Regulation of Caenorhabditis elegans p53/CEP-1-dependent germ cell apoptosis by Ras/MAPK signaling

Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1-dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage-induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1-independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1-dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1-dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution.     

The Fatty Acid Synthase Gene is a Conserved p53 Family Target Gene from Worm to Human

The discovery that the p53 family consists of three members (p53, p63 and p73) in vertebrates and of a single homolog in invertebrates has raised the challenge of understanding the functions of the ancestor and how they have evolved and differentiated within the duplicated genes in vertebrates. Here, we report that the fatty acid synthase (FAS) gene, encoding for a key enzyme involved in the biogenesis of membrane lipids in rapidly proliferating cells, is a conserved target of the p53 family throughout the evolution. We show that CEP-1, the C. elegans p53 homolog, is able to bind the two p53 family responsive elements (REs) identified in the worm fasn-1 gene. Moreover, we demonstrate that fasn-1 expression is modulated by CEP-1 in vivo, by comparing wild-type and CEP-1 knockout worms. In human, luciferase and chromatin immunoprecipitation assays demonstrate that TAp73α and ΔNp63α, but not p53, TAp73β and TAp63α bind the two p53 REs of the human FASN gene. We show that the ectopic expression of TAp73β and ΔNp63α leads to an increase of FASN mRNA levels, while their silencing produces a decrease of FASN expression. Furthermore, we present data showing a correlation between ΔNp63α and FASN expression in cellular proliferation. Of relevant importance is that fasn-1 is the first CEP-1 direct target gene identified so far in C. elegans and our results suggest a new CEP-1 role in cellular proliferation and development, besides the one already described in apoptosis of germ cells. These data confirm the hypothesis that the ancestral functions of the single invertebrate gene may have been spread out among the three vertebrate members, each of them have acquired specific role in cell cycle regulation.     

The EEL-1 ubiquitin ligase promotes DNA damage-induced germ cell apoptosis in C. elegans

E3 ubiquitin ligases target a growing number of pro- and anti-apoptotic proteins, including tumour suppressor p53, caspases, and the Bcl-2 family. The core apoptosis pathway is well conserved between mammals and Caenorhabditis elegans, but the extent to which ubiquitin ligases regulate apoptotic cell death is not known. To investigate the role of E3 ligases in apoptosis, we inhibited 108 of the 165 predicted E3 ubiquitin ligase genes by RNA interference and quantified apoptosis in the C. elegans germline after genotoxic stress. From this screen, we identified the homologous to E6-associated protein C terminus-domain E3 ligase EEL-1 as a positive regulator of apoptosis. Intriguingly, the human homologue of EEL-1, Huwe1/ARF-BP1/Mule/HectH9, has been reported to possess both pro- and anti-apoptotic functions through its ability to stimulate Mcl-1 and p53 degradation, respectively. Here, we demonstrate that eel-1 is required to promote DNA damage-induced germ cell apoptosis, but does not have a role in physiological germ cell apoptosis or developmental apoptosis in somatic tissue. Furthermore, eel-1 acts in parallel to the p53-like gene cep-1 and intersects the core apoptosis pathway upstream of the Bcl-2/Mcl-1 orthologue ced-9. Although ee1-1 mutants exhibit hypersensitivity to genotoxic stress they do not appear to be defective in DNA repair, suggesting a distinct role for EEL-1 in promoting damage-induced apoptosis in the germline.     

The p53 and Mdm2 families in cancer.

Cells within an organism are occasionally exposed to either intracellular or environmental stress. Such stress often has genotoxic potential that enhances the probability of cancer. Two gene families, the p53 family (p53, p63 and p73) and the Mdm2 family (Mdm2 and MdmX), serve as major integrators of the signals generated by genotoxic and oncogenic stress. Their co-ordinated modulation ensures an optimal response to stress and decreases the likelihood of cancer. Work over the past year has provided better understanding of the p53-Mdm2 module that lies in the heart of this regulatory network, and of the intricate interplay between the various members of the network.     

Stress-induced germ cell apoptosis by a p53 independent pathway in Caenorhabditis elegans

In Caenorhabditis elegans, several distinct apoptosis pathways have been characterized in the germline. The physiological pathway is though to eliminate excess germ cells during oogenesis to maintain gonad homeostasis and it is activated by unknown mechanisms. The DNA damage-induced germ cell apoptosis occurs in response to genotoxic agents and involves the proteins EGL-1 and CED-13, and the DNA damage response protein p53. Germ cell apoptosis can also be induced in response to pathogen infection through an EGL-1 dependent pathway. To gain insight into the mechanism and functions of germ cell apoptosis, we investigated whether and how other forms of stress induce this cell death. We found that oxidative, osmotic, heat shock and starvation stresses induce germ cell apoptosis through a p53 and EGL-1 independent pathway. We also learned that the MAPK kinases MEK-1 and SEK-1, and the p53 antagonist protein ABL-1, are essential for stress-induced germ cell apoptosis. We conclude that in C. elegans responses to various stresses that do not involve genotoxicity include an increase in germ cell apoptosis through the physiological pathway.