CEP-1, the sole
p53 family member in C. elegans, is a key regulator of DNA damage-induced germline apoptosis. In response to DNA damage, CEP-1 activates the transcription of EGL-1, resulting in inhibition of the anti-apoptotic protein CED-9 and activation of the pro-apoptotic proteins CED-4 and CED-3. In mammalian cells, members of the E3 ubiquitin ligase family have been shown to target several cell death proteins for degradation, including the
p53 and Bcl-2 family of proteins. To investigate the role of E3 ubiquitin ligases in
cep-1 mediated germline apoptosis, we systematically inhibited the expression of all predicted E3 ligases by RNAi and quantified DNA damage-induced apoptosis in the germline. From this screen we identified the gene
eel-1 as a positive regulator of DNA damage-induced germ cell apoptosis. Animals depleted of
eel-1 by RNAi or mutation had significantly lower levels of germ cell apoptosis in response to ionizing radiation compared with controls. EEL-1 is a member of the HECT domain family of E3 ligases and is homologous to the human protein ARF-BP1. In human osteosarcoma cells, ARF-BP1 has been shown to negatively regulate both the tumour suppressor protein
p53 and the anti-apoptotic protein Mcl-1, a CED-9 ortholog. We found that
egl-1 transcript levels were induced to similar levels in
eel-1(lf) mutants as wild-type controls treated with the same dosage of ionizing radiation. This indicates that
eel-1 does not affect CEP-1 activity and therefore does not target CEP-1 for degradation. Epistasis experiments suggest that
eel-1 acts upstream of
ced-9, and we are currently testing the hypothesis that EEL-1 targets CED-9 for degradation in response to DNA damage. Our results suggest that unlike the mammalian protein ARF-BP1,
eel-1 impinges on
ced-9 downstream of
cep-1 to regulate apoptosis in the C. elegans germline.