EYA-1 is highly conserved and required for normal reproduction:
The structure of EYA-1 has not been solved in either C. elegans or in higher eukaryotic species. To establish greater validity of EYA-1 as an ideal model for studying conserved features of EYA-1 homologues, structural models of C. elegans EYA-1 and homologues in D. melanogaster and H. sapiens were generated (Fig. 1A). As expected, the structures of all three models exhibited similar overall features, with the notable difference of a smaller alpha-helix at the N-terminus in C. elegans compared to the N-terminus in H. sapiens and D. melanogaster. Nonetheless, we next compared the active site which is denoted in C. elegans by two aspartic acid residues at position 237 and 239, which are conserved across all three species (Fig. 1B). Despite the structural variation at the N-terminus, the conserved active site suggests that C. elegans EYA-1 is a valid model for understanding roles conserved in higher eukaryotes.
Having established structural conservation regarding the active site of EYA-1, we next assessed the requirement of eya-1 for normal reproductive capacity. C. elegans hermaphrodites produce up to 300 progeny per animal with little genetic variability, making them an ideal model to assess the requirement of candidate proteins in reproductive tissue [14]. Therefore, we subjected eya-1(ok654) mutants and wild-type animals to a brood size assay to determine if absence of eya-1 impedes normal reproductive capacity (Fig. 2). This was conducted at 20°C and a more permissive temperature of 25°C, and interestingly, brood size was significantly decreased in eya-1(ok654) mutants compared to wild-type animals at both temperatures (Fig. 2A). Moreover, eya-1(ok654) mutants displayed elevated levels of embryonic lethality (Fig. 2B). Absence of EYA proteins in Drosophila are embryonic lethal showing that eya-1(ok654) mutants show a similar, albeit less severe trend. Nonetheless, these findings suggest that eya-1 is required for maintaining normal reproductive capacity in C. elegans.
eya-1 is required for embryonic chromosomal integrity and germline longevity:
Abnormal reproductive capacity and embryonic lethality have been implicated in chromosomal defects, which can be observed in anaphase stage embryos during meiosis II [18]. To investigate whether the germline and embryonic phenotypes observed in eya-1(ok654) mutants were due to any visible chromosome morphological defects, we examined embryos in the 1 to 2 cell stage of division. Strikingly, eya-1(ok654) mutants exhibit a broad range of adverse morphologies, including chromosomal bridges, embryos with missegregated chromosomes, and embryos with abnormal nuclei (Fig. 3A,B). We also assessed the germline nuclei of eya-1(ok654) mutants and interestingly found no abnormalities in pachytene nuclei or diakinetic oocytes (data not shown). Therefore, we cannot determine if these defects are in the preliminary stage in oocytes yet unobservable, or if the observed abnormal embryonic chromosomes are restricted to the anaphase stage events of meiosis II. Nonetheless, the reduced brood size of eya-1(ok654) mutants suggests that germline processes may be impeded in the absence of eya-1.
To investigate this further, eya-1(ok654) mutants were subjected to a mortal brood size assay (Fig. 2C), where individual eya-1(ok654) mutants were transferred from each generation alongside wild-type animals until sterility occurred. The rationale behind is based on the accumulation of inherited germline mutations throughout generations, ultimately render the strain sterile [19, 20]. We observed a marked reduction in brood size that decreased with each generation. This trend persisted until the 10th generation, at which point eya-1(ok654) mutants were sterile, with no recorded change in the brood of wild-type animals. This suggests that eya-1(ok654) mutants accumulate germline defects that are transferred to each generation. This is interesting as eya-1 expression is typified from early embryogenesis and is most active during somatic development [15]. Unfortunately, we were unsuccessful in generating a functional EYA-1 germline antibody to investigate this further. However, it is possible that the absence of EYA-1 activity in the somatic gonad may have a generational accumulative influence of germline processes independent of eya-1 germline expression. Despite this, the absence of eya-1 in C. elegans results in sterility over successive generations, raising novel concerns regarding its influence of germ cell function and longevity.
eya-1(ok654) mutants are hypersensitive to double stranded DNA breaks:
The observation of embryonic lethality and abnormal anaphase chromosomes in eya-1(ok654) mutants suggests a role for eya-1 in DNA damage repair. To determine if these genomic insults were due to single or double stranded DNA breaks, we aimed to induce both forms of DNA damage in eya-1(ok654) mutants and assess the severity of each insult. Therefore, eya-1(ok654) mutants were subjected to hydroxyurea (HU) and camptothecin (CPT) to induce single and double stranded DNA breaks respectively [21, 22] (Fig. 4). This was performed at both 20°C and 25°C, consistent with our prior results, and both temperatures showed the same trend upon exposure to each agent. Single stranded breaks due to HU exposure led to a minor increase in unhatched embryos in brc-1(ok654) mutants similar to the trend observed in wild-type animals (Fig. 4A). However, exposure to CPT led to a more noticeable increase in unhatched embryos (Fig. 4B). This hypersensitivity to CPT, coupled with observed embryonic chromosomal defects suggests a strong requirement of EYA-1 in DSB repair. To ensure that this trend was specific to eya-1 and not a background mutation we performed the same assay on wild-type animals that were subjected to RNAi of eya-1 and observed a similar trend (data not shown). Current understanding of EYA-1 and its role in DSB repair involves H2AX as a substrate for EYA-1 as part of the primary dsDNA break signal [12]. Since C. elegans do not possess an X chromosome and consequently lack H2AX, this might infer that EYA-1 has novel roles in dsDNA break repair.
This correlation between EYA-1 and dsDNA breaks may be more varied than first thought. Increased dsDNA breaks in the absence of H2AX in C. elegans is a significant point of interest as it potentially adds a new role of EYA proteins in DSB repair. In mammals, after the occurrence of DSBs, EYA-1 recruits a series of proteins associated with DSB repair, and the stimulus for this association of EYA-1 is phosphorylation of H2AX [23]. This allows EYA-1 to function as a scaffold for the core DSB machinery to assemble and facilitate DSB repair [24, 25]. Although C. elegans do not possess H2AX, the proteins EYA-1 recruits in this process for DSB repair are highly conserved and therefore present in C. elegans [13]. Due to this, it is possible that EYA-1 may have a conserved role in the repair of DSBs that goes beyond that of H2AX phosphorylation and may participate further upstream in the DSB repair pathway.
EYA-1 is required for the initiation of apoptosis by its association with ceh-34 [17]. Due to this, we cannot rule out the possibility that this hypersensitivity to DSB induction in eya-1 mutants might be due to deficient induction of apoptosis as normally initiated by eya-1 and ceh-34. In this regard, it might well be that cells with DSBs beyond repair that would otherwise undergo apoptosis might be unchecked and allowed to persist. Therefore, our observations may be due to an overaccumulation of DSBs in cells that would otherwise be eradicated in wild-type animals. However, the specific sensitivity to induction of DSBs in eya-1 mutants coupled with the lack of sensitivity to induction of SSBs suggests a correlation between eya-1 and DSB repair independent of H2AX signalling.