SMK-1/PPH-4.1-mediated silencing of the CHK-1 response to DNA damage in early C. elegans embryos

Abstract

During early embryogenesis in Caenorhabditis elegans, the ATL-1-CHK-1 (ataxia telangiectasia mutated and Rad3 related-Chk1) checkpoint controls the timing of cell division in the future germ line, or P lineage, of the animal. Activation of the CHK-1 pathway by its canonical stimulus DNA damage is actively suppressed in early embryos so that P lineage cell divisions may occur on schedule. We recently found that the rad-2 mutation alleviates this checkpoint silent DNA damage response and, by doing so, causes damage-dependent delays in early embryonic cell cycle progression and subsequent lethality. In this study, we report that mutations in the smk-1 gene cause the rad-2 phenotype. SMK-1 is a regulatory subunit of the PPH-4.1 (protein phosphatase 4) protein phosphatase, and we show that SMK-1 recruits PPH-4.1 to replicating chromatin, where it silences the CHK-1 response to DNA damage. These results identify the SMK-1-PPH-4.1 complex as a critical regulator of the CHK-1 pathway in a developmentally relevant context.

Figure 1.

Hyperactivation of CHK-1 by DNA damage in rad-2 mutant embryos. (A–L) Activated CHK-1 was detected by immunostaining with an antibody that recognizes the Ser345-phosphorylated CHK-1 (CHK-1(PO₄-S345)). Embryos were counterstained with Hoechst 33258 to visualize the DNA. The images displayed are representative of a group of ≥10 embryos that were examined per sample. (M) Early embryo extracts were probed by immunoblotting with antibodies against CHK-1 (PO₄-S345), unmodified CHK-1, and PCN-1. (N) Bar diagram summarizes quantitation of the CHK-1(PO₄-S345) band intensity of three independent experiments after image densitometry analysis of the scanned images. +MMS refers to MMS exposure that was accomplished by culturing worms for 20 h on 0.05-mg/ml MMS plates. Error bars represent SD.

Figure 2.

Activated CHK-1 resides in P granules in both N2 and rad-2 embryos. The colocalization of activated CHK-1 with P granules in P cells of four-cell embryos was observed by coimmunostaining with antibodies against activated CHK-1 and the P granule component PGL-1 (OIC1D4). The images displayed are representative of a group of ≥10 embryos that were examined per sample.

Figure 3.

The rad-2 mutation primarily affects the early embryonic DNA damage response but not the checkpoint arrest in the germ line. (A) 50 early embryos collected from gravid worms by bleaching were treated with UV light at the indicated times and doses and were scored for survival to determine embryonic lethality. The data shown were obtained from a representative experiment. See Table S1 (available at http://www.jcb.org/cgi/content/full/jcb.200705182/DC1) for the combined results of three trials with accompanying experimental error. (B) 10 UV light (100 J/m²)–irradiated hermaphrodites were crossed with eight undamaged males harboring a GFP-RNR transgene, and the viability of progeny was assessed. At least 500 eggs were examined per data point. See Materials and methods for experimental details. Error bars represent SD. (C) Gonads were dissected from wild-type N2 and rad-2 hermaphrodites cultured in the absence (control) or presence (UV) of exposure to 100 J/m² UV light and were fixed and stained with Hoechst 33258 to visualize the nuclei in the mitotic zone of the distal tip by fluorescence microscopy. The nuclei within a fixed volume were counted for a minimum of 10 samples per data point as described previously (Holway et al., 2006). These counts ±SD are displayed below each image.

Figure 4.

rad-2 corresponds to mutations in the smk-1 gene. (A) The first embryonic cell cycle was timed in the indicated strains as described previously (Holway et al., 2006). NEB, nuclear envelop breakdown; control, regular media; +MMS, media containing 0.05 mg/ml MMS. (B) Cartoon depicting the construct used to generate the rad-2 (pie-1–smk-1–GFP) strain. The arrow and stop indicate the locations of the start and termination of translation, respectively. (C) Embryonic sensitivity to the indicated DNA-damaging agents was determined as described previously (Holway et al., 2006). Error bars represent SD.

Figure 5.

Identification of mutations in the smk-1 gene from the rad-2 strain. (A) Three mutations (E497G, D580G, and D703G) in genomic DNA sequences for the smk-1 gene isolated from the rad-2 strain were identified. (B) The aspartic acid residue at 703 is highly conserved from yeast to human (SMK-1, worm; PP4R3, human; flfl, fly; and Psy2, yeast). The gray shaded areas indicate similar and identical amino acids. (C) Recombinant myc-tagged SMK-1 or SMK-1 (D703G) was optionally mixed with recombinant untagged PPH-4.1, and the reactions were immunoprecipitated with anti-myc antibodies or nonspecific antibodies (IgG). Input, input material; IP, immunoprecipitated material.

Figure 6.

SMK-1 is recruited to chromatin in a replication-dependent and checkpoint-independent manner. (A–X) Either SMK-1–GFP (A–R) or histone H2B-GFP (S–X) was visualized in fixed samples using fluorescence microscopy. A–L and S–U are otherwise wild-type embryos, whereas M–O and V–X are cdt-1 RNAi embryos, and P–R are atl-1 RNAi embryos. The images displayed are representative of a group of ≥10 embryos that were examined per sample.

Figure 7.

SMK-1 recruits PPH-4.1 to replicating chromatin. (A) A chromatin association assay for early embryos was developed, and the procedure is depicted. The gel shows an immunoblot for α-tubulin, PCN-1, SMK-1–GFP, or PPH-4.1 from the indicated fractions. See Results and Materials and methods for details on the relevant fractions. The tubulin and PCN-1 samples were taken from N2 embryos, and the SMK-1–GFP and PPH-4.1 samples were taken from rad-2 (pie-1–smk-1–GFP) embryos. (B) An immunoblot of either whole embryo extract (WEE) or the chromatin protein–containing fraction C from embryos of the given strain. Animals were cultured in the absence (−) and presence (+) of 0.05 mg/ml MMS, and the blots were probed with anti-GFP antibodies to visualize SMK-1–GFP or anti–PCN-1 antibodies. (C) Same as B except the blots were probed with antibodies recognizing PPH-4.1 or PCN-1. +MMS indicates MMS exposures that were accomplished by culturing worms for 20 h on 0.05-mg/ml MMS plates.