A novel recombination protein C12ORF40/REDIC1 is required for meiotic crossover formation

During meiosis, at least one crossover must occur per homologous chromosome pair to ensure normal progression of meiotic division and accurate chromosome segregation. However, the mechanism of crossover formation is not fully understood. Here, we report a novel recombination protein, C12ORF40/REDIC1, essential for meiotic crossover formation in mammals. A homozygous frameshift mutation in C12orf40 (c.232_233insTT, p.Met78Ilefs*2) was identified in two infertile men with meiotic arrest. Spread mouse spermatocyte fluorescence immunostaining showed that REDIC1 forms discrete foci between the paired regions of homologous chromosomes depending on strand invasion and colocalizes with MSH4 and later with MLH1 at the crossover sites. Redic1 knock-in (KI) mice homozygous for mutation c.232_233insTT are infertile in both sexes due to insufficient crossovers and consequent meiotic arrest, which is also observed in our patients. The foci of MSH4 and TEX11, markers of recombination intermediates, are significantly reduced numerically in the spermatocytes of Redic1 KI mice. More importantly, our biochemical results show that the N-terminus of REDIC1 binds branched DNAs present in recombination intermediates, while the identified mutation impairs this interaction. Thus, our findings reveal a crucial role for C12ORF40/REDIC1 in meiotic crossover formation by stabilizing the recombination intermediates, providing prospective molecular targets for the clinical diagnosis and therapy of infertility.

performed in triplicate and the data represent the mean ± SD. ns, not significant; twotailed unpaired t-test. c Western blot of testicular lysates from 20 dpp wild-type and homozygous Redic1 KI mice using REDIC1 antibody. ACTB served as the internal control. The arrows indicate the target bands. d Immunostaining of spermatocyte spreads from WT and Redic1 KI mice with SYCP3 (magenta) and REDIC1 (green) antibodies. Scale bars, 10 μm.

Supplementary Fig. S18 Quantification of MLH1 foci in oocytes from WT and
Redic1 KI female mice. n, the total number of cells analyzed. Bars represent the mean ± SD. P value was calculated by the Mann-Whitney test. ***P < 0.001.

Supplementary Fig. S19 DSB repair is delayed in Redic1 mutant cells. a
Representative spread spermatocytes from wild-type (WT) and Redic1 KI mice were immunostained for SYCP3 (magenta) and DMC1 (green). Early zygotene, late zygotene, early pachytene, and mid-pachytene spermatocytes are shown. Scale bars, 10 μm. b Quantification of DMC1 foci in spread spermatocytes at the indicated substages.

Cell transfections
HEK293T cells were cultured in plastic dishes in DMEM supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37°C in a 5% CO2 incubator. Cells were plated 12 hours in advance, and plasmid transfection was performed using Lipofectamine 3000 (Thermo Fisher, L300015) at ~70% confluency according to the manufacturer's instructions. After 36 hours of transfection, cells were harvested and directly lysed with 2× SDS sample buffer for Western blot analysis.

Co-immunoprecipitation (co-IP)
HEK293T cells were transfected or co-transfected with the indicated expression vectors.
After 48 hours of transfection, cells were harvested and lysed with 500 µl IP buffer (50 mM Tris pH 7.4, 150 mM NaCl, 0.5% Triton X-100, 3 mM MgCl2. 10% Glycerol, 1 mM DTT, and Protease Inhibitor Cocktail). Take 50 µl of lysate as input sample, add 35 µl of agarose beads conjugated with Flag antibody (Genscript, L00432) to the remaining lysate, and incubate at 4°C with slow rotation for 6 hours. After 4 washes with IP buffer, the protein complexes were directly lysed with 2× SDS sample buffer for Western blot analysis.

Spermatocyte smears preparations and immunofluorescence
Testes from ~20 dpp wild-type and adult Redic1 KI mice were dissected and separated from tunica. The testicular cell suspension was made by a mechanical method. PBS was added to the cell suspension to bring the volume to 5 ml and then centrifuged at 100 g for 5 min at room temperature. Discard the supernatant and gently resuspend the cell pellet with 200 µl of fetal bovine serum. Add 20 µl of the cell suspension to one side of the glass slide and use a coverslip to spread the cell suspension gently to the other side.
After air-drying, the smear slides are ready for immunostaining.
The slides were fixed in 4% PFA for 10 min. The slides were fixed in 4% paraformaldehyde for 10 min and then permeabilized with 0.5% PBST (Triton X-100) for 5 min. After being blocked with 3% BSA for 30 min, the primary antibody was applied and incubated overnight at 4°C. The slides were washed 3 times in 0.03% PBST and the secondary antibodies and Hoechst 33342 were applied and incubated at 37°C for 1 hour. After 3 washes, the slides are applied with the appropriate amount of VECTASHILED and finally covered with a clean coverslip. The images were obtained using a Nikon C2plus laser confocal microscope with five layers captured in a Z-axis step of 0.5 μm and then projected at maximum signal intensity using the NIS-Elements software (Nikon).

Preparation of testicular protein extracts and Western blot
Testicular tissues were homogenized in appropriate amounts of lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.5% Triton X-100, 1 mM EDTA, 10% glycerol, protease inhibitor cocktail) on ice using the tissue grinding tubes. The homogenate was transferred to a 1.5 ml Eppendorf tube for sonication, followed by centrifugation at 16,000 g for 10 min at 4°C. The supernatants were mixed with 1/5 volume 5× SDS sample buffer and boiled for 10 min at 100℃. Protein samples were separated by 9% SDS-PAGE and then blotted onto nitrocellulose membranes, blocked with 5% skim milk, and then primary antibody was applied overnight at 4°C. After three washes in TBST (0.1% Triton X-100), HRP-coupled secondary antibodies were applied for 1 hour at room temperature. After three washes, the signals were obtained on the ImageQuant LAS4000 instrument (GE Healthcare) using the enhanced chemiluminescence method.
The antibodies used are listed in Supplementary Table. S3.