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Caenorhabditis elegans protein arginine methyltransferase PRMT-5 negatively regulates DNA damage-induced apoptosis.

Yang M, Sun J, Sun X, Shen Q, Gao Z, Yang C - PLoS Genet. (2009)

Bottom Line: PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter.Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms.These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Arginine methylation of histone and non-histone proteins is involved in transcription regulation and many other cellular processes. Nevertheless, whether such protein modification plays a regulatory role during apoptosis remains largely unknown. Here we report that the Caenorhabditis elegans homolog of mammalian type II arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. We show that inactivation of C. elegans prmt-5 leads to excessive apoptosis in germline following ionizing irradiation, which is due to a CEP-1/p53-dependent up-regulation of the cell death initiator EGL-1. Moreover, we provide evidence that CBP-1, the worm ortholog of human p300/CBP, functions as a cofactor of CEP-1. PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter. Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms. These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

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cbp-1 RNAi inhibits DNA damage-induced excessive apoptosis in prmt-5(gk357) mutants.(A) cbp-1 RNAi performed in L4-stage animals decreases the mRNA level of cbp-1. Indicated animals at L4 stage were treated with cbp-1 RNAi. RNA was prepared 36 h later and cbp-1 expression level was detected by using semi-quantitative RT-PCR. Actin mRNA was used as internal control. (B) cbp-1 RNAi performed in L4-stage animals suppresses the excessive germ cell apoptosis induced by γ-irradiation in prmt-5(gk357) animals. Representative images of germ cell corpses 36 h post irradiation of 120 Gy in control RNAi- and cbp-1 RNAi-treated prmt-5(gk357) worms are shown. Germ cell corpses are indicated with fragmented circles (top panel) and an arrow (bottom panel). (C, D) Quantitative analyses of cbp-1 RNAi on DNA damage-induced germ cell apoptosis in N2 and prmt-5(gk357) worms. cbp-1 RNAi was performed as above. Indicated animals were treated with different doses of γ-irradiation and germ cell corpses were scored 36 h post irradiation (C), or animals were irradiated with γ-ray of 120 Gy and germ cell corpses were scored at different time points post irradiation (D). Error bars represents SEM. Indicated comparisons were performed by using unpaired t-test. Double asterisks indicate p<0.001. (E) cbp-1 RNAi suppresses egl-1 expression induced by γ-irradiation. cbp-1 RNAi was performed in L4-stage animals. Indicated worms at young adult stage were irradiated with γ-ray of 120 Gy and total RNA were prepared 24 h post irradiation. egl-1 expression was detected by using egl-1 cDNA as probe. α-actin mRNA was probed as loading control of samples. Three independent Northern blot analyses were performed and the representative images are shown. (F) Relative fold induction of egl-1 mRNA by γ-irradiation in cbp-1 RNAi-treated animals. egl-1 fold induction was averaged from three independent Northern blot analyses, which was quantified by using the software ImageQuant 5.2 and normalized with α-actin mRNA. Error bars represent SEM. Comparisons were made between control RNAi and cbp-1 RNAi treatment. Double asterisks indicate p<0.001.
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pgen-1000514-g006: cbp-1 RNAi inhibits DNA damage-induced excessive apoptosis in prmt-5(gk357) mutants.(A) cbp-1 RNAi performed in L4-stage animals decreases the mRNA level of cbp-1. Indicated animals at L4 stage were treated with cbp-1 RNAi. RNA was prepared 36 h later and cbp-1 expression level was detected by using semi-quantitative RT-PCR. Actin mRNA was used as internal control. (B) cbp-1 RNAi performed in L4-stage animals suppresses the excessive germ cell apoptosis induced by γ-irradiation in prmt-5(gk357) animals. Representative images of germ cell corpses 36 h post irradiation of 120 Gy in control RNAi- and cbp-1 RNAi-treated prmt-5(gk357) worms are shown. Germ cell corpses are indicated with fragmented circles (top panel) and an arrow (bottom panel). (C, D) Quantitative analyses of cbp-1 RNAi on DNA damage-induced germ cell apoptosis in N2 and prmt-5(gk357) worms. cbp-1 RNAi was performed as above. Indicated animals were treated with different doses of γ-irradiation and germ cell corpses were scored 36 h post irradiation (C), or animals were irradiated with γ-ray of 120 Gy and germ cell corpses were scored at different time points post irradiation (D). Error bars represents SEM. Indicated comparisons were performed by using unpaired t-test. Double asterisks indicate p<0.001. (E) cbp-1 RNAi suppresses egl-1 expression induced by γ-irradiation. cbp-1 RNAi was performed in L4-stage animals. Indicated worms at young adult stage were irradiated with γ-ray of 120 Gy and total RNA were prepared 24 h post irradiation. egl-1 expression was detected by using egl-1 cDNA as probe. α-actin mRNA was probed as loading control of samples. Three independent Northern blot analyses were performed and the representative images are shown. (F) Relative fold induction of egl-1 mRNA by γ-irradiation in cbp-1 RNAi-treated animals. egl-1 fold induction was averaged from three independent Northern blot analyses, which was quantified by using the software ImageQuant 5.2 and normalized with α-actin mRNA. Error bars represent SEM. Comparisons were made between control RNAi and cbp-1 RNAi treatment. Double asterisks indicate p<0.001.

Mentions: To determine whether PRMT-5 acts through other CEP-1 cofactors, we sought to identify proteins that likely function together with CEP-1. In mammals, p300/CBP was found to act as a transcription coactivator of p53, and the acetylation of p53 by p300 plays an important role in p53 stabilization in response to DNA damage [4]–[6]. In addition, p300-mediated histone acetylation also contributes to the transcription of p53 target genes [27]. In C. elegans, the p300/CBP homolog CBP-1 was shown to regulate the differentiation of some embryonic cell types [28],[29], and CBP-1 may function in concert with the transcription factor LIN-1 to negatively regulate vulva cell specification [30]. However, it is not known whether CBP-1 could act together with CEP-1 to control gene expression in response to DNA damage. We explored this possibility first by checking if CEP-1 could interact with CBP-1. Using GST pull-down assay, we found that GST-CEP-1 fusion protein interacted with 35S-labeled CBP-1(771–1285) and CBP-1(1286–1770), which are within the HAT domain of CBP-1 (Figure 5A and 5B), indicating that CBP-1 and CEP-1 directly interact with one another. Moreover, partial inactivation of cbp-1 by RNAi significantly suppressed DNA damage-induced apoptosis and egl-1 expression in wild-type animals (Figure 6, see below and Materials and Methods), suggesting that CBP-1 is important for CEP-1 transcriptional activity. All together, these findings suggest that CBP-1 likely acts as a cofactor of CEP-1 in C. elegans.


Caenorhabditis elegans protein arginine methyltransferase PRMT-5 negatively regulates DNA damage-induced apoptosis.

Yang M, Sun J, Sun X, Shen Q, Gao Z, Yang C - PLoS Genet. (2009)

cbp-1 RNAi inhibits DNA damage-induced excessive apoptosis in prmt-5(gk357) mutants.(A) cbp-1 RNAi performed in L4-stage animals decreases the mRNA level of cbp-1. Indicated animals at L4 stage were treated with cbp-1 RNAi. RNA was prepared 36 h later and cbp-1 expression level was detected by using semi-quantitative RT-PCR. Actin mRNA was used as internal control. (B) cbp-1 RNAi performed in L4-stage animals suppresses the excessive germ cell apoptosis induced by γ-irradiation in prmt-5(gk357) animals. Representative images of germ cell corpses 36 h post irradiation of 120 Gy in control RNAi- and cbp-1 RNAi-treated prmt-5(gk357) worms are shown. Germ cell corpses are indicated with fragmented circles (top panel) and an arrow (bottom panel). (C, D) Quantitative analyses of cbp-1 RNAi on DNA damage-induced germ cell apoptosis in N2 and prmt-5(gk357) worms. cbp-1 RNAi was performed as above. Indicated animals were treated with different doses of γ-irradiation and germ cell corpses were scored 36 h post irradiation (C), or animals were irradiated with γ-ray of 120 Gy and germ cell corpses were scored at different time points post irradiation (D). Error bars represents SEM. Indicated comparisons were performed by using unpaired t-test. Double asterisks indicate p<0.001. (E) cbp-1 RNAi suppresses egl-1 expression induced by γ-irradiation. cbp-1 RNAi was performed in L4-stage animals. Indicated worms at young adult stage were irradiated with γ-ray of 120 Gy and total RNA were prepared 24 h post irradiation. egl-1 expression was detected by using egl-1 cDNA as probe. α-actin mRNA was probed as loading control of samples. Three independent Northern blot analyses were performed and the representative images are shown. (F) Relative fold induction of egl-1 mRNA by γ-irradiation in cbp-1 RNAi-treated animals. egl-1 fold induction was averaged from three independent Northern blot analyses, which was quantified by using the software ImageQuant 5.2 and normalized with α-actin mRNA. Error bars represent SEM. Comparisons were made between control RNAi and cbp-1 RNAi treatment. Double asterisks indicate p<0.001.
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pgen-1000514-g006: cbp-1 RNAi inhibits DNA damage-induced excessive apoptosis in prmt-5(gk357) mutants.(A) cbp-1 RNAi performed in L4-stage animals decreases the mRNA level of cbp-1. Indicated animals at L4 stage were treated with cbp-1 RNAi. RNA was prepared 36 h later and cbp-1 expression level was detected by using semi-quantitative RT-PCR. Actin mRNA was used as internal control. (B) cbp-1 RNAi performed in L4-stage animals suppresses the excessive germ cell apoptosis induced by γ-irradiation in prmt-5(gk357) animals. Representative images of germ cell corpses 36 h post irradiation of 120 Gy in control RNAi- and cbp-1 RNAi-treated prmt-5(gk357) worms are shown. Germ cell corpses are indicated with fragmented circles (top panel) and an arrow (bottom panel). (C, D) Quantitative analyses of cbp-1 RNAi on DNA damage-induced germ cell apoptosis in N2 and prmt-5(gk357) worms. cbp-1 RNAi was performed as above. Indicated animals were treated with different doses of γ-irradiation and germ cell corpses were scored 36 h post irradiation (C), or animals were irradiated with γ-ray of 120 Gy and germ cell corpses were scored at different time points post irradiation (D). Error bars represents SEM. Indicated comparisons were performed by using unpaired t-test. Double asterisks indicate p<0.001. (E) cbp-1 RNAi suppresses egl-1 expression induced by γ-irradiation. cbp-1 RNAi was performed in L4-stage animals. Indicated worms at young adult stage were irradiated with γ-ray of 120 Gy and total RNA were prepared 24 h post irradiation. egl-1 expression was detected by using egl-1 cDNA as probe. α-actin mRNA was probed as loading control of samples. Three independent Northern blot analyses were performed and the representative images are shown. (F) Relative fold induction of egl-1 mRNA by γ-irradiation in cbp-1 RNAi-treated animals. egl-1 fold induction was averaged from three independent Northern blot analyses, which was quantified by using the software ImageQuant 5.2 and normalized with α-actin mRNA. Error bars represent SEM. Comparisons were made between control RNAi and cbp-1 RNAi treatment. Double asterisks indicate p<0.001.
Mentions: To determine whether PRMT-5 acts through other CEP-1 cofactors, we sought to identify proteins that likely function together with CEP-1. In mammals, p300/CBP was found to act as a transcription coactivator of p53, and the acetylation of p53 by p300 plays an important role in p53 stabilization in response to DNA damage [4]–[6]. In addition, p300-mediated histone acetylation also contributes to the transcription of p53 target genes [27]. In C. elegans, the p300/CBP homolog CBP-1 was shown to regulate the differentiation of some embryonic cell types [28],[29], and CBP-1 may function in concert with the transcription factor LIN-1 to negatively regulate vulva cell specification [30]. However, it is not known whether CBP-1 could act together with CEP-1 to control gene expression in response to DNA damage. We explored this possibility first by checking if CEP-1 could interact with CBP-1. Using GST pull-down assay, we found that GST-CEP-1 fusion protein interacted with 35S-labeled CBP-1(771–1285) and CBP-1(1286–1770), which are within the HAT domain of CBP-1 (Figure 5A and 5B), indicating that CBP-1 and CEP-1 directly interact with one another. Moreover, partial inactivation of cbp-1 by RNAi significantly suppressed DNA damage-induced apoptosis and egl-1 expression in wild-type animals (Figure 6, see below and Materials and Methods), suggesting that CBP-1 is important for CEP-1 transcriptional activity. All together, these findings suggest that CBP-1 likely acts as a cofactor of CEP-1 in C. elegans.

Bottom Line: PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter.Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms.These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Molecular and Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China.

ABSTRACT
Arginine methylation of histone and non-histone proteins is involved in transcription regulation and many other cellular processes. Nevertheless, whether such protein modification plays a regulatory role during apoptosis remains largely unknown. Here we report that the Caenorhabditis elegans homolog of mammalian type II arginine methyltransferase PRMT5 negatively regulates DNA damage-induced apoptosis. We show that inactivation of C. elegans prmt-5 leads to excessive apoptosis in germline following ionizing irradiation, which is due to a CEP-1/p53-dependent up-regulation of the cell death initiator EGL-1. Moreover, we provide evidence that CBP-1, the worm ortholog of human p300/CBP, functions as a cofactor of CEP-1. PRMT-5 forms a complex with both CEP-1 and CBP-1 and can methylate the latter. Importantly, down-regulation of cbp-1 significantly suppresses DNA damage-induced egl-1 expression and apoptosis in prmt-5 mutant worms. These findings suggest that PRMT-5 likely represses CEP-1 transcriptional activity through CBP-1, which represents a novel regulatory mechanism of p53-dependent apoptosis.

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