CXXC1 is not essential for normal DNA double-strand break formation and meiotic recombination in mouse
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ABSTRACT:
PROVIDER: PRJNA478212 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR7442351_1.fastq.gz | Fastqsanger.gz | |||
| SRR7442351_2.fastq.gz | Fastqsanger.gz | |||
| SRR7442352_1.fastq.gz | Fastqsanger.gz | |||
| SRR7442352_2.fastq.gz | Fastqsanger.gz | |||
| SRR7442353_1.fastq.gz | Fastqsanger.gz |
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Project description:In most mammals, including mice and humans, meiotic recombination is determined by the meiosis specific histone methytransferase PRDM9, which binds to specific DNA sequences and trimethylates histone 3 at lysine-4 and lysine-36 at the adjacent nucleosomes. These actions ensure successful DNA double strand break formation and repair that occur on the proteinaceous structure forming the chromosome axis. The process of hotspot association with the axis after their activation by PRDM9 is poorly understood. Previously, we and others have identified CXXC1, an ortholog of S. cerevisiae Spp1 in mammals, as a PRDM9 interactor. In yeast, Spp1 is a histone methyl reader that links H3K4me3 sites with the recombination machinery, promoting DSB formation. Here, we investigated whether CXXC1 has a similar function in mouse meiosis. We created two Cxxc1 conditional knockout mouse to deplete CXXC1 generally in germ cells, and before the onset of meiosis. Surprisingly, male knockout mice were fertile, and the loss of CXXC1 in spermatocytes had no effect on hotspot trimethylation, double-strand break formation or repair. Our results demonstrate that CXXC1 is not an essential link between PRDM9-activated recombination hotspot sites and DSB machinery and that the hotspot recognition pathway in mouse is independent of CXXC1.
2018-08-29 | GSE116336 | GEO
Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are non-randomly distributed across the genome. Here, we use S1Seq mapping to map the distribution of meiotic DSBs in spo11 mutant strains of Saccharomyces cerevisiae.
2020-05-12 | GSE150313 | GEO
Project description:The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5ʹâ??3â?² exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, namely S1Seq, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape. 36 samples total: two biological replicate S1Seq maps of S.cerevisiae for each genetic background and time point in meiosis
2016-08-06 | E-GEOD-85253 | biostudies-arrayexpress
Project description:The DNA double-strand breaks that initiate homologous recombination during meiosis are subject to extensive 5ʹ→3′ exonucleolytic processing. This resection is a central and conserved feature of recombination, yet its mechanism is poorly understood. Using a purpose-made deep-sequencing method, namely S1Seq, we mapped meiotic resection endpoints genome-wide at high spatial resolution in Saccharomyces cerevisiae. Generating full-length resection tracts requires Exo1 exonuclease activity and the DNA-damage responsive kinase Tel1, but not the helicase Sgs1. Tel1 is also required for efficient and timely initiation of resection. We find that distributions of resection endpoints at individual genomic loci display pronounced heterogeneity that reflects a tendency for nucleosomes to block Exo1 in vivo, yet modeling experiments indicate that Exo1 digests chromatin with high apparent processivity and at rates approaching those for naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a determining feature of the meiotic resection landscape.
2016-08-06 | GSE85253 | GEO
Project description:Meiotic double-strand break mapping in spo11 mutants by S1Seq
| PRJNA631754 | ENA
Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates
2007-12-30 | E-GEOD-8981 | biostudies-arrayexpress
Project description:In most organisms, meiotic recombination begins with programmed DNA double strand break (DSB) formation by Spo11. Here, we present evidence that Tel1/Mec1, the budding yeast ATM/ATR, regulate DSB formation by phosphorylating Rec114, an essential Spo11-accessory protein. Analyses of a non-phosphorylatable- or phosphomimetic- alleles of rec114 revealed that DSB-dependent phosphorylation of Rec114 limited its association with DSB-hotspots resulting in reduction in DSB formation. Also observed were the impact of Rec114 phosphorylation on its homolog synapsis-associated removal from chromosomes and NDT80-dependent turnover. Specifically, we found that the synapsis- and NDT80-dependent Rec114 downregulation occurred later in the rec114 mutant with a reduced Spo11-catalysis, but earlier in the other with an enhanced catalysis, strongly implicating the existence of a feedback mechanism coupling the extent of Spo11-catalysis to Rec114 activity. Taken together, these observations suggest that three different mechanisms of down regulating Rec114 contribute to meiotic DSB homeostasis, a feedback mechanism to maintain the number of meiotic DSBs at the developmentally programmed level. 6 genome wide ChIPchip sets: 3 for meiotic DSB formation (Spo11-ChIP) and 3 for protein-DNA association (Rec114-ChIP), each for wild type and two mutants during meiosis (corresponding to the main Figure 3, as well as to Figures S3, S4, S5).
2013-06-01 | E-GEOD-43384 | biostudies-arrayexpress
Project description:DNA double-strand breaks (DSBs) made by SPO11 protein initiate homologous recombination during meiosis. Subsequent to DNA strand breakage, endo- and exo-nucleases process the DNA ends to resect the strands whose 5' termini are at the DSB, generating long 3'-terminal single-stranded tails that serve as substrates for strand exchange proteins. DSB resection is essential for meiotic recombination, but a detailed understanding of its molecular mechanism is currently lacking. Genomic approaches to mapping DSBs and resection endpoints, e.g., S1-sequencing (S1-seq) and similar methods, play a critical role in studies of meiotic DSB processing. In these methods, nuclease S1 or other enzymes that specifically degrade ssDNA are used to trim resected DSB ends, allowing capture and sequencing of the ends of resection tracts. Here, we present optimization of S1-seq that improves its signal:noise ratio and allows its application to analysis of spermatocyte meiosis in adult mice. Furthermore, quantitative features of meiotic resection are evaluated for reproducibility, and we suggest best practices for analysis and interpretation of S1-seq data. We also compare S1-seq to variants that use exonuclease T and or exonuclease VII from Escherichia coli instead of nuclease S1. Detailed step-by-step protocols and suggestions for troubleshooting are provided.
2024-04-30 | GSE265863 | GEO
Project description:SPO11 generates hundreds of DNA double-strand breaks (DSBs) to initiate meiotic recombination. Heritability and genome stability are shaped by the nonrandom distribution of DSBs, but mechanisms molding this landscape remain poorly understood. Here we exploit genome-wide maps of mouse DSBs at unprecedented nucleotide resolution to uncover previously invisible spatial features of recombination. At fine scale, we reveal a stereotyped hotspot structure––DSBs occur within narrow zones between methylated nucleosomes––and identify relationships between SPO11, chromatin, and the histone methyltransferase PRDM9. At large scale, DSB formation is suppressed on non-homologous portions of the sex chromosomes via the DSB-responsive kinase ATM, which also shapes the autosomal DSB landscape at multiple size scales. We also provide the first genome-wide analysis of exonucleolytic DSB resection lengths and elucidate spatial relationships between DSBs and recombination products. Our results paint a comprehensive picture of features that govern successive steps in mammalian meiotic recombination.
2016-08-13 | GSE84689 | GEO
Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose
2007-12-31 | GSE8981 | GEO