS-Myc (shown as “Act^TS>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 <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/UAS-p35 (shown as “Act^TS>p35”) and <i>lacZ</i> #2/Tub-Gal80^TS; Actin-Gal4/UAS-Myc, UAS-p35 (shown as “Act^TS>Myc+p35”). These are statistically significantly different (p<0.001, Log-rank test).</p

Myc overexpression increases the number of γ-H2A.X positive cells.

<p>(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).</p