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Meiotic DNA break formation requires the unsynapsed chromosome axis-binding protein IHO1 (CCDC36) in mice

Abstract

DNA double-strand breaks (DSBs) are induced by SPO11 during meiosis to initiate recombination-mediated pairing and synapsis of homologous chromosomes. Germline genome integrity requires spatiotemporal control of DSB formation, which involves the proteinaceous chromosome axis along the core of each meiotic chromosome. In particular, a component of unsynapsed axes, HORMAD1, promotes DSB formation in unsynapsed regions where DSB formation must occur to ensure completion of synapsis. Despite its importance, the underlying mechanism has remained elusive. We identify CCDC36 as a direct interactor of HORMAD1 (IHO1) that is essential for DSB formation. Underpinning this function, IHO1 and conserved SPO11-auxiliary proteins MEI4 and REC114 assemble chromatin-bound recombinosomes that are predicted activators of DSB formation. HORMAD1 is needed for robust recruitment of IHO1 to unsynapsed axes and efficient formation and/or stabilization of these recombinosomes. Thus, we propose that HORMAD1–IHO1 interaction provides a mechanism for the selective promotion of DSB formation along unsynapsed chromosome axes.

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Figure 1: IHO1, interactor of HORMAD1, localizes to unsynapsed axes.
Figure 2: SC formation and alignment of chromosome axes is defective in Iho1−/− spermatocytes.
Figure 3: Recombination foci and SPO11-oligonucleotides are effectively absent from Iho1−/− spermatocytes and testes, respectively.
Figure 4: IHO1 has HORMAD1-independent DSB-formation-promoting activity.
Figure 5: IHO1 is not required for ATR, BRCA1 or γH2AX accumulation on unsynapsed chromatin.
Figure 6: Robust MEI4 focus formation requires IHO1.
Figure 7: MEI4 and IHO1 co-localize on unsynapsed chromosome axes.
Figure 8: IHO1 is a key component of putative DSB-promoting recombinosomes.

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Acknowledgements

We thank R. Jessberger (Institute of Physiological Chemistry, Faculty of Medicine at the TU Dresden, Germany) for sharing ideas, antibodies (anti-SYCP3 and anti-STAG3) and departmental support; M. Stevense for discussion, and revising and proofreading the manuscript; G. Vader (Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany) for sharing budding yeast protocols, E. Marcon (Donnelly Centre, University of Toronto, Canada) for the anti-RPA antibody; F. Schwarz (Genomics and Proteomics Core Facilities, German Cancer Research Center-DKFZ, Germany) and the protein interaction unit of Genomics and Proteomics Core Facility at the German Cancer Research Center—DKFZ for yeast two-hybrid vectors and for performing yeast two-hybrid screens; M. P. Thelen (Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, USA) for the anti-SPO11 antibody, M. A. Handel (The Jackson Laboratory, USA) for histone H1T antibody; D. Drechsel (Max Planck Institute of Molecular Cell Biology and Genetics, Germany) and the EMBL protein expression and purification facility for protein expression vectors; K. Daniel (Biotechnology Center TU Dresden-BIOTEC, Germany) for testis and somatic tissue cDNAs. We thank the DIGS-BB programme for supporting M.S. The Deutsche Forschungsgemeinschaft (DFG; grants: TO421/3-2, SPP1384:TO421/4-2, TO421/5-1, TO421/6-1, TO421/8-1 and 8-2) supported M.S., M.B., F.P., I.D., A.R., D.T. and A.T.; The JSPS Research Fellowship supported H.S., Y.W.; B.d.M. was funded by grants from the Centre National pour la Recherche Scientifique (CNRS) and the European Research Council Executive Agency under the European Community’s Seventh Framework Programme (FP7/2007-2013 Grant Agreement no. [322788]); the NIH (R01 GM105421) supported J.L., M.J. and S.K.; J.L. was supported in part by American Cancer Society fellowship PF-12-157-01-DMC. S.K. is an Investigator of the Howard Hughes Medical Institute.

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Contributions

Most experiments were carried out by M.S. (Figs 1a–c, 4 and 68a, d, g and Supplementary Figs 1b, c, 2b, d, 3, 5b–e, 6 and 7a–d) and M.B. (Figs 1e and 3a–f and Supplementary Figs 1a, d, e, 2a, c and 5a, f, h). Further experiments were performed by F.P. (Figs 2 and 5 and Supplementary Figs 4 and 5f, g), I.D. (Figs 1d and 8b, c, e, f and Supplementary Fig. 7e–l) and J.L. (Fig. 3g, h). Knockout embryonic stem cells were generated by D.T. with the supervision of A.R.; M.J. and S.K. contributed to SPO11-oligonucleotide measurements and S.K. supplied REC114 antibodies; B.d.M. supplied MEI4 antibodies, experimental material and critical unpublished information, H.S. and Y.W. contributed with critical supporting IHO1 localization data. A.T. and M.S. wrote the manuscript. All authors were involved in discussions and commented on the manuscript.

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Correspondence to Attila Tóth.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 IHO1 expression and localization in wild-type spermatocytes.

(a) RT-PCR was used to test expression of Iho1, a meiosis marker (Hormad1) and a “house-keeping” gene (S12) in testis and a somatic tissue mix. cDNAs were prepared from four RNA mixtures: (1) somatic tissue mix: 1 μg of RNA mix of 59 ng total RNAs from 17 somatic tissues (see Materials and Methods for the tissue list). (2) testis: 59 ng total testis RNAs from adult. (3) somatic + testis: 1 μg of RNA mix of 59 ng total testis RNAs and 941 ng of somatic tissue mix. (4) somatic + 5xtestis: 1 μg of RNA mix of 295 ng total testis RNAs and 705 ng of somatic tissue mix. (5) no RT: no RT control with somatic + 5xtestis. Iho1 specific PCR-products were amplified only from templates that contain testis cDNA. (b) Testis cell suspensions were incubated in the presence of EdU to label cells with ongoing DNA replication. An EdU-labelled preleptotene cell is shown, where SYCP3, IHO1 and HORMAD1 were detected by immunofluorescence. HORMAD1 and IHO1 form foci that often colocalize. The red channel is shifted three pixels to the right to allow better visualization of colocalizing signals in the enlarged inset of the overlay image. (c,d) SYCP3 and IHO1 were detected on nuclear surface spreads of wild-type spermatocytes (c) and oocytes (d) at the indicated stages. Oocytes were collected from ovaries either at 14.5 (preleptotene, leptotene and zygotene) or 20.5 (pachytene, diplotene) days post coitum (dpc). Arrow and arrowhead mark synapsed and unsynapsed axes in zygotene cells, respectively. Asterisks (c) mark the largely non-homologous X and Y chromosomes, which synapse only in their short homologous PAR regions in spermatocytes. Note that IHO1 is depleted from X and Y chromosomes in early-pachytene, but it reappears on them in late-pachytene spermatocytes. Scale bars, 10 μm. (e) Indicated proteins were detected in protein extracts from 14.5, 17.5 and 20.5 dpc ovaries, which are rich in leptotene-to-zygotene, pachytene and diplotene-dictyate oocytes, respectively. Histone H3 and GAPDH are loading controls. See Supplementary Fig. S8 for unprocessed gel-scans.

Supplementary Figure 2 IHO1 localization from late-zygotene to diplotene.

(a,d) SYCP3, IHO1 and histone H1T variant (marker of mid-pachytene onwards, a) or γH2AX (d) were detected by immunofluorescence on nuclear surface spreads of wild-type spermatocytes. The images show high levels of IHO1 on sex chromosome axes at the zygotene-to-pachytene transition (a: I; d) and late-pachytene (a: IV; d) while IHO1 levels are much lower in early- (a: II, d) and mid-pachytene (a: III). (d) Asterisk marks sex chromosomes. (a,d) Enlarged insets show higher magnification images of sex chromosomes. P marks the PAR region, where IHO1 is detectable during the zygote-to-pachytene transition. Arrow in d points at the gap between X and Y PAR regions during late-pachytene. Scale bars, 10 μm. (b) Drawings of sex chromosome axes in the four marked cells of a. (c) Quantification of H1T signal on chromatin (upper panel), and IHO1signal on autosomal axes (middle panel, measured on unsynapsed axes in zygotene and diplotene, measured on synapsed axes in other stages) and unsynapsed sex chromosomal (lower panel) axes during late-zygotene (l-zy), zygotene-to-pachytene transition (zy-pa), early-pachytene (e-pa), mid-pachytene (m-pa), late-pachytene (l-pa) and diplotene (di). Median values are shown by red horizontal bars. Numbers of nuclei analyzed are indicated (n). Quantification is shown from a single experiment, the conclusions of which were reconfirmed in further four experiments without in silico quantification of protein-signal intensities in images. Primary source data of graphs are shown in Supplementary Table 2.

Supplementary Figure 3 Iho1 targeting strategy.

(a) Schematics of the targeting construct, the wild-type (WT) and modified Iho1 genomic locus. Black boxes represent exons (not to scale). Recombination at the homology arms (HA) of the targeting construct modifies intron 2 by introducing: 1) an additional exon (SA-IRES-LacZ) that contains a strong splice acceptor site (SA) and poly-adenylation site (left grey box), 2) a transcriptional unit that contains the strong housekeeping human β-Actin promotor (BactP) driving the neomycin (Neo) resistance gene as a selection marker. This modification of intron 2 disrupts the Iho1 open reading frame after the 80th codon (Iho1insertion allele). Recombination catalyzed by FLPo at FRT sites removes the SA-IRES-LacZ exon and the BactP-Neo gene, and restores the IHO1 ORF (Iho1restored). Iho1restored is a functional allele that can be disrupted by Cre-mediated recombination between loxP sites (Iho1deletion). Excision of exon 3 causes a frameshift after the 80th codon. The positions of PCR-genotyping primers are indicated. Red bars mark the 3′ and the internal Southern blot probes; the predicted length of restriction fragments is indicated. (b) Southern blot of DNA from wild-type (+/+), Iho1+/insertion (+/i), Iho1insertion/insertion (i/i) mice derived from the targeted embryonic stem cell clones (ES16). DNA was digested with BclI (both upper and lower panel). The blots indicate a single integration of the targeting cassette in the Iho1 locus. (c) Western blot analysis of extracts prepared from testes of wild-type (WT), Iho1deletion/deletion (Iho1del/del) and Iho1insertion/insertion (Iho1i/i) mice. Rabbit antibody against the first 144 amino acid of IHO1 did not detect full length IHO1 protein in Iho1deletion/deletion (del/del), Iho1insertion/insertion (i/i) testes (upper panel). Actin was used as a loading control (lower panel). Arrowheads mark bands that were not detected reproducibly under distinct blotting and blocking conditions. Hence, these bands are considered non-specific. The pattern of these non-specific bands is distinct in testis extracts of adult wild-type and Iho1-deficient mice, which is likely caused by severely altered cellularity in the testis of Iho1-deficient mice. Unprocessed gel-scans are shown in Supplementary Fig. S8.

Supplementary Figure 4 Iho1−/− mice are deficient in germ cells from late meiotic prophase onwards in both sexes.

(a) Cryosections of testes from 16-week-old wild-type (WT) and Iho1−/− mice. DNA was detected by DAPI, apoptotic cells were detected by nuclear cleaved PARP1 immunostaining. (upper panel) We show a stage VIII wild-type testis tubule, which contains several layers of germ cells at distinct spermatogenic stages: preleptotene (pl) and late-pachytene (pa) spermatocytes and post-meiotic spermatids (sd) and spermatozoa (sp). (lower panel) Iho1−/− meiocytes underwent apoptosis at a stage corresponding to wild-type mid-pachytene in stage IV tubules. Consequently, spermatocytes were not found in the inner layers of testis tubules beyond stage IV, and post-meiotic spermatids and spermatozoa were also missing from Iho1−/− testes. To illustrate this, stage IV, V-VI and VII-VIII tubules of Iho1−/− mice are shown. Apoptotic spermatocytes (ap) are detected in the stage IV tubule, which was identified by the presence of mitotic intermediate spermatogonia (m) and intermediate spermatogonia (In). Stage V-VI and VII-VIII tubules contain somatic Sertoli cells (st) and spermatogonia B (SgB) or preleptotene (pl) spermatocytes, respectively, but more advanced spermatogenic cells are missing. (b) Nobox (oocyte marker) was detected by immunofluorescence on cryosections of ovaries from 6-week-old mice. DNA was stained by DAPI. Oocytes in primordial (pd) and antral (a) follicles are shown in the section of a wild-type ovary. In contrast, oocytes are not detected in the shown Iho1−/− ovary section. Quantification of oocytes revealed much fewer oocytes in Iho1−/− than in wild-type ovaries at 6 weeks of age. Oocytes were counted on every tenth section of ovaries: a total of 624 and 724 oocytes in one ovary from each of two wild-type mice, and 3, 4, 2 and 9 oocytes in one ovary of each of four Iho1−/− mice. Scale bars, 50 μm.

Supplementary Figure 5 Synaptonemal complex formation, but not axis formation, is defective in Iho1−/− mice.

(a,b) SYCP3 and either STAG3 cohesin (a) or HORMAD1 (b) were detected on nuclear spreads of wild-type (WT) and Iho1−/− spermatocytes. Axis morphology, axis association of STAG3 and enrichment of HORMAD1 on unsynapsed axes are similar in wild type and Iho1−/−. (b) The same Iho1−/− spermatocyte is shown in Fig. 2b. Scale bars, 10 μm. (c) Indicated proteins were detected in extracts of wild-type and Iho1−/− testes. See Supplementary Fig. S8 for unprocessed gel-scans. (df) Axis and synaptonemal complex development were assessed by detecting SYCP3 (axis) and SYCP1 (synaptonemal complex) in nuclear spreads of spermatocytes from litter-mate pairs of wild-type and Iho1−/− mice. Ages of mice are indicated in d and e (days postpartum-dpp). Results of two (f) or three (d,e) experiments were pooled; counted cell numbers are indicated (n); source data are available in Supplementary Table 2. (d) Fractions of spermatocytes belonging to four different axis-development category are shown: (blue) punctate axes, preleptotene; (green) short stretches of axis, leptotene; (yellow) relatively long but incomplete axes, early-zygotene; (orange) fully formed axes, late-zygotene and pachytene. Axis development did not differ significantly in Iho1−/− and wild type. (e) shows fractions of spermatocytes with either (blue) no synaptonemal complex, or (orange) variable levels of synapsis ranging from short synaptonemal complex stretches to complete autosomal synaptonemal complex (the latter is observed only in wild type). Fractions of spermatocytes with synaptonemal complex is significantly lower in Iho1−/− than in wild-type mice at 13dpp (p = 0.245 at 9dpp, p = 0.039 at 13dpp, paired t-test). (f) Numbers of SYCP1 stretches are shown in early-zygotene wild-type and Iho1−/− spermatocytes at 13dpp. The median number (bars) of SYCP1 stretches is 5.5-fold lower in Iho1−/− than in wild type (Mann-Whitney test). Thus, synapsis initiation appears less efficient in Iho1−/− than in wild type. (g,h) SYCP3 and SYCP1 were detected on nuclear spreads of wild-type and Iho1−/− spermatocytes (g, early-zygotene) or oocytes (h, 14.5dpc littermate fetuses). Iho1−/− oocytes display similar synaptonemal complex formation defects as Iho1−/− spermatocytes (Fig. 2).

Supplementary Figure 6 IHO1 is required for the accumulation of γH2AX on the chromatin of leptotene and early-zygotene spermatocytes.

(a) SYCP3 (chromosome axis) and γH2AX were detected by immunofluorescence in nuclear spreads of leptotene and early-zygotene wild-type (WT), Hormad1−/−, Iho1−/− and Spo11−/− spermatocytes. Matched exposure images of γH2AX are shown. Bars, 10 μm. (b) Quantification of total nuclear γH2AX immunofluorescence signal in nuclear spreads of wild-type (WT), Hormad1−/−, Spo11−/− and Iho1−/− leptotene and early-zygotene spermatocytes. Arbitrary units are shown. Median γH2AX intensities are marked, and n shows analyzed spermatocyte number. Combined results from three (wild type and Iho1−/−) or two (Hormad1−/− and Spo11−/−) experiments are shown. Primary source data of graphs are available in Supplementary Table 2. The γH2AX-specific signal is significantly lower in Iho1−/− than in wild-type (14-fold) and Hormad1−/− (4.6-fold) spermatocytes, and it is not higher than the signal in Spo11−/− spermatocytes, which are completely deficient in programmed DSB formation (Mann-Whitney test). This indicates that DSB formation is similarly defective in Spo11−/− and Iho1−/− spermatocytes, and more defective than in Hormad1−/− spermatocytes. Note that leptotene and early-zygotene cells are not homogenous populations with respect to DSB numbers in DSB formation-proficient backgrounds, because DSBs are actively introduced into the genome during these stages. Variation in γH2AX levels of wild-type and Hormad1−/− spermatocytes probably reflects this heterogeneity.

Supplementary Figure 7 REC114 interacts with IHO1 and forms co-foci with MEI4 on chromatin in spermatocytes.

(ad) Yeast two-hybrid assays testing interactions of indicated proteins. 10 μl of yeast cell suspensions of indicated optical densities were plated onto indicated dropout plates. (a) The MEI4-REC114 interaction served as a positive control. (b) The single colony in the SPO11/HORMAD1 lane of the –Trp–Leu–Ade–His plate was not reproducible, hence we do not consider it as a sign of true interaction. (b,c) The IHO1 bait weakly self-activated (first and last rows in b and c, respectively). Nevertheless, enhanced growth of experimental yeast strains in comparison to controls indicates IHO1-IHO1 interaction, and provided a reciprocal reconfirmation of REC114-IHO1, and HORMAD1-IHO1 interactions from a. (eg,j) Indicated proteins were detected by immunofluorescence in nuclear spreads of wild-type (WT) and Hormad1−/− spermatocytes at indicated stages. Scale bars, 10 μm. REC114 foci are present along chromosome axes in zygotene (e), but essentially absent in pachytene (f). (g,j) Frequent colocalization of REC114 and MEI4 foci was observed in wild-type (g) and Hormad1−/− (j) spermatocytes. The green channel is shifted three pixels to the right in the enlarged insets to allow better visualization of colocalization. (h,i,k,l) Quantification of colocalization between REC114 and MEI4 in wild-type preleptotene and leptotene (h,i), or Hormad1−/− (k,l) leptotene spermatocytes. Data points show fractions of REC114 foci that colocalize with MEI4 (h,k), and fractions of MEI4 foci that colocalize with REC114 (i,l). Medians are marked, and numbers of analyzed cells are indicated (n); results from two independent experiments were pooled; source data of graphs are shown in Supplementary Table 2. To control for random overlap between foci of examined proteins, colocalization frequencies were measured with (red, 90° turn) and without (blue, unturned) rotating the images of MEI4 and REC114 by 90° relative to each other. Significantly lower (Mann-Whitney test) colocalization frequencies were found after rotation in all cases.

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Stanzione, M., Baumann, M., Papanikos, F. et al. Meiotic DNA break formation requires the unsynapsed chromosome axis-binding protein IHO1 (CCDC36) in mice. Nat Cell Biol 18, 1208–1220 (2016). https://doi.org/10.1038/ncb3417

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