Cyclin B3 and dynein heavy chain cooperate to increase fitness in the absence of mdf-1/MAD1 in Caenorhabditis elegans

Abstract

Spindle assembly checkpoint (SAC) ensures genome stability by delaying anaphase onset until all the chromosomes have achieved proper spindle attachment. Once correct attachment has been achieved, SAC must be silenced. In the absence of mdf-1/MAD1, an essential SAC component, Caenorhabditis elegans cannot propagate beyond 3 generations. Previously, in a dog-1(gk10)/FANCJ mutator background, we isolated a suppressor of mdf-1(gk2) sterility (such-4) which allowed indefinite propagation in the absence of MDF-1. We showed that such-4 is a Cyclin B3 (cyb-3) duplication. Here we analyze mdf-1 such-4; dog-1, which we propagated for 470 generations, with freezing of samples for long time storage at F170 and F270. Phenotypic analysis of this strain revealed additional suppression of sterility in the absence of MDF-1, beyond the effects of such-4. We applied oligonucleotide array Comparative Genomic Hybridization (oaCGH) and whole genome sequencing (WGS) and identified a further amplification of cyb-3 (triplication) and a new missense mutation in dynein heavy chain (dhc-1). We show that dhc-1(dot168) suppresses the mdf-1(gk2), and is the second cloned suppressor, next to cyb-3 duplication, that does not cause a delay in anaphase onset. We also show that amplification of cyb-3 and dhc-1(dot168) cooperate to increase fitness in the absence of MDF-1.

Keywords: APC/C, anaphase promoting complex/cyclosome; CIN, chromosome instability; Cyclin B3 (cyb-3); EMS, ethyl methanesulfonate; Him, high incidence of males; SAC, spindle assembly checkpoint; WGS, whole genome sequencing; dynein heavy chain (dhc-1); genome stability; mdf-1/MAD1; oaCGH, oligonucleotide array Comparative Genomic Hybridization; spindle assembly checkpoint (SAC).

Figure 1.

Long-term propagation of unc-46 mdf-1 such-4; dog-1 homozygotes for 470 generations results in significant fitness recovery. (A) A schematic representation of the long-term propagation experiment. First, we generated a P0 strain of the following genotype: unc-46(e177) mdf-1(gk2) +/+ + nT1[let-X]; dog-1(gk10)/dog-1(gk10). Since mdf-1(gk2) results in lethality, gk2 is kept balanced over nT1 which is a reciprocal translocation between Chromosomes IV (depicted in red) and V (depicted in blue), and serves as an effective recombination suppressor along the translocated portions of each chromosome. dog-1(gk10) on Chromosome I is depicted in brown. We picked F1 unc-46 mdf-1; dog-1 homozygotes (n = 40) and plated them individually. We isolated a single plate containing fertile worms that survived beyond 3 generations as a suppressor candidate and named it such-4.⁹ The rest of the plates had no surviving progeny after 3 generations (depicted in light gray). We backcrossed one worm from F4 unc-46 mdf-1 such-4; dog-1 to N2 in order to remove dog-1(gk10) and thus avoid further accumulation of dog-1 induced mutations (depicted with light green). A second clone from F4 unc-46 mdf-1 such-4; dog-1 was maintained at 20°C for 470 generations to allow further accumulation of mutations. The worms were frozen for long term storage at the following points: F170 (depicted in dark green), F270 (depicted in purple) and F470 (depicted in blue). (B) Quantification of fitness, measured as the percent of fertile hermaphrodite progeny. Error bars represent SEM (n = 8 trials for each strain and 10 worms per trial). Please note that our initial analysis revealed that F2 unc-46(e177) mdf-1(gk2); dog-1 did not differ significantly from F2 unc-46(e177) mdf-1(gk2) homozygotes (t-test p = 0.845) and F4 unc-46(e177) mdf-1(gk2) such-4(h2168); dog-1 homozygotes did not differ significantly from F4 unc-46(e177) mdf-1(gk2) such-4(h2168) homozygotes (t-test p = 0.357). For that reason we performed multiple trials using F2 unc-46(e177) mdf-1(gk2) (depicted in white) and F4 unc-46(e177) mdf-1(gk2) such-4(h2168) (depicted in light green) as controls.

Figure 2.

dhc-1(dot168) suppresses lethality and sterility in the absence of MDF-1. (A) Tetra-primer ARMS-PCR20 analysis of dhc-1(+), unc-46 (e177) mdf-1(gk2) such-4(h2168) and unc-46 (e177) mdf-1(gk2) such-4(h2168); dog-1(gk10) mutation accumulation lines at F170, F270 and F470. The 439 bp product of the 2 outer primers was present in all strains; the 199 bp product of the wild-type allele was present in WT and unc-46 (e177) mdf-1(gk2) such-4(h2168); the 299 bp product of the dhc-1(dot168) mutant allele was present in F170, F270 and F470. (B) dhc-1(dot168) changed serine to leucine at position 3708 within the strut/buttress motif of the fifth AAA ATPase domain. Other organisms also have serine or threonine in this position (or glutamic acid for budding yeast). Organisms: Ce (Caenorhabditis elegans), Dd (Dictyostelium discoideum), Dm (Drosophila melanogaster), Hs (Homo sapiens), Mm (Mus musculus) and Sc (Saccharomyces cerevisiae). (C) Graphic representation of the dynein heavy chain motor domain organization based on the D. discoideum structure.¹⁸ We used T-Coffee (http://www.tcoffee.org) to align the C. elegans DHC-1 sequence to D. discoideum Dhca and based on homology extrapolated the location of the important motifs and domains in C. elegans DHC-1. (D) 3D model of the motor domain with arrows pointing to locations of dot168 and or283ts. The model was made based on the previously published model.³¹ (E) Phenotypic analysis of dhc-1(dot168) and dhc-1(or283ts) at 20°C and 25°C. The graph represents percent of progeny that develop into adults, while error bars represent SEM.

Figure 3.

dhc-1(dot168) does not delay anaphase onset. (A) Time-lapse images (in seconds) of one-cell stage embryos that carry ruIs32, an integrated H2B::GFP transgene;²² wild-type [ruIs32], dhc-1 [dhc-1(dot168); ruIs32], mdf-1 [unc-46(e177) mdf-1(gk2); ruIs32] and mdf-1; dhc-1 [unc-46(e177) mdf-1(gk2); dhc-1(dot168); ruIs32] embryos are shown. (B) Summary of the timing measurements of the interval from complete nuclear envelope breakdown (NEBD) to anaphase onset (measured in seconds). Error bars represent standard error of the mean (SEM) for n = 5 measurements for each strain.

Figure 4.

dhc-1(dot168) substitution and cyb-3 amplification work together to increase fitness in the absence of MDF-1. (A) oaCGH plots of 190 kb duplication located on Chromosome V. The y axes represent the log2 ratio of signal intensity of the region in F170 (green) versus N2, F270 (purple) versus N2, and F470 (blue) versus N2. The X axes represent genomic position on Chromosome V. The reference line intersecting at the point 0 reflects wild-type copy number, 2x depicts duplication (F170), while 3x is indicative of triplication (F270 and F470). (B) qRT-PCR was used to confirm the copy number variations affecting the cyb-3 locus in our strains. (C) cyb-3 amplification and dhc-1 act together to increase fitness in the absence of MDF-1. Fitness was measured as the percent of progeny that develop into fertile hermaphrodites. Error bars represent SEM (n = 8 trials for unc-46 mdf-1 such-4 and unc-46 mdf-1 cyb-3trip strains; n = 5 trials for the dhc-1-containing strains).