Meiotic recombination initiation hotspots in Arabidopsis genes and transposons (SPO11-1-oligonucleotides-seq)
Ontology highlight
ABSTRACT:
INSTRUMENT(S): NextSeq 500
ORGANISM(S): Arabidopsis thaliana
SUBMITTER: Xiaohui Zhao
PROVIDER: E-MTAB-5041 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. This study tests the hypothesis that DSB control involves a network of intersecting regulatory circuits. We show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. This counterintuitive result suggests that homologous chromosomes that have successfully engaged one another stop making DSBs, and provides new insight into phenotypes of zmm and other recombination-defective mutants. A genetically distinct pathway ties DSB formation to meiotic progression through the Ndt80 transcription factor. High-resolution genome-wide DSB maps generated by sequencing short oligonucleotides covalently bound to Spo11 (Spo11 oligos) demonstrate that feedback tied to ZMM function contributes in unexpected ways to spatial patterning of the recombination landscape. Four samples total: two wild type and two zip3 mutant (each an independent culture)
2014-04-23 | E-GEOD-48299 | biostudies-arrayexpress
Project description:The nonrandom distribution of meiotic recombination shapes patterns of inheritance and genome evolution, but chromosomal features governing this distribution are poorly understood. Formation of the DNA double-strand breaks (DSBs) that initiate recombination results in accumulation of Spo11 protein covalently bound to small DNA fragments. We show here that sequencing these fragments provides a genome-wide DSB map of unprecedented resolution and sensitivity. We use this map to explore the influence of large-scale chromosome structures, chromatin, transcription factors, and local sequence composition on DSB distributions. Our analysis supports the view that the recombination terrain is molded by combinatorial and hierarchical interaction of factors that work on widely different size scales. Mechanistic aspects of DSB formation and early processing steps are also uncovered. This map illuminates the occurrence of DSBs in repetitive DNA elements, repair of which can lead to chromosomal rearrangements. We discuss implications for evolutionary dynamics of recombination hotspots. We deep sequenced 4 samples of Spo11 oligos on Roche 454 platform. Three samples are technical replicates of Spo11 oligo products prepared from one meiotic culture and the fourth sample was prepared from an independent culture.
2011-03-07 | E-GEOD-26449 | biostudies-arrayexpress
Project description:The Spo11-generated double-strand breaks (DSBs) that initiate meiotic recombination are dangerous lesions that can disrupt genome integrity, so meiotic cells regulate their number, timing, and distribution. Here, we use Spo11-oligonucleotide complexes, a byproduct of DSB formation, to examine the contribution of the DNA damage-responsive kinase Tel1 (ortholog of mammalian ATM) to this regulation in Saccharomyces cerevisiae. A tel1Î mutant had globally increased amounts of Spo11-oligonucleotide complexes and altered Spo11-oligonucleotide lengths, consistent with conserved roles for Tel1 in control of DSB number and processing. A kinase-dead tel1 mutation also increased Spo11-oligonucleotide levels, but mutating known Tel1 phosphotargets on Hop1 and Rec114 did not. Deep sequencing of Spo11 oligonucleotides from tel1Î mutants demonstrated that Tel1 shapes the nonrandom DSB distribution in ways that are distinct but partially overlapping with previously described contributions of the recombination regulator Zip3. Finally, we uncover a context-dependent role for Tel1 in hotspot competition, in which an artificial DSB hotspot inhibits nearby hotspots. Evidence for Tel1-dependent competition involving strong natural hotspots is also provided. Sixteen samples total: The first 12 are two biological replicate Spo11-oligo maps from each of the following: wild type and tel1 each collected at 4, 5, and 6 hours after meiotic induction; the next 4 are one biological replicate Spo11-oligo map from each of the following: wild type and tel1 bearing an artificial hotspot insertion either on Chr III or on Chr IX.
2016-07-22 | E-GEOD-84696 | biostudies-arrayexpress
Project description:Meiotic recombination starts with the formation of DNA double-strand breaks (DSBs) made by Spo11. In Saccharomyces cerevisiae, the nonrandom distribution of meiotic DSBs along the genome can be attributed to the combined influence of multiple factors on Spo11 cleavage. One factor is higher-order chromatin structure, particularly the loop-axis organization of meiotic chromosomes. Axial element proteins Red1 and Hop1 provide the basis for meiotic loop-axis organization and are implicated in diverse aspects of meiotic recombination. Mek1 is a meiotic-specific kinase associated with Red1 and Hop1. Red1, Hop1, and Mek1 are required for normal DSB levels, but their effects on the DSB distribution has not been examined, and exactly how these proteins influence DSB levels and distribution is unknown. Here, we examined the contributions of Red1, Hop1, and Mek1 to the DSB distribution by deep sequencing and mapping Spo11-associated oligonucleotides from red1, hop1, and mek1 mutant strains, thereby generating genome-wide meiotic DSB maps.
2018-09-01 | GSE84859 | GEO
Project description:Spo11-mediated DNA double strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To shed light on this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S-phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea (HU) treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thereby we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation. Keywords: ChIP-chip â?¢ The goal of the experiment Genome-wide localization of Spo11, Mre11, Rec8, and DSB sites on meiotic chromosomes in Saccharomyces cerevisiae â?¢ Keywords Meiosis, Meiotic homologous recombination, Premeiotic DNA replication, cohesin, Saccharomyces cerevisiae, Genome tilling array (chromosome III, IV, V, VI), Spo11, Mre11, Rec8, DSB (Double strand break) â?¢ Experimental factor Distribution of Spo11, Mre11, and Rec8 in wild type in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium) Distribution of Spo11 in rec8delta cells in early meiosis (1.5 hrs, 2 hrs, 3 hrs, 4 hrs, and 5 hrs in sporulation medium) Distribution of Spo11 in wild type in the presence of HU (2hrs and 4 hrs in sporulation medium containing HU) Distribution of DSB sites in rad50S mutant cells at 7 hrs in sporulation medium Distribution of DSB sites in rec8delta rad50S mutant cells at 7 hrs in sporulation medium â?¢ Experimental design ChIP analyses: SK1 background cells expressing FLAG tagged protein were used for the ChIP using anti-FLAG M2 antibody. ChIP-chip analyses: In all cases, hybridization data for ChIP fraction was compared with WCE (whole cell extract) fraction. Saccharomyces cerevisiae affymetrix genome tiling array (SC3456a520015F for chromosome III, IV, V, VI and rikDACF for chromosome VI) were used. Mapping of DSB sites: DSB rich fraction was concentrated by ChIP of Spo11-FLAG in rad50S mutant without crosslinking. In the mutant, DSBs ramain unrepaired with covalently attached Spo11.Meiotic cells (at 7 hours in sporulation medium) were used for the analyses. â?¢ Quality control steps taken Confirmation of several loci by quantitative real time PCR. Southern blotting of several DSB sites.
2010-05-18 | E-GEOD-8422 | biostudies-arrayexpress
Project description:Meiotic recombination promotes genetic diversification as well as pairing and segregation of homologous chromosomes, but the double-strand breaks (DSBs) that initiate recombination are dangerous lesions that can cause mutation or meiotic failure. How cells control DSBs to balance between beneficial and deleterious outcomes is not well understood. This study tests the hypothesis that DSB control involves a network of intersecting regulatory circuits. We show that DSBs form in greater numbers in Saccharomyces cerevisiae cells lacking ZMM proteins, a suite of recombination-promoting factors traditionally regarded as acting strictly downstream of DSB formation. This counterintuitive result suggests that homologous chromosomes that have successfully engaged one another stop making DSBs, and provides new insight into phenotypes of zmm and other recombination-defective mutants. A genetically distinct pathway ties DSB formation to meiotic progression through the Ndt80 transcription factor. High-resolution genome-wide DSB maps generated by sequencing short oligonucleotides covalently bound to Spo11 (Spo11 oligos) demonstrate that feedback tied to ZMM function contributes in unexpected ways to spatial patterning of the recombination landscape.
2014-04-23 | GSE48299 | GEO
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 initiation hotspots in Arabidopsis genes and transposons (SPO11-1-oligonucleotides-seq)
| PRJEB15185 | ENA
Project description:Spo11-mediated DNA double strand breaks (DSBs) that initiate meiotic recombination are temporally and spatially controlled. The meiotic cohesin Rec8 has been implicated in regulating DSB formation, but little is known about the features of their interplay. To shed light on this point, we investigated the genome-wide localization of Spo11 in budding yeast during early meiosis by chromatin immunoprecipitation using high-density tiling arrays. We found that Spo11 is dynamically localized to meiotic chromosomes. Spo11 initially accumulated around centromeres and thereafter localized to arm regions as premeiotic S-phase proceeded. During this stage, a substantial proportion of Spo11 bound to Rec8 binding sites. Eventually, some of Spo11 further bound to both DSB and Rec8 sites. We also showed that such a change in a distribution of Spo11 is affected by hydroxyurea (HU) treatment. Interestingly, deletion of REC8 influences the localization of Spo11 to centromeres and in some of the intervals of the chromosomal arms. Thereby we observed a lack of DSB formation in a region-specific manner. These observations suggest that Rec8 would prearrange the distribution of Spo11 along chromosomes and will provide clues to understanding temporal and spatial regulation of DSB formation. Keywords: ChIP-chip
2009-05-29 | GSE8422 | GEO
Project description:Resection, nucleolytic processing of DSB ends is necessary to generate 3' single-stranded DNA tails (ssDNA) for homologous recombination (HR). Meiotic recombination initiated by Spo11 induced double-strand breaks (DSBs) is essential for the accurate segregation of homologous chromosomes. After cleavage, Spo11 stays covalently linked to the DSB ends, which requires MRX/Sae2 incision on broken molecules to allow following Exo1-mediated resection. Exonuclease I activity is inhibited by nucleosome bound DNA in vitro. To ensure resection proceeds, resection machineries must overcome chromatin barrier. Here we show that DSB dependent enrichment of Fun30 proteins at hotspots and meiotic axes.
2023-05-01 | GSE221033 | GEO