Abstract
DNase II enzymes are acidic endonucleases that have been implicated in mediating apoptotic DNA degradation, a critical cell death execution event. C. elegans genome contains three DNase II homologues, NUC-1, CRN-6, and CRN-7, but their expression patterns, acting sites, and roles in apoptotic DNA degradation and development are unclear. We have conducted a comprehensive analysis of three C. elegans DNase II genes and found that nuc-1 plays a major role, crn-6 plays an auxiliary role, and crn-7 plays a negligible role in resolving 3′ OH DNA breaks generated in apoptotic cells. Promoter swapping experiments suggest that crn-6 but not crn-7 can partially substitute for nuc-1 in mediating apoptotic DNA degradation and both fail to replace nuc-1 in degrading bacterial DNA in intestine. Despite of their restricted and largely non-overlapping expression patterns, both CRN-6 and NUC-1 can mediate apoptotic DNA degradation in many cells, suggesting that they are likely secreted nucleases that are retaken up by other cells to exert DNA degradation functions. Removal or disruption of NUC-1 secretion signal eliminates NUC-1's ability to mediate DNA degradation across its expression border. Furthermore, blocking cell corpse engulfment does not affect apoptotic DNA degradation mediated by nuc-1, suggesting that NUC-1 acts in apoptotic cells rather than in phagocytes to resolve 3′ OH DNA breaks. Our study illustrates how multiple DNase II nucleases play differential roles in apoptotic DNA degradation and development and reveals an unexpected mode of DNase II action in mediating DNA degradation.
Highlights
Programmed cell death, or apoptosis, is a conserved cellular process critical for animal development and tissue homeostasis
We found that the numbers of apoptotic cell corpses in various embryonic stages were similar among N2, nuc1(e1392), crn-6(tm890), and crn-7(ok866) single mutant, and the crn7 crn-6; nuc-1 triple mutant (Figure 1C), suggesting that loss of all three DNase II genes in C. elegans does not affect the activation or progression of cell death and that these three DNase II genes likely act at a late stage of apoptosis
Since nuc-1 and crn-6 are expressed in different regions of embryos, we examined whether they mediate apoptotic DNA degradation in the regions where they are expressed, by scoring transferase dUTP nick end labeling (TUNEL) signals in the head and tail regions of nuc-1(e1392), crn-6(tm890), and crn-6; nuc-1 double mutant embryos
Summary
Programmed cell death, or apoptosis, is a conserved cellular process critical for animal development and tissue homeostasis. In animals carrying a high copy number extrachromosomal array (smEx4085) that contains Pnuc-1crn-7::gfp, we observed bright CRN-7::GFP signals in the anterior region of the transgenic embryos, we failed to detect rescue of the nuc-1 TUNEL defect (Figure 4A and Table S1) These results indicate that CRN-7 cannot substitute for NUC-1 to mediate apoptotic DNA degradation. Even in the line with a lower copy number of Pcrn-6nuc-1::gfp (smIs199, 2–3 copies), partial rescue of the TUNEL defect of nuc-1(e1392) embryos was detected (Figure 4B) These results indicate that NUC-1 can still function to resolve 39 OH DNA breaks in apoptotic cells of the whole embryo, even though its expression is restricted to intestine cells at the posterior region of the embryo under the crn-6 promoter. NUC-1 can act in apoptotic cells to digest 39OH DNA breaks
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