Description

Dosage compensation is the mechanism by which organisms correct the sex chromosome imbalance between sexes (e.g. females having two X chromosomes compared to one X in males). In C. elegans, dosage compensation is achieved by the downregulation by half of both X chromosomes in hermaphrodites (Albritton & Ercan, 2018). This downregulation is accomplished by the Dosage Compensation Complex (DCC), which is comprised of a condensin I^DC subcomplex interacting with other accessory proteins. Condensin I^DC has a similar structure to canonical condensins (condensin I and condensin II), which function to compact chromosomes during mitosis and meiosis (Csankovszki et al., 2009). The DPY-27 subunit is unique to condensin I^DC, MIX-1 is present in all three condensins, while the proteins CAPG-1, DPY-26, and DPY-28 are found both in condensin I and condensin I^DC (Figure 1A).

DCC mutants show maternal effect lethality, since all subunits of condensin I^DC and several of the accessory proteins are maternally contributed to oocytes (Plenefisch et al., 1989; Csankovszki et al., 2009). Homozygous DCC mutants derived from heterozygous mothers survive to adulthood due to the maternally provided RNA and/or protein. These mutants are referred to as maternal positive, zygotic negative (M+Z-). M+Z- hermaphrodites are unable to produce a functional gene product; therefore, their progeny have no maternal or zygotic contribution of these proteins (M-Z-). As a consequence, very few M-Z- hermaphrodites survive past the L1 stage; however, males do not require the DCC to survive. It is possible, then, to recover M-Z- male progeny from self-fertilizing hermaphrodites in these conditions.

A previous study (Csankovszki et al. 2009) showed that M+Z- capg-1 null mutants (tm1514) are sterile and have severe developmental defects. This phenotype is different from and more severe than what was previously seen for genes encoding other condensin I^DC members (Plenefisch et al., 1989). It raised the possibility that the sterility and more severe developmental phenotypes of capg-1(tm1514) is the result of another role of CAPG-1 outside of condensin I and I^DC function. We acquired another capg-1 allele (tm5770) from the Japanese National Bioresource Project. This allele deletes a smaller portion of the coding sequence than capg-1(tm1514) but is also predicted to be null due to the resulting frameshift mutation. Also of interest, the capg-1(tm5770) allele removes only the terminal nucleotide from a short piRNA gene deleted entirely in capg-1(tm1514) (Figure 1B). If the absence of the CAPG-1 protein function was responsible for the sterility phenotype observed in capg-1(tm1514), the M+Z- hermaphrodites from the capg-1(tm5770) strain should also be sterile.

We first confirmed via fluorescence microscopy that the DCC is not recruited to the X chromosome in the capg-1(tm1514) adult hermaphrodites, consistent with previously published results (Csankovszki et al., 2009). For additional control, we used a mutation in another condensin I^DC member, dpy-28(tm3535), which has similar defects (Hernandez et al., 2018). Fluorescent antibodies specific to CAPG-1 and another condensin I^DC subunit, DPY-27, were used to visualize localization of the DCC to the X chromosome compared to wild type (N2) (Figure 1C). N2 hermaphrodites have overlapping signals of CAPG-1 and DPY-27 on both X chromosomes. The X localization of these two condensin I^DC proteins is also absent in capg-1(tm5770). This indicates that the capg-1(tm5770) mutation also disrupts DCC localization to the X to a similar degree as capg-1(tm1514) or dpy-28(tm3535).

The capg-1(tm5770) M+Z- hermaphrodites, unlike the capg-1(tm1514) mutants, were observed laying embryos, some of which hatched then arrested in L1, showing more phenotypic similarity to the dpy-28(tm3535) mutants than the capg-1(tm1514) mutants. To quantify this observation, we conducted embryo and lethality counts in capg-1(tm1514), capg-1(tm5770), and dpy-28(tm3535) mutants to assess both the number of embryos laid and the number of embryos hatched (Figure 1D). Our results show that while capg-1(tm1514) M+Z- mutants did not lay any eggs, the capg-1(tm5770) M+Z- and the dpy-28(tm3535) M+Z- mutants produced significant numbers of embryos. Many of the embryos laid by the capg-1(tm5770) and dpy-28(tm3535) mutants hatched then arrested in the L1 stage. Interestingly, there is a high amount of variability in numbers of embryos laid between individual worms in the dpy-28(tm3535) and capg-1(tm5770) strains. The dpy-28(tm3535) and capg-1(tm5770) mutants produced a small percentage of M-Z- progeny that survived until adulthood. Phenotypically, these were either males or very Dpy hermaphrodites that had severe developmental defects and were sterile. The capg-1(tm5770) mutants (n=10) laid an average of 58 embryos per worm, ranging between 0 and 195. Of the 580 total embryos laid, 297 hatched, of which 291 arrested in the L1 stage, with 1 male and 5 hermaphrodites surviving to adulthood. The dpy-28(tm3535) mutants (n=7) laid an average of 122 embryos per worm, ranging between 8 and 255. Of the 853 total embryos laid, 354 hatched, of which 340 arrested in the L1 stage, with 9 males and 5 hermaphrodites surviving to adulthood. The appearance of males in the M-Z- progeny is consistent with a weak Him phenotype reported previously for condensin I mutants (Plenefisch et al., 1989; Hernandez et al., 2018). Overall, these results indicate that the capg-1(tm5770) mutation results in phenotypes resembling the phenotypes caused by dpy-28(tm3535) and other mutations in condensin I^DC subunits (Plenefisch et. al, 1989). These condensin I^DC mutant phenotypes are different from the phenotypes resulting from the capg-1(tm1514) mutation.

These data suggest that the more severe phenotype in the capg-1(tm1514) mutants is not due to disruption of CAPG-1 function. There are several potential alternative explanations. The phenotype may be due to the deletion of the piRNA gene near the 5’ end of the capg-1 gene (Figure 1B). It is also possible that the severe phenotype observed in capg-1(tm1514) is due to a disruption of the trans-splice site between genes. The capg-1 gene is last in its operon, and the capg-1(tm1514) deletion includes a trans-spliced acceptor site (Worm Base). This would result in defective trans-splicing between capg-1 and the upstream gene, F26A3.1. Our data does suggest, however, that the severity of the capg-1(tm1514) phenotype is not due to an alternative role of CAPG-1 outside of condensin I and condensin I^DC function.