Genome-wide control of the distribution of meiotic recombination

Abstract

Meiotic recombination events are not randomly distributed in the genome but occur in specific regions called recombination hotspots. Hotspots are predicted to be preferred sites for the initiation of meiotic recombination and their positions and activities are regulated by yet-unknown controls. The activity of the Psmb9 hotspot on mouse Chromosome 17 (Chr 17) varies according to genetic background. It is active in strains carrying a recombinant Chr 17 where the proximal third is derived from Mus musculus molossinus. We have identified the genetic locus required for Psmb9 activity, named Dsbc1 for Double-strand break control 1, and mapped this locus within a 6.7-Mb region on Chr 17. Based on cytological analysis of meiotic DNA double-strand breaks (DSB) and crossovers (COs), we show that Dsbc1 influences DSB and CO, not only at Psmb9, but in several other regions of Chr 17. We further show that CO distribution is also influenced by Dsbc1 on Chrs 15 and 18. Finally, we provide direct molecular evidence for the regulation in trans mediated by Dsbc1, by showing that it controls the CO activity at the Hlx1 hotspot on Chr 1. We thus propose that Dsbc1 encodes for a trans-acting factor involved in the specification of initiation sites of meiotic recombination genome wide in mice.

Figure 1. CO and NCO Events at Psmb9 Are Activated by a Long Distance–Acting Element from the wm7 Haplotype

The origin of the Chr 17 fragments in the different recombined lines is represented: black, B10 (B10 or B6 for R115) (haplotype b); blue, A (haplotype a); and orange, SGR (haplotype wm7). The 95% confidence intervals for recombinant product frequencies are calculated as described in [38]. NCOs were detected either at the BsrFI site or the site “38,” depending on which is heterozygous in each hybrid. Values for B10xB10.A and B10xR209 are from [38].

Figure 2. MLH1 Foci at Psmb9 Correlate with CO Activity

(A–D) Immuno-FISH assay on adult pachytene spermatocyte spreads. SYCP3 (blue), MLH1 (red), Chr 17 probe (mix of BACs RP23-10B20 and RP24-67L15; blue), Psmb9 PCR fragments probe (green), and DAPI staining (white). (A) Whole nucleus. (B and C) Chr 17 from nuclei showing colocalization between MLH1 and Psmb9, with distances of 70 nm and 200 nm, respectively. (D) Chr 17 without MLH1/Psmb9 colocalization, with four Psmb9 foci corresponding to the four chromatids.

Figure 3. Different Distributions of MLH1 Foci on Chr 17 in B10xB10.A, RB2xB10.A, and SGRxSGR

(A) Pachytene spermatocyte labeled with SYCP3 (green), MLH1 (red), and Chr 17 probe (mix of BACs RP23-10B20 and RP24-67L15; white) and stained with DAPI (blue). The region of Chr 17 is enlarged in the inset. (B) Percentage of MLH1 foci per 5% SC length intervals on Chr 17 in B10xB10.A and RB2xB10.A hybrids (n = 245 and 320 foci, respectively). (C) Percentage of MLH1 foci per 5% SC length intervals on Chr 17 in B10xB10.A and SGRxSGR hybrids (n = 245 and 226 foci, respectively).

Figure 4. Different Distributions of γH2AX Foci on Chr 17 in B10xB10.A and RB2xB10.A

(A) Chr 17 of a pachytene spermatocyte labeled with SYCP3 (blue), γH2AX (red), Chr 17 probe (mix of BACs RP23-10B20, and RP24-67L15; green), and stained with DAPI (white). (B) Percentage of γH2AX foci per 5% SC intervals on Chr 17 in B10xB10.A and RB2xB10.A hybrids (n = 549 and 513 foci, respectively).

Figure 5. Correlation between Variations of γH2AX and MLH1 Distributions Due to the Dsbc1 Locus

The ratios between focus densities in RB2xB10.A and B10xB10.A hybrids per 5% Chr 17 SC length intervals (values of intervals 0%–25%, 25%–35%, and 35%–45% have been pooled) are represented for γH2AX (x-axis) and MLH1 (y-axis). The line resulting from the linear regression analysis between these ratios, and the coefficient of regression R ², are shown.

Figure 6. Distributions of MLH1 Foci on Different Chromosomes in B10xB10.A and RB2xB10.A Hybrids

(A) Chr 2 divided into 35 intervals of 2.85% SC length (n = 269 and 263, respectively), (B) Chr 15 divided into 20 intervals of 5% SC length (n = 325 and 336, respectively), and (C) Chr 18 divided into 20 intervals of 5% SC length (n = 231 and 265, respectively).

Figure 7. Dsbc1 Controls CO Activity at the Hlx1 Hotspot on Chr 1

COs (recombinant molecules B10-CAST) were detected at the Hlx1 hotspot by allele-specific PCR on pools containing the indicated number (n) of amplifiable molecules, extracted from testes of 18-d-old mice of various genotypes for Chr 17 (b/c, Chr 17 of B10 and CAST origins; b/wm7, Chr 17 of B10 and R209 origins; b/b, both Chr 17 of B10 origin). The results from a subset of pools are shown. No recombinant molecules among 24,458 amplifiable genomes was observed in mice with the b/b genotype on Chr 17.