Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1

ABSTRACT: Mcm2-7 ChIP in pre-meiotic and pre-mitotic cells, axis factor ChIP in wild-type and replication compromised strains in meiosis Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots.

ORGANISM(S): Saccharomyces cerevisiae

SUBMITTER: Stephen Bell

PROVIDER: E-GEOD-35658 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Separation of DNA replication from the assembly of break-competent meiotic chromosomes.

Blitzblau Hannah G HG Chan Clara S CS Hochwagen Andreas A Bell Stephen P SP

PLoS genetics 20120517 5

The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA rep ...[more]

PMID: 22615576

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Project description:The meiotic cell division reduces the chromosome number from diploid to haploid to form gametes for sexual reproduction. Although much progress has been made in understanding meiotic recombination and the two meiotic divisions, the processes leading up to recombination, including the prolonged pre-meiotic S phase (meiS) and the assembly of meiotic chromosome axes, remain poorly defined. We have used genome-wide approaches in Saccharomyces cerevisiae to measure the kinetics of pre-meiotic DNA replication, and to investigate the interdependencies between replication and axis formation. We found that replication initiation was delayed for a large number of origins in meiS compared to mitosis, and that meiotic cells were far more sensitive to replication inhibition, most likely due to the starvation conditions required for meiotic induction. Moreover, replication initiation was delayed even in the absence of chromosome axes, indicating replication timing is independent of the process of axis assembly. Finally, we found that cells were able to install axis components and initiate recombination on unreplicated DNA. Thus, although pre-meiotic DNA replication and meiotic chromosome axis formation occur concurrently, they are not directly coupled. The functional separation of these processes reveals a modular method of building meiotic chromosomes, and predicts that any crosstalk between these modules must occur through superimposed regulatory mechanisms. Multiple studies of meiotic chromosomes were undertaken. To study DNA replication, the locations of replicative helicase (Mcm2-7) were mapped in pre-meiotic and pre-mitotic cells, and DNA replication profiles were created for pre-meiotic S (meiS) and pre-mitotic S (mitS) phases. Early origins were mapped in hydroxyurea for wild-type cells in mitS + 200mM HU, and meiS +20mM HU for wild-type, sml1, rec8 and spo11 deletion cells. Rec8, Hop1 and Red1 binding to meiotic chromosomes was evaluated using ChIP-chip in wild-type cells with and without 20 mM HU, and in cdc6-mn and clb5 clb6 delete cells. Finally, meiotic DNA double-strand breaks (DSBs) were mapped in cdc6-mn dmc1 delete cells by measuring the ssDNA that accumulates at DSB hotspots. This SuperSeries is composed of the following subset Series: GSE35658: Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1 GSE35662: S phase and HU profiles in wild-type and mutant cells GSE35666: DSB formation in replication compromised cells

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.

Dataset Information

Chromatin IP for Mcm2-7, Rec8, Hop1 and Red1

Publications

Separation of DNA replication from the assembly of break-competent meiotic chromosomes.

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