Trans-regulation of mouse meiotic recombination hotspots by Rcr1

Abstract

Meiotic recombination is required for the orderly segregation of chromosomes during meiosis and for providing genetic diversity among offspring. Among mammals, as well as yeast and higher plants, recombination preferentially occurs at highly delimited chromosomal sites 1-2 kb long known as hotspots. Although considerable progress has been made in understanding the roles various proteins play in carrying out the molecular events of the recombination process, relatively little is understood about the factors controlling the location and relative activity of mammalian recombination hotspots. To search for trans-acting factors controlling the positioning of recombination events, we compared the locations of crossovers arising in an 8-Mb segment of a 100-Mb region of mouse Chromosome 1 (Chr 1) when the longer region was heterozygous C57BL/6J (B6) x CAST/EiJ (CAST) and the remainder of the genome was either similarly heterozygous or entirely homozygous B6. The lack of CAST alleles in the remainder of the genome resulted in profound changes in hotspot activity in both females and males. Recombination activity was lost at several hotspots; new, previously undetected hotspots appeared; and still other hotspots remained unaffected, indicating the presence of distant trans-acting gene(s) whose CAST allele(s) activate or suppress the activity of specific hotspots. Testing the activity of three activated hotspots in sperm samples from individual male progeny of two genetic crosses, we identified a single trans-acting regulator of hotspot activity, designated Rcr1, that is located in a 5.30-Mb interval (11.74-17.04 Mb) on Chr 17. Using an Escherichia coli cloning assay to characterize the molecular products of recombination at two of these hotspots, we found that Rcr1 controls the appearance of both crossover and noncrossover gene conversion events, indicating that it likely controls the sites of the double-strand DNA breaks that initiate the recombination process.

Figure 1. Crosses Used for Recombination Map on Chr1

Crosses of B6xCAST (interstrain) and B6xB6.CAST-1T (congenic): B6 sequences are black; CAST sequences are red. The region within which recombination was measured is boxed; the crossovers are detected in the final progeny.

Figure 2. Recombination Map of the Chr 1 Region between 183.5 and 191.5 Mb

(A) Comparison between the female maps of B6xB6.CAST-1T (blue line) and B6xCAST (red line). (B) Comparison between the male maps of the two crosses using the same colors. The maps of B6xCAST are taken from previous experiments [10]. Recombination rates are expressed in centimorgans per megabase for the intervals between adjacent markers. Hotspots included in Table 1 are shown with their names and positions indicated by arrows. Red arrows mark hotspots where recombination was present only in the B6xCAST cross; blue arrows mark regions where recombination was present only in the B6xB6.CAST-1T cross. The numbers show actual centimorgans per megabase values for hotspots exceeding the limits of the figure. The insets expand the map in the 50-kb region between 189.750 and 189.800 Mb; the red arrow marks the Esrrg-1 hotspot that is active only in the B6xCAST cross.

Figure 3. Crosses for Mapping of Trans-Acting Genes

(B6.CAST-1TxCAST)xB6 (A) and B6xCAST (B) crosses used for mapping trans-acting genes. B6 sequences are in black, and CAST in red. The region where the recombination is studied is boxed. Segregating loci are located in the white regions. Note that any recombination within the congenic region between the B6.CAST-1T and CAST chromosomes does not alter the allelic composition of this region.

Figure 4. Allele-Specific PCR for Hotspot Hlx1

Gel electrophoresis of F2 samples from the interstrain cross after two rounds of allele-specific PCR. M is a 100-bp ladder; 1–20 are F2 samples; the last three lanes contain negative and positive controls as described.

Figure 5. QTL Analysis of Phenotyping and Genotyping Data

(A) A total of 211 samples from the first mapping and (B) 98 samples from the second mapping cross were used in the analysis. The LOD scores are the result of 10,000 permutations per analysis. (C) Further mapping on Chr 17 of recombinants in the interval 5–25 Mb, including eight recombinants from the congenic backcross and two from the interstrain cross. The borders of the critical region within which Rcr1 must lie are marked with dashed vertical lines.