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Calibrated ChIP-seq for meiotic-specific cohesin (Rec8) and mitotic-specific cohesin (Scc1) in budding yeast cells arrested in metaphase of mitosis

ABSTRACT: Calibrated ChIP-seq for meiotic-specific cohesin (Rec8) and mitotic-specific cohesin (Scc1) in budding yeast cells arrested in metaphase of mitosis

PROVIDER: PRJNA594025 | ENA |

REPOSITORIES: ENA

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Genome-wide binding of the meiosis specific Rec8 cohesin subunit in metaphase I

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Genome-wide binding of the meiosis specific Rec8 cohesin subunit in metaphase I

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Project description:The mitotic cohesin complex necessary for sister chromatid cohesion and chromatin loop formation shows local and global association to chromosomes in response to DNA double-strand breaks (DSBs). Here, by genome-wide binding analysis of the meiotic cohesin with Rec8 as a kleisin, we found that Rec8 shows dynamic localization from middle to late meiotic prophase I with cleavage-independent global dissociation along chromosomes, driven by meiotic DSB formation. Each Rec8 binding site on the chromosome axis follows distinct dynamics with dissociation and association. Centromeres also showed reduced Rec8 binding in late prophase I relative to mid-prophase I, implying chromosome remodeling of the regions. Rec8 dissociation profile per chromosome is largely correlated with meiotic DSB density. Indeed, the spo11 mutant deficient in meiotic DSB formation did not show the cleavage-independent dissociation of Rec8 in late meiotic prophase I. These suggest the presence of a regulatory pathway for global Rec8-cohesin dynamics in response to meiotic DSBs.

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Rec8 cohesin-mediated loop-axis chromatin architecture is required for meiotic recombination

Project description:During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, functional correlation between these structure formation and cohesion remains elusive. Here we examined formation of the cohesin-dependent axial structure in fission yeast, which forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the central elements, and found that Rec8 cohesin is crucial for the formation of the loop-axis structure within the atypical SC. Furthermore, the Rec8-mediated loop-axis structure is formed in the absence of LinEs, and provides a structural platform for aligning homologous chromosomes. We also succeeded to identify a rec8 mutant that lost the ability of assembling the loop-axis structure without losing cohesion. Remarkably, this mutant showed defects in LinE assembly, resulting in great reduction of meiotic recombination. Collectively, our results demonstrate an essential role of the Rec8-dependent loop-axis structure in LinE assembly, facilitating meiotic recombination.

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Project description:Genome binding/occupancy profiling of the cohesin subunit Scc1 by high throughput sequencing. S. pombe is included as the calibration genome.

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Project description:Cohesin is essential for genome folding and chromosome inheritance through cell divisions. In somatic cells, both of these functions are mediated by Scc1-cohesin, which in mitosis is released from chromosomes by Wapl and separase. In mammalian oocytes, cohesion is mediated by Rec8-cohesin. Scc1 is also expressed but neither required nor sufficient for cohesion, and its function in oocytes is unknown. Likewise, it is unknown whether Wapl regulates one or both cohesin complexes and chromosome segregation in mature oocytes. Using conditional mouse mutagenesis, we show that in meiosis I Wapl is required for proper chromosome segregation, predominantly releases Scc1-cohesin from chromosomes and promotes production of euploid eggs. Using single-nucleus Hi-C, we found that Scc1 is essential for chromatin loops and topologically associating domains (TADs) in oocytes. Increasing Scc1 residence time on chromosomes by Wapl depletion leads to vermicelli formation and intra-loop structures but, unlike in mitotic cells, does not increase loop size. This implies that loop size has reached a maximum in oocytes, possibly because it is limited by barriers such as cohesive cohesin. We conclude that distinct cohesin complexes generate loops and cohesion in mammalian oocytes and propose that the same principle applies to all cell types and species.

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Rec8 ChIP-Seq experiment to analyse cohesin localisation in the Ctf19c mutants during meiotic prophase.

Project description:Ctf19c mutants were shown to specifically impair cohesin localisation at the pericentromere during mitosis. The goal of this experiment is to confirm whether this mutant has the same effect on cohesin localisation during meiotic prophase. To do so, diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication) Diploid S. cerevisiae SK1 cells were constructed containing Rec8-3HA in an ndt80-d background. Cells were allowed to sporulate in sporulation media for 5 hours after which formaldehyde was added to the cultures and cells were fixed for 2hr. Samples were then processed according to the standard lab ChIP-Seq protocol (with 3 times 30cycles of 30sec of sonication)

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