MPK-1/ERK pathway regulates DNA damage response during development through DAF-16/FOXO

Abstract

Ultraviolet (UV) induces distorting lesions to the DNA that can lead to stalling of the RNA polymerase II (RNAP II) and that are removed by transcription-coupled nucleotide excision repair (TC-NER). In humans, mutations in the TC-NER genes CSA and CSB lead to severe postnatal developmental defects in Cockayne syndrome patients. In Caenorhabditis elegans, mutations in the TC-NER genes csa-1 and csb-1, lead to developmental growth arrest upon UV treatment. We conducted a genetic suppressor screen in the nematode to identify mutations that could suppress the developmental defects in csb-1 mutants. We found that mutations in the ERK1/2 MAP kinase mpk-1 alleviate the developmental retardation in TC-NER mutants, while constitutive activation of the RAS-MAPK pathway exacerbates the DNA damage-induced growth arrest. We show that MPK-1 act via insulin/insulin-like signaling pathway and regulates the FOXO transcription factor DAF-16 to mediate the developmental DNA damage response.

Figure 1.

mpk-1 mutations suppress UV-induced developmental growth arrest in TC-NER deficient csb-1, csa-1 and in completely NER-defective xpa-1 mutants. (A) csb-1(ok2335) worms were mutagenized with EMS (1 mM) and F2 generation synchronized by bleaching. After egg-laying for 2–3 h F2 adults were backed up in 96-well plate liquid culture. F3 larvae were irradiated with 60 mJ/cm² UVB and screened for bypass developmental arrest 48 h post-irradiation. Phenotype was confirmed by using the worms from the 96-well backup plate. (B) Representation of the genomic architecture of mpk-1. Blue boxes represent exons, black lines represent introns and untranslated regions are in gray. ga111 and sbj10 alleles are indicated. sbj10 is a missense mutation changing Cytosine to Guanine and ga111 is a missense mutation changing Thymine to Guanine. (C and D) L1 larvae were treated with UV and grown at 15, 20 or 25°C. Larval stages were determined 48 h post-treatment for the animals at 20 and 25°C and 72 h post-treatment for the animals at 15°C. (E and F) L1 larvae were treated with UV and grown at 25°C. Larval stages were determined 48 h post-treatment. Error bars represent standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test comparing the fraction of each larval stage of two different mutants for the same treatment condition. mpk-1(ga111);csb-1 and mpk-1(sbj10);csb-1 were compared to csb-1 in (C), mpk-1(ga111) was compared to WT (D), mpk-1;csa-1 was compared to csa-1 (E) and mpk-1;xpa-1 to xpa-1 (F). Statistical analysis of additional comparison and Fisher’s Exact test for analyzing the distribution of larval stages see Supplementary Tables S1 and 8. Each treatment was conducted in triplicate with a minimum of 30 animals for each sample. Representative of three different experiments shown.

Figure 2.

mpk-1 mutation suppress csb-1 mutant sensitivity to transcription blocking lesions. (A) L1 larvae were treated 48 h with different concentrations of illudinM, in liquid culture, then plated and larval stages were determined immediately. (B) L1 larvae were treated 24 h with different concentrations of actinomycin D, in liquid culture, then plated and larval stages were determined 24 h later at 25°C. (C) L1 larvae were treated 24 h with different concentrations of α-amanitin, in liquid culture, then plated and larval stages were determined 24 h later at 25°C. Error bars represent standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test comparing the fraction of each larval stage of two different mutants for the same treatment condition. mpk-1(ga111);csb-1 and mpk-1(sbj10);csb-1 were compared to csb-1. Statistical analysis of additional comparison and Fisher’s Exact test for analyzing the distribution of larval stages see Supplementary Tables S2 and 8. Each treatment was conducted in triplicate with a minimum of 30 animals for each sample. Representative of three different experiments shown.

Figure 3.

Modulation of MPK-1/ERK signaling pathway affects the developmental arrest upon UV treatment of csb-1. (A and B) L1 larvae were treated with UV and grown at 25°C. Larval stages were determined 48 h post-treatment. Error bars represent standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test comparing the fraction of each larval stage of two different mutants for the same treatment condition. mpk-1;csb-1, lip-1;csb-1 and let-60;csb-1 were compared to csb-1. Statistical analysis of additional comparison and Fisher’s Exact test for analyzing the distribution of larval stages see Supplementary Tables S3 and 8. Each treatment was conducted in triplicate with a minimum of 30 animals for each sample. Representative of three different experiments shown.

Figure 4.

MPK-1 regulates DAF-16 in the developmental DDR. (A) DAF-16::GFP cellular localization after UV treatment in L1 larvae. WT, csb-1, mpk-1 and csb-1;mpk-1 strains carrying the DAF-16::GFP transgene were irradiated with 0, 40 or 80 mJ/cm² and kept at 20°C (average of n = 3 independent experiments per strain and dose is shown, 10 individuals analyzed per experiment; error bars show standard deviation). The graph shows the percentage of worms showing cytosolic, nuclear and partially nuclear DAF-16::GFP localization following UV irradiation. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test comparing each type of localization of two different mutants for the same treatment condition. mpk-1 was compared to WT and mpk-1;csb-1 to csb-1. Statistical analysis of additional comparison and Fisher’s Exact test for analyzing the distribution of larval stages see Supplementary Tables S4 and 8. (B) Representative pictures exemplifying the cytosolic, partially nuclear and nuclear categories of DAF-16::GFP localization, shown 24 h after UV treatment 80 mJ/cm² in mpk-1(ga111);DAF-16::GFP c. L1 larvae were treated with UV and grown at 25°C. Larval stages were determined 48 h post-treatment. Error bars represent standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t-test comparing the fraction of each larval stage of two different mutants for the same treatment condition. mpk-1;csb-1 and daf-16;csb-1 was compared to csb-1 and daf-16;mpk-1;csb-1 to mpk-1;csb-1. Statistical analysis of additional comparison and Fisher’s Exact test for analyzing the distribution of larval stages see Supplementary Tables S4 and 8. Each treatment was conducted in triplicate with a minimum of 30 animals for each sample. Representative of three different experiments shown.