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Genome-wide expression analysis identifies a modulator of ionizing radiation-induced p53-independent apoptosis in Drosophila melanogaster.

van Bergeijk P, Heimiller J, Uyetake L, Su TT - PLoS ONE (2012)

Bottom Line: Key data from microarrays were confirmed by quantitative RT-PCR.The results solidify the central role of p53 in IR-induced transcriptome changes, but also show that nearly all changes are made of both p53-dependent and p53-independent components. p53 is found to be necessary not just for the induction of but also for the repression of transcript levels for many genes in response to IR.These and other results support the emerging notion that there is not a single dominant mechanism but that both positive and negative inputs collaborate to induce p53-independent apoptosis in response to IR in Drosophila larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.

ABSTRACT
Tumor suppressor p53 plays a key role in DNA damage responses in metazoa, yet more than half of human tumors show p53 deficiencies. Therefore, understanding how therapeutic genotoxins such as ionizing radiation (IR) can elicit DNA damage responses in a p53-independent manner is of clinical importance. Drosophila has been a good model to study the effects of IR because DNA damage responses as well as underlying genes are conserved in this model, and because streamlined gene families make loss-of-function analyses feasible. Indeed, Drosophila is the only genetically tractable model for IR-induced, p53-independent apoptosis and for tissue regeneration and homeostasis after radiation damage. While these phenomenon occur only in the larvae, all genome-wide gene expression analyses after irradiation to date have been in embryos. We report here the first analysis of IR-induced, genome-wide gene expression changes in wild type and p53 mutant Drosophila larvae. Key data from microarrays were confirmed by quantitative RT-PCR. The results solidify the central role of p53 in IR-induced transcriptome changes, but also show that nearly all changes are made of both p53-dependent and p53-independent components. p53 is found to be necessary not just for the induction of but also for the repression of transcript levels for many genes in response to IR. Furthermore, Functional analysis of one of the top-changing genes, EF1a-100E, implicates it in repression of IR-induced p53-independent apoptosis. These and other results support the emerging notion that there is not a single dominant mechanism but that both positive and negative inputs collaborate to induce p53-independent apoptosis in response to IR in Drosophila larvae.

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Validation by quantitative RT-PCR of 10 candidate genes identified in microarray analysis.Total RNA was isolated from wing imaginal discs of third instar larvae at 2 or 18 hr after exposure to 0 or 4000R of X-rays. RNA was reverse-transcribed into cDNA and subjected for quantitative RT-PCR analysis as described in Methods. a-tubulin levels were used to normalize the graphs. Error bar = 1 STD. (A) A schematic representation of data comparisons used to select candidates for validation. The gray box denotes the time at which apoptosis becomes detectable in p53 mutants. (B) Q-RT-PCR results in p53 mutant wing imaginal discs (B) Q-RT-PCR results in wild type (yw) wing imaginal discs.
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pone-0036539-g001: Validation by quantitative RT-PCR of 10 candidate genes identified in microarray analysis.Total RNA was isolated from wing imaginal discs of third instar larvae at 2 or 18 hr after exposure to 0 or 4000R of X-rays. RNA was reverse-transcribed into cDNA and subjected for quantitative RT-PCR analysis as described in Methods. a-tubulin levels were used to normalize the graphs. Error bar = 1 STD. (A) A schematic representation of data comparisons used to select candidates for validation. The gray box denotes the time at which apoptosis becomes detectable in p53 mutants. (B) Q-RT-PCR results in p53 mutant wing imaginal discs (B) Q-RT-PCR results in wild type (yw) wing imaginal discs.

Mentions: Generally speaking, p53-dependent mechanisms that respond to DNA damage are better characterized than p53-independent mechanisms. In order to better understand p53-independent mechanisms, we identified genes that show a response profile similar to that of Drosophila pro-apoptotic Smac/DIABLO orthologs. At 2 hr after irradiation, rpr, hid and skl, are induced in yw discs but are either not induced or induced to a lesser degree in p53 mutant discs (Table 2, bold). At this time point, yw discs are about to undergo apoptosis but p53 mutant discs are not. At 18 hr after irradiation, when p53-independent apoptosis occurs, these genes are induced in p53 mutants relative either to age-matched non-irradiated controls (p18− vs. p18+ in Table 2) or to p53 mutants at 2 hr after irradiation (p2+ vs. p18+ in Table 2). Therefore, we identified genes whose expression in p53 mutants (1) increased significantly at 18 hr after irradiation compared to un-irradiated p53 mutants, and (2) showed a significant increase in IR+18 hr p53 mutant compared to IR+2 hr p53 mutant (≥1.5-fold, p<0.005; arrows in Figure 1A). Of ∼13,000 genes analyzed, 87 fulfilled these criteria (Table S4). None of these were induced in 18 hr-IR samples compared to 2 hr-IR samples using similar cut-offs; that is, induction in 18 hr+IR samples relative to 2 hr+IR samples is not due to aging of larvae. Of the 87 genes, 7 genes were also induced by IR at 2 hr after irradiation in p53 mutants (p2+ vs. p2−, ≥1.5-fold, p<0.005, in bold in Table S4). The level of induction, however, was less than that at 18 hr+IR such that p18+IR level was significantly higher than p2+IR level (fulfilling criteria #2). We reasoned that these genes may be induced in a p53-independent manner at 2 hr after irradiation but their levels climbed higher at longer times, and thus could contribute to the delayed apoptotic response.


Genome-wide expression analysis identifies a modulator of ionizing radiation-induced p53-independent apoptosis in Drosophila melanogaster.

van Bergeijk P, Heimiller J, Uyetake L, Su TT - PLoS ONE (2012)

Validation by quantitative RT-PCR of 10 candidate genes identified in microarray analysis.Total RNA was isolated from wing imaginal discs of third instar larvae at 2 or 18 hr after exposure to 0 or 4000R of X-rays. RNA was reverse-transcribed into cDNA and subjected for quantitative RT-PCR analysis as described in Methods. a-tubulin levels were used to normalize the graphs. Error bar = 1 STD. (A) A schematic representation of data comparisons used to select candidates for validation. The gray box denotes the time at which apoptosis becomes detectable in p53 mutants. (B) Q-RT-PCR results in p53 mutant wing imaginal discs (B) Q-RT-PCR results in wild type (yw) wing imaginal discs.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362589&req=5

pone-0036539-g001: Validation by quantitative RT-PCR of 10 candidate genes identified in microarray analysis.Total RNA was isolated from wing imaginal discs of third instar larvae at 2 or 18 hr after exposure to 0 or 4000R of X-rays. RNA was reverse-transcribed into cDNA and subjected for quantitative RT-PCR analysis as described in Methods. a-tubulin levels were used to normalize the graphs. Error bar = 1 STD. (A) A schematic representation of data comparisons used to select candidates for validation. The gray box denotes the time at which apoptosis becomes detectable in p53 mutants. (B) Q-RT-PCR results in p53 mutant wing imaginal discs (B) Q-RT-PCR results in wild type (yw) wing imaginal discs.
Mentions: Generally speaking, p53-dependent mechanisms that respond to DNA damage are better characterized than p53-independent mechanisms. In order to better understand p53-independent mechanisms, we identified genes that show a response profile similar to that of Drosophila pro-apoptotic Smac/DIABLO orthologs. At 2 hr after irradiation, rpr, hid and skl, are induced in yw discs but are either not induced or induced to a lesser degree in p53 mutant discs (Table 2, bold). At this time point, yw discs are about to undergo apoptosis but p53 mutant discs are not. At 18 hr after irradiation, when p53-independent apoptosis occurs, these genes are induced in p53 mutants relative either to age-matched non-irradiated controls (p18− vs. p18+ in Table 2) or to p53 mutants at 2 hr after irradiation (p2+ vs. p18+ in Table 2). Therefore, we identified genes whose expression in p53 mutants (1) increased significantly at 18 hr after irradiation compared to un-irradiated p53 mutants, and (2) showed a significant increase in IR+18 hr p53 mutant compared to IR+2 hr p53 mutant (≥1.5-fold, p<0.005; arrows in Figure 1A). Of ∼13,000 genes analyzed, 87 fulfilled these criteria (Table S4). None of these were induced in 18 hr-IR samples compared to 2 hr-IR samples using similar cut-offs; that is, induction in 18 hr+IR samples relative to 2 hr+IR samples is not due to aging of larvae. Of the 87 genes, 7 genes were also induced by IR at 2 hr after irradiation in p53 mutants (p2+ vs. p2−, ≥1.5-fold, p<0.005, in bold in Table S4). The level of induction, however, was less than that at 18 hr+IR such that p18+IR level was significantly higher than p2+IR level (fulfilling criteria #2). We reasoned that these genes may be induced in a p53-independent manner at 2 hr after irradiation but their levels climbed higher at longer times, and thus could contribute to the delayed apoptotic response.

Bottom Line: Key data from microarrays were confirmed by quantitative RT-PCR.The results solidify the central role of p53 in IR-induced transcriptome changes, but also show that nearly all changes are made of both p53-dependent and p53-independent components. p53 is found to be necessary not just for the induction of but also for the repression of transcript levels for many genes in response to IR.These and other results support the emerging notion that there is not a single dominant mechanism but that both positive and negative inputs collaborate to induce p53-independent apoptosis in response to IR in Drosophila larvae.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Colorado, United States of America.

ABSTRACT
Tumor suppressor p53 plays a key role in DNA damage responses in metazoa, yet more than half of human tumors show p53 deficiencies. Therefore, understanding how therapeutic genotoxins such as ionizing radiation (IR) can elicit DNA damage responses in a p53-independent manner is of clinical importance. Drosophila has been a good model to study the effects of IR because DNA damage responses as well as underlying genes are conserved in this model, and because streamlined gene families make loss-of-function analyses feasible. Indeed, Drosophila is the only genetically tractable model for IR-induced, p53-independent apoptosis and for tissue regeneration and homeostasis after radiation damage. While these phenomenon occur only in the larvae, all genome-wide gene expression analyses after irradiation to date have been in embryos. We report here the first analysis of IR-induced, genome-wide gene expression changes in wild type and p53 mutant Drosophila larvae. Key data from microarrays were confirmed by quantitative RT-PCR. The results solidify the central role of p53 in IR-induced transcriptome changes, but also show that nearly all changes are made of both p53-dependent and p53-independent components. p53 is found to be necessary not just for the induction of but also for the repression of transcript levels for many genes in response to IR. Furthermore, Functional analysis of one of the top-changing genes, EF1a-100E, implicates it in repression of IR-induced p53-independent apoptosis. These and other results support the emerging notion that there is not a single dominant mechanism but that both positive and negative inputs collaborate to induce p53-independent apoptosis in response to IR in Drosophila larvae.

Show MeSH
Related in: MedlinePlus