ATM and PRDM9 Regulate SPO11-bound Recombination Intermediates During Meiosis
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ABSTRACT:
PROVIDER: PRJNA577718 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR10286900.fastq.gz | Fastqsanger.gz | |||
| SRR10286901.fastq.gz | Fastqsanger.gz | |||
| SRR10286902.fastq.gz | Fastqsanger.gz | |||
| SRR10286903.fastq.gz | Fastqsanger.gz | |||
| SRR10286904.fastq.gz | Fastqsanger.gz |
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Project description:Meiotic recombination is initiated by genome-wide SPO11-induced double-strand breaks (DSBs) that are processed by MRE11-mediated release of SPO11-bound oligonucleotides (SPO11-oligos). The DSB is then resected and loaded with DMC1/RAD51 filaments that invade homologous chromosome templates. In most mammals, DSB locations (“hotspots”) are determined by the DNA sequence specificity of the PRDM9 DNA binding zinc finger array. Here, we demonstrate the first direct detection of meiotic DSBs in vertebrates by performing END-seq on mouse spermatocytes using low sample input. We find that DMC1 limits both the minimum and maximum length of ssDNA produced at all hotspots, whereas 53BP1, BRCA1 and EXO1 play a surprisingly minimal role in meiotic resection. Through enzymatic modifications to the END-seq protocol that mimic the in vivo processing of SPO11, we identify a novel meiotic recombination intermediate (RI) with SPO11 still bound to the 3’ end via a small stretch of dsDNA (“SPO11-RI”) that is dependent on the presence of PRDM9. We propose that SPO11-RI is generated because chromatin-bound PRDM9 blocks MRE11 from releasing SPO11 via 3’-5’ resection. SPO11-RI is present at all hotspots and correlates with the localization and frequency of crossovers and noncrossovers. In Atm–/– spermatocytes, multiple DSBs at the same hotspot reduces SPO11-RI formation, while unresected DNA-bound SPO11 accumulates because of defective MRE11 initiation. Thus in addition to their global roles in governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.
2020-02-20 | GSE138915 | GEO
Project description:Meiotic recombination is initiated by SPO11-induced double-strand breaks (DSBs). In most mammals, the methyltransferase PRDM9 guides SPO11 targeting, and the ATM kinase controls meiotic DSB numbers. Following MRE11 nuclease removal of SPO11, the DSB is resected and loaded with DMC1 filaments for homolog invasion. Here, we demonstrate the direct detection of meiotic DSBs and resection using END-seq on mouse spermatocytes with low sample input. We find that DMC1 limits both minimum and maximum resection lengths, whereas 53BP1, BRCA1 and EXO1 play surprisingly minimal roles. Through enzymatic modifications to END-seq, we identify a SPO11-bound meiotic recombination intermediate (SPO11-RI) present at all hotspots. We propose that SPO11-RI forms because chromatin-bound PRDM9 asymmetrically blocks MRE11 from releasing SPO11. In Atm-/- spermatocytes, trapped SPO11 cleavage complexes accumulate due to defective MRE11 initiation of resection. Thus, in addition to governing SPO11 breakage, ATM and PRDM9 are critical local regulators of mammalian SPO11 processing.
| S-EPMC7016097 | biostudies-literature
Project description:Homologous recombination is required for proper segregation of homologous chromosomes during meiosis. It predominantly occurs at recombination hotspots that are defined by the DNA binding specificity of the PRDM9 protein. PRDM9 contains three domains which are typically different families of proteins involved in regulation of transcription in other proteins, yet, the role of PRDM9 in gene expression control has not been evaluated. Here we analyze the germline transcriptome of the Prdm9-/- male mice in comparison to Prdm9+/+ males and find no apparent differences in the mRNA and miRNA profiles. We further explore the role of PRDM9 in meiosis by analyzing the activity of PRDM9 lacking effect of the KRAB, SSXRD and post-SET zinc finger domains deletion mutants in a cell expression system and the KRAB domain deletion in mice. We found that although the post-SET zinc finger and the KRAB domains are not essential for the methyltransferase activity of PRDM9 in a cell culture expression systemin vitro, mice lacking the KRAB domain mutant mice domain show only residual PRDM9 methyltransferase activity and undergo meiotic arrest. In aggregate, our data indicate that these domains that are classically involved in gene regulation do not serve that role in PRDM9, but instead are involved in PRDM9 function has likely diverged from the typical function of a transcription factor and adapted the new role in setting the proper chromatin environment for initiation and completion of homologous recombination.
2018-04-27 | GSE104850 | GEO
Project description:Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes – normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions. Detection of meiotic double strand breaks in mice with a hypomorphic Spo11 allele.
2013-09-11 | E-GEOD-48493 | biostudies-arrayexpress
Project description:Meiotic recombination starts with the formation of DNA double-strand breaks (DSBs) at specific genomic locations that correspond to PRDM9-binding sites. The molecular steps occurring from PRDM9 binding to DSB formation are unknown. Using proteomic approaches to find PRDM9 partners, we identified HELLS, a member of the SNF2-like family of chromatin remodelers. Upon functional analyses during mouse male meiosis, we demonstrated that HELLS is required for PRDM9 binding and DSB activity at PRDM9 sites. However, HELLS is not required for DSB activity at PRDM9-independent sites. HELLS is also essential for 5-hydroxymethylcytosine (5hmC) enrichment at PRDM9 sites. Analyses of 5hmC in mice deficient for SPO11, which catalyzes DSB formation, and in PRDM9 methyltransferase deficient mice reveal that 5hmC is triggered at DSB-prone sites upon PRDM9 binding and histone modification, but independent of DSB activity. These findings highlight the complex regulation of the chromatin and epigenetic environments at PRDM9-specified hotspots.
2020-11-19 | GSE145768 | GEO
Project description:A hallmark of meiosis is the rearrangement of parental alleles to assure genetic diversity in gametes. These chromosome rearrangements are mediated by the repair of programmed DNA double-strand-breaks (DSBs) as genetic crossovers between parental homologs. In mice, humans, and many other mammals, meiotic DSB occur primarily at hotspots, determined by sequence-specific binding of the PRDM9 protein. Without PRDM9, meiotic DSBs occur near gene promoters and other functional sites. Studies in a limited number of mouse strains showed that functional PRDM9 is required to complete meiosis, but despite its apparent importance, Prdm9 has been repeatedly lost across many animal lineages. Both the reason for mouse sterility in the absence of PRDM9 and the mechanism by which Prdm9 can be lost remain unclear. Here, we explore if mice can tolerate the loss of Prdm9. By generating Prdm9 functional knockouts in an array of genetic backgrounds, we observe a wide range of fertility phenotypes and ultimately demonstrate that PRDM9 is not required for completion of meiosis. Although DSBs still form at a common subset of functional sites in all mice lacking PRDM9, meiotic outcomes differ substantially. We speculate that DSBs at functional sites are difficult to repair as a crossover and that by increasing the efficiency of crossover formation at these sites, genetic modifiers of recombination rates can allow for meiotic progression. This model implies that species with a sufficiently high recombination rate may lose Prdm9 yet remain fertile.
2019-06-07 | GSE109874 | GEO
Project description:Vertebrate recombination concentrates in meiotic chromatin regions (hotspots) that are opened in some species by the DNA-sequence-specific-binding histone H3 trimethyltransferase PRDM9, while other species recombine in regions with already opened chromatin and other function. Inactivation of the mouse Prdm9 gene induces the shift of hotspots to functional regions, gross fertility reduction in males, and sterility in females. In contrast, the other vertebrate species lacking PRDM9 remain fertile. To resolve this discrepancy, we generated Prdm9 deletions in the Rattus norvegicus genome and generated the first rat genome-wide maps of recombination-initiating double-strand break hotspots. Rat strains carrying the same wild-type Prdm9 allele shared 88% hotspots but strains with different Prdm9 alleles only 3%. After Prdm9 deletion, rat hotspots relocated to functional regions, 40% to positions corresponding to Prdm9-independent mouse hotspots. Despite of hotspot relocation and of decreased fertility, Prdm9-deficient rats of the SHR/OlaIpcv strain produced apparently normal offspring. Rat PRDM9 thus makes recombination landscape unique, but it is unnecessary for recombination. This peculiarity is likely similar for human PRDM9 and may resolve the paradox between the apparently species-specific functions. PRDM9 is known to play a role in speciation, as it causes mouse hybrid sterility via meiotic asynapsis. Besides the expected mild meiotic arrest, we also detected apoptosis of postmeiotic spermatids, suggesting that PRDM9 has an additional role during spermatogenesis and perhaps also in speciation.
2021-03-25 | GSE163474 | GEO
Project description:Homologous recombination is the key process that generates genetic diversity and drives evolution. SPO11 protein triggers recombination by introducing DNA double stranded breaks at discreet areas of the genome called recombination hotspots. The hotspot locations are largely determined by the DNA binding specificity of the PRDM9 protein in human, mice and most other mammals. In budding yeast Saccharomyces cerevisae, which lacks a Prdm9 gene, meiotic breaks are formed opportunistically in the regions of accessible chromatin, primarily at gene promoters. The genome-wide distribution of hotspots in this organism can be altered by tethering Spo11 protein to Gal4 recognition sequences in the strain expressing Spo11 attached to the DNA binding domain of the Gal4 transcription factor. To establish whether similar re-targeting of meiotic breaks can be achieved in PRDM9-containing organisms we have generated a Gal4BD-Spo11 mouse that expresses SPO11 protein joined to the DNA binding domain of yeast Gal4. We have mapped the genome-wide distribution of the recombination initiation sites in the Gal4BD-Spo11 mice. More than two hundred of the hotspots in these mice were novel and were likely defined by Gal4BD, as the Gal4 consensus motif was clustered around the centers in these hotspots. Surprisingly, meiotic DNA breaks in the Gal4BD-Spo11 mice were significantly depleted near the ends of chromosomes. The effect is particularly striking at the pseudoautosomal region of the X and Y chromosomes – normally the hottest region in the genome. Our data suggest that specific, yet-unidentified factors influence the initiation of meiotic recombination at subtelomeric chromosomal regions.
2013-09-11 | GSE48493 | GEO
Project description:PRDM9 specifies the sites of meiotic DNA double strand break that initiate meiotic recombination in mice and humans. PRDM9 is known to bind to specific DNA sequences with its DNA binding domain, to induce H3K4me3 and H3K36me3 to adjacent nucleosomes through its methyltransferase activity, and to recruit or activate the meiotic DSB machinery. To understand how PRDM9 executes these various steps, we set up to identify its partners. This was performed by a proteomic approach where protein extracts from mouse testis were immunoprecipitated with anti-PRDM9 antibody for mass spectrometry analysis.
2020-12-03 | PXD017337 | Pride
Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use Spo11-oligonucleotide complexes to map the distribution of meiotic DSBs in a spo11 mutant strain of Saccharomyces cerevisiae.
2020-05-12 | GSE150314 | GEO