MRG-1 is required for genomic integrity in Caenorhabditis elegans germ cells

Abstract

During meiotic cell division, proper chromosome synapsis and accurate repair of DNA double strand breaks (DSBs) are required to maintain genomic integrity, loss of which leads to apoptosis or meiotic defects. The mechanisms underlying meiotic chromosome synapsis, DSB repair and apoptosis are not fully understood. Here, we report that the chromodomain-containing protein MRG-1 is an important factor for genomic integrity in meiosis in Caenorhabditis elegans. Loss of mrg-1 function resulted in a significant increase in germ cell apoptosis that was partially inhibited by mutations affecting DNA damage checkpoint genes. Consistently, mrg-1 mutant germ lines exhibited SPO-11-generated DSBs and elevated exogenous DNA damage-induced chromosome fragmentation at diakinesis. In addition, the excessive apoptosis in mrg-1 mutants was partially suppressed by loss of the synapsis checkpoint gene pch-2, and a significant number of meiotic nuclei accumulated at the leptotene/zygotene stages with an elevated level of H3K9me2 on the chromatin, which was similarly observed in mutants deficient in the synaptonemal complex, suggesting that the proper progression of chromosome synapsis is likely impaired in the absence of mrg-1. Altogether, these findings suggest that MRG-1 is critical for genomic integrity by promoting meiotic DSB repair and synapsis progression in meiosis.

Figure 1

Loss of mrg-1 function induces germ cell apoptosis. (A) mrg-1 RNAi enhances the number of germ cell corpses in vps-18(tm1125) mutants. (B) Representative DIC images of germ cell corpses in wild-type (N2), mrg-1(qa6200), mrg-1(tm1227) and mrg-1(RNAi) worms. Arrows indicate cell corpses. Bars, 10 μm. (C) Time course analyses of germ cell corpses in wild-type and smIs34 worms treated with mrg-1 RNAi. In wild type, cell corpses were viewed by formation of disc-like structures under DIC optics. In smIs34 worms, cell corpses were viewed by formation of CED-1::GFP rings. (D) mrg-1 deletion mutants show increased germ cell apoptosis. (E, F) Quantification of germ cell apoptosis induced by γ-irradiation (E) and ENU (F) in mrg-1 mutants. Cell corpses were scored and analyzed as described in Materials and Methods. Error bars represent standard error of the mean (SEM). Comparisons were performed using unpaired t-test. *P < 0.05, **P < 0.001.

Figure 2

Mutations affecting DNA damage checkpoint genes partially suppress germ cell apoptosis induced by mrg-1 RNAi. (A) Germ cell corpses in mrg-1 RNAi-treated mutants including hus-1(op244), mrt-2(e2663), clk-2(mn159), cep-1(gk138), spo-11(ok79) and cep-1(gk138);spo-11(ok79) were quantified and analyzed at the adult ages of 36 h (left) and 48 h (right) post L4 stage, respectively. Comparisons were made between N2 and other strains treated with mrg-1 RNAi using unpaired t-test. *P < 0.05, **P < 0.001. (B) Quantification of germ cell corpses in N2, cep-1(gk138) and double mutants of cep-1(gk138) with mrg-1(tm1227), mrg-1(qa6200) and mrg-1(ok1262) at 36 and 48 h post L4 stage. *P < 0.05, **P < 0.001.

Figure 3

mrg-1 mutants accumulate RAD-51 foci in the germ line. (A) Representative images of RAD-51 foci and merged images of RAD-51 foci and DAPI staining are shown for early, mid and late pachytene nuclei of N2, mrg-1(qa6200), mrg-1(tm1227), mrg-1(RNAi), and spo-11(ok79);mrg-1(RNAi) worms. Bars, 5 μm. (B) Quantification of RAD-51 foci in early, mid and late pachytene stages of germ lines in worms as indicated. For each region, at least 20 nuclei per gonad arm were analyzed for a total of ≥ 3 gonads.

Figure 4

γ-irradiation induced chromosome fragmentation in N2 and mrg-1(lf) worms. (A) Representative images of chromosomes in oocytes of N2 and mrg-1(lf) worms treated with γ-irradiation of 70 Gy. Bars, 5 μm. (B) Quantification of chromosome fragmentation in oocyte nuclei with γ-irradiation of 70 and 90 Gy, respectively. (C) Quantification of chromosome fragmentation in oocyte nuclei of met-2 RNAi-treated worms as indicated after exposure to γ-irradiation of 70 and 90 Gy, respectively. The percentages of nuclei with > 6 DAPI-stained bodies are shown and the total numbers of nuclei scored are shown in parentheses.

Figure 5

mrg-1 mutants are defective in meiosis. (A) Quantification of brood sizes, hatching and male percentages of N2, mrg-1(RNAi), mrg-1(qa6200), mrg-1(tm1227), ced-3(n717) and mrg-1(qa6200);ced-3(n717) worms. (B) mrg-1 mutant oocytes show > 6 DAPI-stained bodies. Bars, 5 μm. (C) Quantification of mrg-1 mutant oocytes with > 6 DAPI-stained bodies.

Figure 6

The synapsis checkpoint gene pch-2 and DNA damage checkpoint genes are required for increased apoptosis in mrg-1(RNAi) worms. (A) Quantification of germ cell apoptosis in mrg-1 RNAi-treated N2, pch-2(tm1458), pch-2(tm1458);spo-11(ok79), cep-1(gk138);pch-2(tm1458), and egl-1(n1084n3082) worms. Germ cell corpses in RNAi-treated worms at adult ages of 36 h (left) and 48 h (right) post L4 stage were scored and analyzed as described in Materials and Methods. Comparisons were made between N2 and other mutants treated with mrg-1 RNAi. *P < 0.05, **P < 0.001. (B) A schematic summary of the signaling pathways leading to apoptosis in the absence of mrg-1.

Figure 7

mrg-1 mutants are defective in progression of meiotic prophase. Representative images of DAPI-stained nuclei of whole-mount gonads from age-matched worms of the following strains are shown: (A, B) N2, (C, D) mrg-1(qa6200), (E, F) mrg-1(tm1227), (G, H) syp-2(ok307), and (I, J) brc-1(tm1145). Boxed regions in A, C, E, G and I representing mid to late pachytene stages are magnified and shown in B, D, F, H and J, respectively. ≥ 19 gonads for each strain were analyzed. Bars, 10 μm.

Figure 8

Analysis of chromosome pairing, sister chromatid cohesion, and chromosomal loading of the SC in mrg-1 mutants. (A) Representative image of a wild-type germ line with regions of meiotic progression indicated. (B) Representative FISH images of mid pachytene nuclei in N2 (left) and mrg-1(qa6200) (middle) germ lines using 5S rDNA (green) on chromosome V. DAPI staining is shown in red. Arrowheads indicate nuclei with unpaired FISH signals. Bars, 10 μm. Quantification of nuclei with paired FISH signals in different germline regions is indicated in the right panel. For each strain, 7 gonads were examined and a total of ≥ 74 nuclei were analyzed for each zone. Comparisons were performed between N2 and mrg-1(qa6200) mutants using unpaired t-test. *P < 0.05, **P < 0.001. (C) Representative images of REC-8 antibody staining (red) in pachytene and diakinesis nuclei in N2 and mrg-1(qa6200) worms. DAPI staining is shown in blue. Bars, 5 μm. (D) Representative images of HTP-3 (red) and SYP-2 (green) antibody staining in mid pachytene nuclei in N2 and mrg-1(qa6200) germ lines. DAPI staining is indicated in blue. Bars, 5 μm.

Figure 9

mrg-1 and syp-2 mutants show increased H3K9me2 in the germ line. (A) Representative images of H4K5ac (left) and H4K8ac (right) of nuclei in the pachytene regions of N2 and mrg-1(qa6200) germ lines. Bars, 5 μm. (B) Representative images of H3K9me2 patterns in N2, mrg-1(qa6200), syp-2(ok307), and spo-11(ok79) germ lines. The boxed regions in the left panels are magnified and shown in the right panels. Bars, 10 μm. (C) Representative images of H3K9me2 in nuclei of late pachytene regions in N2 and mrg-1(qa6200) germ lines treated with RNAi of met-2 and ego-1. Bars, 5 μm. In all panels DAPI-stained chromatins are shown in blue and antibody staining is shown in red.