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A quantitative assay for crossover and noncrossover molecular events at individual recombination hotspots in both male and female gametes.

ABSTRACT: Meiotic recombination is a fundamental process in all eukaryotes. Among organisms in which recombination initiates prior to synapsis, recombination preferentially occurs in short 1-to 2-kb regions, known as recombination hotspots. Among mammals, genotyping sperm DNA has provided a means of monitoring recombination events at specific hotspots in male meiosis. To complement these current techniques, we developed an assay for amplifying all copies of a hotspot from the DNA of male and female germ cells, cloning the products into Escherichia coli, and SNP genotyping the resulting colonies using fluorescence technology. This approach examines the molecular details of crossover and noncrossover events of individual meioses directly at active hotspots while retaining the simplicity of using pooled DNA. Using this technique, we analyzed recombination events at the Hlx1 hotspot located on mouse chromosome 1, finding that the results agree well with a prior genetic characterization of 3026 male and 3002 female meioses.

SUBMITTER: Ng SH

PROVIDER: S-EPMC2610674 | biostudies-other | 2008 Oct

REPOSITORIES: biostudies-other

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A quantitative assay for crossover and noncrossover molecular events at individual recombination hotspots in both male and female gametes.

Ng Siemon H SH Parvanov Emil E Petkov Petko M PM Paigen Kenneth K

Genomics 20080812 4

Meiotic recombination is a fundamental process in all eukaryotes. Among organisms in which recombination initiates prior to synapsis, recombination preferentially occurs in short 1-to 2-kb regions, known as recombination hotspots. Among mammals, genotyping sperm DNA has provided a means of monitoring recombination events at specific hotspots in male meiosis. To complement these current techniques, we developed an assay for amplifying all copies of a hotspot from the DNA of male and female germ c ...[more]

PMID: 18656531

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Project description:In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we suggest that it may play the same role in meiosis.

Project description:Background:The frequency of recombination events varies across the genome and between individuals, which may be related to some genomic features. The objective of this study was to assess the frequency of recombination events and to identify QTL (quantitative trait loci) for recombination rate in two purebred layer chicken lines. Methods:A total of 1200 white-egg layers (WL) were genotyped with 580?K SNPs and 5108 brown-egg layers (BL) were genotyped with 42?K SNPs (single nucleotide polymorphisms). Recombination events were identified within half-sib families and both the number of recombination events and the recombination rate was calculated within each 0.5?Mb window of the genome. The 10% of windows with the highest recombination rate on each chromosome were considered to be recombination hotspots. A BayesB model was used separately for each line to identify genomic regions associated with the genome-wide number of recombination event per meiosis. Regions that explained more than 0.8% of genetic variance of recombination rate were considered to harbor QTL. Results:Heritability of recombination rate was estimated at 0.17 in WL and 0.16 in BL. On average, 11.3 and 23.2 recombination events were detected per individual across the genome in 1301 and 9292 meioses in the WL and BL, respectively. The estimated recombination rates differed significantly between the lines, which could be due to differences in inbreeding levels, and haplotype structures. Dams had about 5% to 20% higher recombination rates per meiosis than sires in both lines. Recombination rate per 0.5?Mb window had a strong negative correlation with chromosome size and a strong positive correlation with GC content and with CpG island density across the genome in both lines. Different QTL for recombination rate were identified in the two lines. There were 190 and 199 non-overlapping recombination hotspots detected in WL and BL respectively, 28 of which were common to both lines. Conclusions:Differences in the recombination rates, hotspot locations, and QTL regions associated with genome-wide recombination were observed between lines, indicating the breed-specific feature of detected recombination events and the control of recombination events is a complex polygenic trait.

Dataset Information

A quantitative assay for crossover and noncrossover molecular events at individual recombination hotspots in both male and female gametes.

Publications

A quantitative assay for crossover and noncrossover molecular events at individual recombination hotspots in both male and female gametes.

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