Fig. 7: IHO1-HORMAD1 complex and ANKRD31 are redundantly necessary for meiotic DSBs.

A, B, D Numbers of small MEI4-REC114 co-clusters (A), MEI4 intensities in MEI4-REC114 co-clusters (B, data points show median cluster intensities per cell), and DMC1 focus numbers (D) in spermatocytes of adult mice. Zygo-pachytene (D), only in SC-defective backgrounds) is equivalent to a mix of late-zygotene and early pachytene stages which are indistinguishable if SC is defective. Pooled data is shown from 6 (A, B) or 2 (D) mice of each genotype. Bars are medians, n=cell numbers. Two-tailed Mann Whitney U-Test, ns=P > 0.05, *=P < 0.05, **=P < 0.01, ***=P < 0.001, ****=P < 0.0001. Exact P values: (A), wild type (wt) versus Ankrd31^−/− pre-leptotene P = 5.35e-13, early zygotene P = 0.0001596, all the others P < 2.2e-16, (B), all comparisons in pre-leptotene and leptotene P < 2.2e-16, early zygotene, wt vs. Ankrd31^−/− P = 0.3047, Ankrd31^−/− vs. Iho1^C7Δ/C7Δ P = 0.001186, Ankrd31^−/− vs. Hormad1^−/− P = 7.19e-5, (D), leptotene, wt vs. Ankrd31^−/− P = 1.07e-6, Ankrd31^−/− vs. Iho1^C7Δ/C7Δ P = 0.01621, Iho1^C7Δ/C7Δ vs. Ankrd31^−/− Iho1^C7Δ/C7Δ P = 5.28e-5, Ankrd31^−/− vs. Hormad1^−/− P = 0.1945, Hormad1^−/− vs. Ankrd31^−/− Hormad1^−/− P = 0.175, early zygotene, wt vs. Ankrd31^−/− P = 5.82e-10, Ankrd31^−/− vs. Iho1^C7Δ/C7Δ P = 0.00041, Iho1^C7Δ/C7Δ vs. Ankrd31^−/− Iho1^C7Δ/C7Δ P = 1.47e-13, Ankrd31^−/− vs. Hormad1^−/− P = 0.0002882, Hormad1^−/− vs. Ankrd31^−/− Hormad1^−/− P < 2.2e-16, late zygotene, wt vs. Ankrd31^−/− P = 0.0004372, Ankrd31^−/− vs. Iho1^C7Δ/C7Δ P = 1.48e-13, Iho1^C7Δ/C7Δ vs. Ankrd31^−/− Iho1^C7Δ/C7Δ P = 5.35e-16, Ankrd31^−/− vs. Hormad1^−/− P < 2.2e-16, Hormad1^−/− vs. Ankrd31^−/− Hormad1^−/− P = 2.49e-11, early pachytene, wt vs. Ankrd31^−/− P = 0.0001234, Ankrd31^−/− vs. Iho1^C7Δ/C7Δ P = 1.1e-10. C Immunostaining in leptotene spermatocytes from adult mice. Bars, 10 µm. E Radiograph of immunoprecipitated and radioactively labeled SPO11-oligo complexes from testes of adult mice. Bar, SPO11-specific signal, asterisk, nonspecific labelling, and arrowhead, immunoglobulin heavy-chain. Radioactive signals were background-corrected (Iho1^−/−, signal=0) and normalized to wild-type control (1). Means and standard deviations are from n = 2 biological replicates. F Schematic summary of phenotypes caused by the disruption of IHO1-HORMAD1 complex and/or ANKRD31. G Model for the assembly of DSB-factor clusters on axis. Black arrows represent promotion of (i) IHO1 phosphorylation and (ii) seeding or (iii) growth of DSB-factor clusters by CDC7-DBF4, IHO1 (in particular, phosphorylated IHO1 C-terminus) and ANKRD31, respectively. See also related Supplementary Fig. 10 and 11 and Supplementary Table 6. Source data are provided as a Source Data file.