"You have made your way from worm to man, and much within you is still a worm" (Nieztsche)
A genetic pathways that acts parallel or downstream of cep-1/p53 or affect DNA damage induced apoptosis, unexpected roles of C. elegans SIR-2
One of the highlights of the last years led by my PhD student Sebastian related to our analysis of the C. elegans Sir-2 histone deacetylase. We found that C. elegans sir-2.1, previously known to have a role in aging and stress responses is essential for DNA damage induced apoptosis. We showed and recently published in Genes and Development that sir-2.1 is essential for the execution of apoptosis in response to DNA damage and that sir-2.1 genetically acts in parallel to the worm p53 like gene cep-1 pointing towards a new cep-1 independent apoptosis pathway (link to paper). Cytological analysis of Sir-2.1 suggests a novel mechanism of apoptosis induction. During apoptosis Sir-2.1 translocates from the nucleus to the cytoplasm of dying cells and transiently co-localizes with the C. elegans Apaf-1 homologue CED-4 at the nuclear periphery. SIR-2.1 translocation is an early event in germ cell apoptosis and occurs independently of apoptosis execution raising the possibility that SIR-2.1 nuclear export is linked to the induction of DNA damage induced apoptosis.
Before establishing that a non nuclear function of SIR-2 might be required for DNA damage induced apoptosis we also asked if transcriptional regulation by SIR-2 might impinge on apoptosis regulation. These studies were based on the established role of SIR-2 in transcriptional silencing in budding yeast. As to this we did transcriptional arrays and indeed found that ~100 genes are derepressed in sir-2 mutants. We are currently following up on this and we collaborated with Barbara Meyer (Berkeley), trying to establish if SIR-2 associates with chromosomes and if so whether this occurs near genes regulated by SIR-2 by CHIP-chip analysis.
Novel components of the C. elegans germ cell apoptosis pathway
Over the past year it has become clear that several other genes might act in the SIR-2 pathway to affect DNA damage induced apoptosis. One such gene, isolated by the lab of Brent Derry (Toronto), which like SIR-2 has been implicated in aging. We could show that SIR-2 nucleo-cytolplasmic translocaton is largely reduced in Brent's mutants, hinting towards the possibility that kri-1 might act upstream of SIR-2 to regulate germ cell apoptosis. In addition in collaboration with Dimitirs Xirodimas we found a further gene also specifically needed for DNA damage induced apoptosis.
In summary, we have found several genes that are required for DNA damage induced apoptosis and that act independently of cep-1/p53 transcriptional induction. At the same time we found that there are only very view genes transcriptionally induced by cep-1/p53 (link to paper). At present, based on the biochemical nature of those proteins we can not build up a coherent biochemical pathway, but this is a long term undertaking which without doubt will require us to identify more genes required for DNA damage induced apoptosis. Indeed, my PhD student Ehsan is currently attempting backcrossing two C. elegans mutants that are largely defective in DNA damage induced apoptosis and that were identified in a screen for mutants hypersensitive to treatment with ionizing irradiation.
The C. elegans cell death machinery might be much more complex than anticipated
Using apoptosis occurring during the somatic development of the worm, seminal studies by Horvitz and colleagues lead to the discovery of the basic apoptosis machinery needed for nearly all apoptosis. This includes the single cell death protecting Bcl-2-like molecule CED-9, and the pro-apoptotic worm Apaf-1 like molecule CED-4 as well as the caspase CED-3. Follow up studies led to the notion that CED-4 and CED-9 localize at mitochondria in surviving cells and that in cells destined to die apoptosis activation is mediated by breaking the direct CED-4::CED-9 interaction through the transcriptional induction of EGL-1 which displaces CED-4 form the mitochondrial CED-9 complex. CED-4 would than oligomerize and trigger apoptosis induction by activating the CED-3 caspase.
In contrast to these studies which were done on developmental apoptosis, we focus on C. elegans germ cell apoptosis. While the above three proteins are similarly required for germ cell apoptosis regulation, we found that the cellular localization of CED-4 and CED-9 may not overlap in healthy germ cells, indicating the germ cell apoptosis regulation might be more similar to mammalian apoptosis regulation where Bcl-2 like molecules and Apaf-1 do not directly interact. We are currently following up on the above notion aim at better understanding how CED-4 works using both Biochemical and Genetic approaches.
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