C. elegans strains
All strains were maintained under standard conditions at 20 °C. The strains used were N2 (Bristol; wild type), CB4108 fog-2(q71)BC784 spe-8(hc50)RB1067 his-24(ok1024)MT13971 hpl-1(n4317)DW102 brc-1(tm1145)FX1524 cku-70(tm1524)FX2026 polq-1(tm2026)BJS1017 his-24(ok1024); fog-2(q71)BJS1018 hpl-1(n4317); fog-2(q71)BJS1019 brc-1(tm1145); fog-2(q71)BJS1020 cku-70(tm1524); fog-2(q71)BJS1021 polq-1(tm2026); fog-2(q71).
Measurement of offspring mortality caused by ionizing radiation
For the feminized mutants, synchronized L4 females and males were separated and maintained overnight. On the second day, adult females or males were either left untreated or irradiated with the indicated dose of ionizing radiation delivered by a cesium 137 radiation source (Biobeam GM 8000, Eckert & Ziegler, Gamma-Service Medical). Then, ≥3 irradiated adult worms and ≥3 non-irradiated adult worms of the opposite sex were immediately transferred to 3 new plates serving as 3 biological replicates and allowed to lay eggs for 2 h. Males were then removed and females were allowed to continue laying eggs for another 4 h. The females were then removed and the number of eggs was counted. The number of surviving offspring was characterized 24 h later to examine offspring mortality of the P0 generation. We then transferred ≥3 surviving male offspring (F1) or ≥3 surviving female offspring (F1) into new dishes serving as a single biological replicate and placed them with the three untreated adults of the opposite sex and allowed them to lay eggs for one day. At least three biological replicates were included in each experiment. The adults were then removed and the number of eggs laid was counted. Twenty-four hours later, surviving offspring were counted to characterize offspring mortality of the F1 generation.
For hermaphrodite worms, synchronized hermaphrodite late L4s were separated from the remaining worms by picking, and ≥3 late L4 hermaphrodites were either untreated or irradiated with the indicated dose of ionizing radiation. Three irradiated hermaphrodites were transferred to 3 separate plates as 3 biological replicates and allowed to lay eggs for 6 h. Adults were then removed and the number of eggs was counted 24 h later, the hatched progeny quantified as P0 generation progeny mortality. Surviving worms are transferred to three plates and allowed to lay eggs for one day. Adults were removed and offspring mortality of subsequent generations was quantified 24 h later.
Development analysis
Synchronized L1 worms from control and paternally treated F1s were generated by standard hypochlorite treatment. Arrested L1 worms were plated on NGM agar plates with OP50, fed with bacteria and incubated at 20 °C for 48 h. The larval stage of the worms was then characterized under a Zeiss discovery.V8 microscope. For each experiment, >30 L1 larvae were included for each replicate, n = 3 biological replicates were used.
Quantification of apoptotic germ cell corpses
Day-1 adult female worms were immobilized using 5 mM levamisole (AppliChem A431005) and mounted on a 2% agarose pad on a microscope slide. The number of apoptotic corpses was assessed by Nomarski DIC microscopy on a Zeiss Axio Imager M1/2 based on the refractive morphological changes occurring in apoptotic germ cells within the gonadal loop50.
RNAi treatment
RNAi feeder clones were obtained from the library of J. Ahringer. The E. coli feeder strain HT115 (DE3) with RNAi clones were cultured with LB medium containing ampicillin (100 μg ml-1) overnight. IPTG (1 mM) was added to the culture to induce RNAi product before plating on RNAi agar plates (NGM agar with ampicillin and IPTG). For the RNAi feeding assay, >30 synchronized L1 larvae as P0 generation were plated on RNAi agar plates seeded with E. colifood strain HT115 (DE3) containing specific RNAi or an empty vector control. Three days later, adult males and females were separated and transferred to fresh RNAi plates to maintain RNAi efficiency until further experiments were performed. Subsequent experiments were performed as described in “Measurement of progeny mortality induced by ionizing radiation”.
Immunofluorescence staining
Adult worms were picked from plates and transferred into a drop of M9 buffer onto a 0.3% polylysine-treated three-well slide (3 × 14 mm printed well slides from Fisher Scientific). Germline dissection was performed with two syringe needles, followed by fixation with 3.7% formaldehyde for 1 h. A 24 × 24 mm coverslip was then placed over the drop and the slide was placed in a −80 °C freezer for 10 min to perform the freeze-cracking procedure. The slide was then quickly transferred to -20 °C methanol for less than 1 min. To visualize nuclei, slides were washed once with PBS and once with PBST (0.2% Tween in PBS) and mounted with DAPI Fluoromount-G mounting medium (Southern Biotech) and sealed with nail polish. For the other staining, after fixation, the slides were washed 1 time with 1 × PBS and 2 times with 1 × PBT (0.5% Triton X-100 in PBS). To improve signal quality, slides were first blocked for 20 min with Image-iT FX signal enhancer (Thermo Fisher) before blocking with 1 × PBT containing 10% donkey serum for another 20 min. Then, primary antibodies diluted in 1× PBT containing 5% donkey serum were applied to the slides and incubated at 4 °C overnight. After washing 3 times with 1× PBT, the slides were incubated with secondary antibodies diluted in 1× PBT at 37 °C for 30 min. Slides were then washed with 1 × PBT 3 times, mounted with DAPI Fluoromount-G mounting medium (Southern Biotech), and sealed with nail polish. Slides were stored at 4 °C in the dark before imaging.
Primary antibodies used for immunofluorescence staining were rabbit polyclonal anti-phospho-RNAPII (Ser2) antibody (Thermo Fisher, A300-654A; dilution 1:500 in PBT); mouse monoclonal anti-H3K9me2 antibody (Abcam, ab1220; dilution 1:100 in PBT); rabbit polyclonal anti-HIM-8 (Novus Biologicals, 41980002; dilution 1:100 in PBT); rabbit anti-RAD-51 antibody (a gift from the laboratory of A. Gartner; dilution 1:2000 in PBT). The secondary antibodies used were AlexaFluor 488 donkey anti-mouse IgG (Thermo Fisher, A21202; dilution 1:500 in PBT) and AlexaFluor 594 donkey anti-rabbit IgG (Thermo Fisher A21207; dilution 1:500 in PBT).
Fluorescence images for quantification were taken with a Zeiss Meta 710 confocal laser scanning microscope. For quantification, a fixed exposure time was set for different treatments and strains. For H3K9me2, RNAPII p-Ser2 and RAD-51 staining, z -stack images were obtained with a Zeiss Meta 710 confocal microscope, and H3K9me2 and RNAPII p-Ser2 signal intensity and the number of RAD-51 foci per nucleus were quantified with Imaris x64 9.1.2 software. Fluorescence intensities were normalized to DAPI signal.
SILAC analysis
The stable isotope labeling procedure has been described in a previous study51. ET505 in short E. coli(lysine auxotrophy, from Coli Genetic Stock Center) were grown in M9 minimal medium (Na2HPO4 5.8 g l-1, KH2PO4 3 g l-1, NaCl 0.5 g l-1, NH4Cl2 1 g l-1, glucose 0.2% (w /v), MgSO4 1 mM, thiamine 0.01% (w/v) and 40 µg ml-1 13C6-labeled lysine (Cambridge Isotope Laboratory) or 40 µg ml-1 normal l-lysine and incubated at 37 ° C overnight to reach A600 = 1. Bacteria are concentrated to A600 = 50 and plated in NGM-N plates (3 g NaCl and 12 g agarose dissolved in 970 ml deionized water).
Synchronized embryos generated by hypochlorite treatment were hatched and maintained in M9 buffer, and then L1 worms were transferred to NGM-N plates seeded with heavy isotope-labeled lysine (heavy lysine)- or normal lysine (light lysine)-labeled ET505 E. coli . Worms were fed labeled bacteria for two generations to reach >97% engraftment, and then L4 late-stage worms were selected to be irradiated with ionizing radiation or mock-treated. Four replicates were included in this experiment, two of which were the heavy lysine group treated with ionizing radiation and the light lysine group treated with ionizing radiation, while the other two replicates were the heavy lysine group treated with ionizing radiation and the light lysine group treated with ionizing radiation.
Equal numbers of F1 adult worms were washed from heavy lysine plates and light lysine plates with M9 buffer and combined. After removal of M9 buffer, lysis buffer was added to the worm pellet (6 M guanidinium chloride (GuCl), 10 mM TCEP, 40 mM CAA, 100 mM Tris-HCl). Heat the sample at 95 °C for 10 min and sonicate it with the Bioruptor (30 s sonication, 30 s break, 10 cycles, high throughput). The heating and sonication were repeated once more. The samples were then centrifuged at 20,000gfor 20 min and the supernatant collected. Five microliters of the protein solution was diluted with 20 mM Tris to reduce the guanidinium chloride concentration to below 0.6 M. 50 mM TEAB was added to dilute the samples to 100 µl and then 1 µg Lys-C was added for incubation at 37 °C for 4 h. Samples were further diluted with 180 µl TEAB and treated with 2 µg Lys-C at 37 °C overnight. Enzymatic digestion was stopped by addition of formic acid to 1%, and StageTip sample purification was performed according to the CECAD/CMMC Proteomics Core facility standard protocol (Mass spectrometry proteomics data have been deposited in the ProteomeXchange Consortium through the PRIDE partner repository with the identifier of dataset PXD031873.
Single worm WGS
Single L4 male and female fog-2, and hermaphrodite wild-type with the indicated treatment were transferred to plates with UV-killed OP50 bacteria to reduce bacterial DNA contamination. On the second day, a single adult worm was collected in 10 µl of M9 buffer,…
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