Developmental arrest of Caenorhabditis elegans BRAP-2 mutant exposed to oxidative stress is dependent on BRC-1

Abstract

Oxidative damage by reactive oxygen species is believed to be a contributor to the development of cancer and the physiological deterioration associated with aging. In this report, we describe the effect of reactive oxygen species exposure to a developing Caenorhabditis elegans organism containing a deletion in the homolog of BRCA1-associated protein 2 (BRAP-2). A mutant containing a deletion of brap-2 was highly sensitive to oxidizing conditions and demonstrated early larval arrest and lethality at low concentrations of the oxidative stress-inducing drug paraquat compared with the wild-type. This developmental arrest occurred early in the L1 stage and was dependent specifically on the function of the C. elegans ortholog of BRCA-1 tumor suppressor brc-1. We also show that developmental arrest in brap-2 mutants when exposed to oxidative stress was due to enhanced expression levels of the cell cycle inhibitor cki-1, and this increase in the expression levels of cki-1 requires brc-1 in brap-2 mutant animals. Our findings demonstrate that BRAP-2 is necessary for preventing an inappropriate response to elevated levels of reactive oxygen species by countering premature activation of BRC-1 and CKI-1.

FIGURE 1.

Domain structure of brap-2 gene and characterization of ok1492 deletion allele. A, schematic representation of the domain structure of the C. elegans BRAP-2 protein, as predicted by the SMART modular architecture program. B, sequence the ok1492 allele. Four individual worms that were homozygous for the ok1492 allele underwent single worm PCR using the primers as stated under “Experimental Procedures.” The PCR products were purified using a standard PCR purification kit from Invitrogen and sequenced by The Centre for Applied Genomics situated at the Hospital for Sick Children in Toronto, Ontario, Canada. The sequence was compared with cosmid EEED8; the numbers refer to the nucleotides of this cosmid, and the deleted region is illustrated. The predicted protein sequence from expression of the ok1492 is also listed. The deletion results in the removal of 259 amino acids near the C-terminal portion of the BRAP-2 protein. C, genomic structure of the brap-2 locus. The boxes represent exons, and the introns are represented by the spaces between the exons. The region of brap-2 deleted in the ok1492 allele (exons 5–8) is represented by the thick line below the genomic structure.

FIGURE 2.

Developmental arrest of brap-2(ok1492) arrest upon oxidative stress. A, DIC image (10× magnification) of a 2-day-old N2 worm grown on an NGM plate containing 2 mm t-butyl hydroperoxide. B, DIC image (20× magnification) of a 2-day-old brap-2(ok1492) mutant worm grown on a plate containing 2 mm t-butyl hydroperoxide. The N2 strain was able to grow to full adulthood, whereas the brap-2(ok1492) mutants arrested at the L1 larval stage. C, concentration dependence of brap-2(ok1492) to paraquat and transgenic rescue by cosmid containing brap-2. Adult N2 and brap-2(ok1492) nematodes were placed on plates containing different concentrations of paraquat, allowed to lay eggs and then were removed. Over 7 days, the progeny were scored for the proportion of animals dead (black), arrested in development (striped) or adult (gray). N2 and brap-2 mutant worms containing the transgene tqEx34 (containing EEED8 cosmid and pF25B3.3::GFP as co-injection marker) were scored at a concentration of 0.1 mm paraquat.

FIGURE 3.

brap-2(ok1492) arrest during the L1 stage. A, differentiation of the M-cell lineage during development (19). B, hlh-8::GFP expression of in two M-cells after 7 days of the brap-2(ok1492) L1-arrested nematode placed on a paraquat containing a plate. C, table of percentage of brap-2 mutant worms under oxidative stress conditions with specific number of cells from the M lineage.

FIGURE 4.

brc-1-dependent brap-2 expression of cki-1 upon oxidative stress induces developmental arrest. Worms carrying an integrated cki-1::GFP reporter gene were crossed into N2 (A), brap-2(ok1492) (B) and brap-2(ok1492) (C); brc-1(tm1145) worms and allowed to hatch in the presence of 0.1 mm paraquat. One-day-old larvae were then removed and placed under 60× magnification. Expression of cki-1::GFP is present in N2 and mutant animals in some neurons in the head and tail, whereas strong expression is visible in the blast cells of brap-2(ok1492) L1s (white arrows), but this up-regulation is reduced in the brap-2;brc-1 double mutant.