Homologous pairing preceding SPO11-mediated double-strand breaks in mice

Abstract

How homologous chromosomes (homologs) find their partner, pair, and recombine during meiosis constitutes the central phenomenon in eukaryotic genetics. It is widely believed that, in most organisms, SPO11-mediated DNA double-strand breaks (DSBs) introduced during prophase I precede and are required for efficient homolog pairing. We now show that, in the mouse, a significant level of homolog pairing precedes programmed DNA cleavage. Strikingly, this early chromosome pairing still requires SPO11 but is not dependent on its ability to make DSBs or homologous recombination proteins. Intriguingly, SUN1, a protein required for telomere attachment to the nuclear envelope and for post-DSB synapsis, is also required for early pre-DSB homolog pairing. Furthermore, pre-DSB pairing at telomeres persists upon entry into prophase I and is most likely important for initiation of synapsis. Our findings suggest that the DSB-triggered homology search may mainly serve to proofread and stabilize the pre-DSB pairing of homologous chromosomes.

Figure 1. Overview of morphological classification of cell types and criteria to define pairing

(A), Schematic illustration of meiosis I in male mice. Type B spermatogonia divide into preleptotene spermatocytes, which carry out one round of DNA replication before entering prophase I. At the onset of prophase I, SPO11 introduces the DSBs that trigger homolog recombination, synapsis and ultimately, the formation of crossover DNA products (chiasmata) that ensures the accurate reductional segregation of chromosomes in the first meiotic division. We used incorporation of the thymidine analog EdU to mark preleptotene spermatocytes. (B), Schematic illustration of the experimental set-up, mouse spermatogenesis and the criteria used for cell classification. Immunofluorescence staining of spermatogonia, preleptotene, leptotene and zygotene spermatocytes is shown. Nuclei are stained with a 488-azide (EdU), synaptonemal complex protein SYCP3, a germ cell specific marker (DAZL) and DAPI. (C), Illustrative image of a preleptotene spermatocyte analyzed by combined Immunofluorescence-Fluorescent in situ hybridization (IF-FISH) showing a Chr3-INT probe, 488-azide (EdU), SYCP3 and DAPI stained nucleus in a structurally preserved preleptotene (PreLep) spermatocyte. (D), The frequency of non-homologous pairing between mouse chr3 and chr7 determined by co-FISH is 8% (n = 50). PreLep (top) and pachytene (bottom) spermatocytes are shown. Chr3-INT probes (arrows), Chr7-INT probes (arrowheads). Scale bar: 10 μm. See also Figure S1.

Figure 2. A significant level of homolog pairing is detected in preleptotene spermatocytes prior to programmed DSBs

(A), Assessment of pairing during early spermatogenesis, using chr1, chr3 and chr7 interstitial probes in either structurally preserved nuclei (SPN) from prepuberal (8–12 dpp) mice or frozen tissue sections of 21 dpp mice. Statistical significance of the difference between samples was assessed by Fisher’s exact test. The p-values for difference in pairing between preleptotene spermatocytes and spermatogonia are given for each probe (^† P < 0.0001, *** P < 0.001, ** P < 0.01, n = 321 to 609). The error bar is an estimation of the standard deviation (SD), based on the counting error (square root of n). (B), IF-FISH on frozen testis tissue sections of 21 dpp EdU-injected mice: Frozen sections were stained with 488-azide (EdU), DAZL (marker for germ cells), and hybridized with a Chr3-INT probe. Shown is a representative image taken with 40X magnification. Images of sections taken with 100X magnification (see inserts) were used for the analysis. Preleptotene (PreLep), and zygotene (Zyg)/pachytene (Pach) spermatocytes. Scale bar, 10 μm. (C), Four cell populations isolated by FACS (see Figures S2B and S2C) enriched in either spermatogonia (spermatogonia, SPGN + seminal germ cells, SGC) or early, mid and late preleptotene spermatocytes, were analyzed by Chr1-INT FISH. Statistical significance of the difference between samples was assessed by Fisher’s exact test and the p-values indicated. The error bar is an estimation of the standard deviation (SD), based on the counting error. For each data point, 234 to 450 total number of cells were analyzed. See also Figure S2.

Figure 3. A DSB-independent activity of SPO11 and the telomere tethering protein SUN1 are both required for preleptotene pairing but the meiotic homologous recombination machinery is dispensable

(A, B), Preleptotene pairing is disrupted in Spo11^−/− and Sun1^−/− but unaffected in Spo11^FF/FF or Hop2^−/− mice, as determined with a Chr1-INT probe (A) and a Chr3-INT probe (B). *** P < 0.001 (n = 282 to 551, where n = total number of cells analyzed), applies to the difference in pairing between wild-type or Spo11^FF/FF and Spo11^−/− or Sun1^−/− mice. Statistical significance of difference between samples was assessed by Fisher’s Exact Test. The error bar is an estimation of the standard deviation (SD), based on the counting error. (C), Spo11^FF/FF spermatocytes are defective in DSB formation, meiotic recombination and synapsis. IF analysis of surface-spread preparations shows that leptotene spermatocytes of both Spo11^FF/FF and Spo11^−/− are devoid of γH2AX staining (a marker for DSBs) and RAD51 foci (a marker for homologous recombination intermediates). Zygotene-like spermatocytes only show minimal co-localization of SYCP3 and SYCP1 staining (indicative of defective synapsis). Scale bar, 10 μm. (D), Spo11^FF/FF mice, like other mutants arrested in prophase I, express primarily the SPO11-β polypeptide. Total testis extracts from wild-type, Spo11^−/−, Dmc1^−/−, Hop2^−/−, and Spo11^FF/FF mice (5 mg of total protein) were precipitated/blotted with anti-SPO11 antibody. (E), The mutated Spo11::YY137,138FF allele is transcribed. In order to verify expression of the mutated allele, total Spo11 transcripts were quantified by reverse transcription-qPCR (RT-qPCR) of total RNA from adult mice testes, (see Extended Experimental Procedures). As previously reported, Spo11 heterozygous knockout (+/−) testes carry half the amount of transcripts when compared to WT mice (because only one allele is being transcribed) (Bellani et al., 2010). Given that homozygous Spo11^FF/FF mice are arrested in prophase, their total Spo11 transcript levels are very low (because the majority of Spo11 transcripts correspond to Spo11-α, which is expressed after mid prophase) (Bellani et al., 2010). Nevertheless, heterozygous knockin mice (+/FF) showed comparable levels of total transcripts to WT mice, indicating that both the WT and mutant (FF) alleles are being transcribed. Error bars represent the SD. (F), Homozygous Spo11^FF/FF mice synthesize primarily Spo11-β transcripts. Total testis RNA from Spo11^FF/FF mice, and from several mutants arrested in either prophase I (Hop2^−/−, Dmc1^−/−) or metaphase I (Mlh1^−/−) were analyzed by RT-qPCR, using Taqman assays targeting isoform-specific exon junctions in order to quantify Spo11-α and Spo11-β transcripts Bars represent the ratios of Spo11-α or -β transcripts in homozygous mutants relative to a wild-type littermate. Thus, Spo11^FF/FF mice synthesize primarily Spo11-β transcripts at levels comparable to those of other prophase I arrested mutant mice. In contrast a mutant mouse arrested in metaphase I express both isoforms. Error bars represent the SD. See also Figure S3.

Figure 4. SUN1 is required for telomere attachment to the nuclear envelope in preleptotene spermatocytes

(A), Telomere re-localization to the nuclear envelope in late preleptotene spermatocytes requires SUN1: IF with antibodies against telomere repeat binding factor 2 (TRF2), and axial/lateral element protein SYCP3, counter stained with DAPI and 488-azide (EdU, for cell classification) in WT and Sun1^−/− mice at indicated stages, showing that telomeres (TRF2) relocalize to the nuclear periphery in late preleptotene spermatocytes of WT mice but not in Sun1^−/− mice. (B), Control showing that TRF2 binding to the telomeres is not impaired in Sun1^−/− mice. (C), IF with CREST antiserum labeling centromeres and antibodies against the axial/lateral element protein SYCP3 and germ cell marker DAZL in Sun1^−/− mice at indicated stages, showing the distribution of CREST foci localization in both the nuclear periphery and nuclear lumen at all stages in Sun1^−/− mice. (D), In contrast to (C) the distribution of CREST foci is restricted to nuclear peripheral localization in preleptotene in WT mice. (E), WT zygotene spermatocyte with the centromeres clustered into a bouquet. 2.4% of 1008 total WT leptotene/zygotene prophase spermatocytes displayed a bouquet configuration, consistent with previous reports (see (Liebe et al., 2006) and references therein). In contrast, bouquets are not observed either in Sun1^−/− mice (C) or in preleptotene of WT mice (D) of over 500 nuclei analyzed at each stage per mice. Scale bar, 10 μm. See also Figure S4.

Figure 5. Pre-DSB pairing at telomeres persists upon entry into prophase I and is most likely important for initiation of synapsis at chromosomal ends

(A), Chr1 ideogram showing the loci position of the interstitial (Chr1-INT), subtelomeric (Chr1-TEL) and subcentromeric (Chr1-CEN) probes (see Extended Experimental Procedure). (B), Sample IF-FISH images showing the terminal position of the Chr1-TEL loci in a pachytene surface spread preparation (left panel), and Chr1-TEL foci in preleptotene (top two cells, EdU+, green) and zygotene/pachytene (bottom cell, EdU−) cells in a SPN preparation (right panel). Scale bar, 10 μm. (C), Terminal pairing achieved in preleptotene spermatocytes is preserved upon entry into leptotene, whereas interstitial pairing is lost. Terminal versus interstitial homolog pairing was assessed on either structurally preserved nuclei (SPN) or frozen tissues sections (FS) of wild type mice, probed with interstitial (Chr1-INT), subtelomeric (Chr1-TEL) or subcentromeric (Chr1-CEN) probes. Notice that the preleptotene homologous telomeric pairing was 35–50% (n = 457 to 495; Table S1A), depending on the probe used, and is significantly higher than the experimentally determined average non homologous telomeric pairing between chr1 and chr3 of 14% in SPN, (n = 77, P < 0.0001) (see Extended Experimental Procedures). Note that the experimentally determined average non homologous telomeric pairing between chr1 and chr3 in frozen sections was 12% (Figure S5). The error bar is an estimation of the standard deviation (SD), based on the counting error. (D), Maintaining terminal pairing upon entry into leptotene requires DSB formation and meiotic recombination. Frozen tissue sections of wild type (WT), Dmc1^−/−, Mnd1^−/−, Spo11^−/−, Mei1^−/− and Spo11^FF/FF mice were probed with Chr1-TEL probe. Notice that telomeric pairing is also significantly disrupted during preleptotene in Spo11^−/− but not in DSB or HR impaired mutants (Table S1B). HR^−/−, homologous recombination impaired; DSB^−/−, DSB impaired. The error bar is an estimation of the standard deviation (SD), based on the counting error. (E), Synapsis initiates from the terminal ends of chromosomes in mice. Sample images of chromosome spreads from WT spermatocytes stained with antibodies against the central element protein SYCP1, the axial/lateral element protein SYCP3 and the telomeric protein TRF1 or centromere marker, CREST are shown, indicating that in early zygotene spermatocytes TRF1/CREST signals lie adjacent or associate with (but not necessarily co-localize with) the ends of most short SYCP1-stretches. Arrows: terminal synapsis, arrowheads: interstitial synapsis. Scale bar, 10 μm. (F), Quantification of (E), the proportion of synapsed homologs per nuclei in early zygotene (10–30% synapsis) and in mid-zygotene (40–60% synapsis) spermatocytes, as assessed by SYCP1/SYCP3 co-localization, either adjacent to (terminal synapsis) or apart from (interstitial synapsis) TRF1/CREST foci. For each classification, about 100 nuclei were analyzed. Note that the early to mid-zygotene (10–60% synapsis) classification is a pool of the above two classifications. To determine the resolution of this analysis, we first estimated the threshold length of SYCP1 short stretches from 126 measurements to be 2.8 μm (median). Similarly, we determined the mean overall length of all chromosomes to be 13.4 μm and computed the resolution or estimated fraction of chromosome carrying SYCP1 short stretches to be about 20% (2.8 μm/13.4 μm). The error bar is an estimation of the standard deviation (SD), based on the counting error. See also Figure S5.

Figure 6. Preleptotene DSB-independent pairing in mice

Our data argues for a progressive increase in telomeric homolog pairing which serves to promote interstitial pairing at multiple loci along the whole chromosome, prior to DSB formation occurring at the onset of meiotic prophase I (leptotene). Preleptotene pairing is DSB-independent but requires the topoisomerase II-like protein SPO11, and the protein anchoring telomeres to the nuclear envelope (NE), SUN1. We propose that the tethering of telomeres to the nuclear envelope in late preleptotene (Chehrehasa et al., 2009; Scherthan et al., 1996) (Figures 4 and S4), facilitates the initiation of homolog pairing at subtelomeric regions by simplifying the search for the cognate partner. Upon entry into prophase, DSB-independent pairing at interstitial (non-telomeric) sites is lost, presumably to allow for the removal of unwanted associations and entanglements. However, telomeric pairing is maintained at least at one end, as long as the homologous recombination (HR) machinery is functional. While interstitial interactions are lost, we cannot rule out the possibility that the homologs stay in close proximity. Also, this reversibility in interstitial pairing may permit or promote strand invasion mediated by the HR machinery. Thus the HR may only serve to proofread the initial pairing established prior to DSB formation and as a checkpoint to ensure that ectopic associations are disrupted. Subsequently, “validated” interactions would be stabilized via the polymerization of the synaptonemal complex (synapsis). Furthermore, DSB repair and synapsis, initiating at the preserved preleptotene homologously paired telomeric sites, extends into the chromosome to restore pairing at interstitial sites, ultimately leading to a progressive synapsis (almost zipper-like) of homologs later in prophase I.