Genome-wide binding site of Rec10 and Rec15 during meiosis
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ABSTRACT: Higher-order chromosome structure is assumed to control various DNA-templated reactions in eukaryotes. Meiotic chromosomes implement developed structures called M-bM-^@M-^\axesM-bM-^@M-^] and M-bM-^@M-^\loopsM-bM-^@M-^]; both are suggested to tether each other, activating Spo11 to catalyze meiotic DNA double-strand breaks (DSBs) at recombination hotspots. We found that the Schizosaccharomyces pombe Spo11 homolog Rec12 and its partners form two distinct subcomplexes, DSBC (Rec6-Rec12-Rec14) and SFT (Rec7-Rec15-Rec24). Additionally, Mde2, whose expression is strictly regulated by the replication checkpoint, interacts with a component of each subcomplex. The SFT subcomplex binds to both axes via direct interaction of Rec15 with Rec10 in axes and DSB sites, hence axial Rec10 can partially tether DSB sites located in loops. Importantly, this multiprotein-based tethered axis-loop complex is destabilized in the absence of Mde2. We therefore propose a novel mechanism by which Mde2 functions as a recombination initiation mediator to tether axes and loops, in liaison with the meiotic replication checkpoint. ChIP-chip analyses of Rec10 (in wild type), Mde2 (in wild type and rec15M-bM-^HM-^F), and Rec15 (in wild type, rec10M-bM-^HM-^F, rec24M-bM-^HM-^F and mde2M-bM-^HM-^F) at meiosis 4 hours.
Project description:Yersinia pestis, the etiological agent of plague, is able to sense cell density by quorum sensing. The function of quorum sensing in Y. pestis is not clear. Here, the process of quorum sensing was investigated by comparing transcript profiles when three quorum-sensing synthase genes are knocked out. Two strains, M-bM-^HM-^Fpgm (pigmentation-negative) mutant R88 as treatment and quorum sensing null strain R115 with mutations (M-bM-^HM-^Fpgm, M-bM-^HM-^FypeIR, M-bM-^HM-^FyspIR, and M-bM-^HM-^FluxS) as control, are used in this analysis. Six independent RNA samples from R115 cultures were paired with six independent RNA samples from R88 cultures for hybridization to six two-color microarrays. A dye-swap design was used to remove the Cy5 and Cy3 dye bias.
Project description:Higher-order chromosome structure is assumed to control various DNA-templated reactions in eukaryotes. Meiotic chromosomes implement developed structures called “axes” and “loops”; both are suggested to tether each other, activating Spo11 to catalyze meiotic DNA double-strand breaks (DSBs) at recombination hotspots. We found that the Schizosaccharomyces pombe Spo11 homolog Rec12 and its partners form two distinct subcomplexes, DSBC (Rec6-Rec12-Rec14) and SFT (Rec7-Rec15-Rec24). Additionally, Mde2, whose expression is strictly regulated by the replication checkpoint, interacts with a component of each subcomplex. The SFT subcomplex binds to both axes via direct interaction of Rec15 with Rec10 in axes and DSB sites, hence axial Rec10 can partially tether DSB sites located in loops. Importantly, this multiprotein-based tethered axis-loop complex is destabilized in the absence of Mde2. We therefore propose a novel mechanism by which Mde2 functions as a recombination initiation mediator to tether axes and loops, in liaison with the meiotic replication checkpoint.
Project description:Genome-wide ChIP data of CTCF and Rad21 binding in Rag1M-bM-^HM-^R/M-bM-^HM-^R pro-B cells CTCF and Rad21 binding in Rag1M-bM-^HM-^R/M-bM-^HM-^R pro-B
Project description:Meiotic DSB, catalyzed by the Spo11 transesterase protein and accessory DSB proteins, form in the nucleosome depleted regions (NDR) at promoters, preferentially those located on the chromosome loops that shape meiotic chromosomes, whereas the DSB proteins are located on chromosome axes at the basis of these loops. Mechanisms bridging these two chromosomal regions for DSB formation have remained elusive. Here we show that Spp1, a conserved member of the histone H3K4 methyltransferase Set1 complex, is required for normal levels of DSB formation and is associated with chromosome axes in the DSB-rich domains during meiosis. Moreover, Spp1 physically interacts with the Mer2 axis-associated DSB protein, and uses its PHD finger as a magnet to read H3K4 trimethylation close to promoters, tether these regions to chromosome axes and activate cleavage in the nearby promoter by the DSB proteins. We further show that in the absence of Spp1 or the Set1 complex, DSB are introduced at a few new sites, located in promoters of transcriptionally induced genes, suggesting another selection mechanism of preferred DSB sequences. This paper provides the molecular mechanism linking H3K4me3 to the DSB forming machinery, by the meiosis-specific specialization of Spp1 as an active member of the DSB complex and a reader of H3K4me3, and opens perspectives for the study of DSB formation at mammalian recombination hotspots that are also enriched in H3K4me3. ChIP-chip experiment in vegetative or meiotic diploid SK1 yeast cells - two biological replicates
Project description:We employed PAR-CLIP, a recently developed method based on RNA-protein crosslinking, to identify Cirbp and Rbm3 binding sites at transcriptome-level. Genome-wide RNA-seq analysis indicated that cold temperature leads to extensive 3M-bM-^@M-^YUTR lengthening whereas the loss of Cirbp or Rbm3 resulted in 3M-bM-^@M-^YUTR shortening. Combining with PAR-CLIP results, we found that these two RBPs repress the polyadenylation adjacent to their binding sites. Examination of the binding sites of Cirbp and Rbm3 by PAR-CLIP and their influence on the transcriptome by RNA-seq. PARCLIP was performed as in Hafner et al. 2010 Cell 141, 129M-bM-^@M-^S141, with MEFs cell lines stably expressing FLAG-tagged Cirbp and Rbm3. We used 4-thiouridine (4SU) to enhance the crosslink. For RNA-seq, polyadenylated RNA from the mock-transfected cells at 37M-BM-0C or 32M-BM-0C with two replicates, siCirbp-1, siCirbp-2, siRbm3-1 and siRbm3-2 MEFs at 37M-BM-0C were sequenced on Solexa GAII using 76bp single-end kits according to the manufacturerM-bM-^@M-^Ys instructions.
Project description:Yersinia pestis, the etiological agent of plague, is able to sense cell density by quorum sensing. The function of quorum sensing in Y. pestis is not clear. Here, the process of autoinducer 2 (AI-2) quorum sensing was investigated by comparing transcript profiles when luxS gene was knocked out. The luxS gene encodes S-ribosylhomocysteinase which can produce DPD, a precursor of AI-2. The strain M-bM-^HM-^Fpgm (pigmentation-negative) mutant R88 was called wild type. The M-bM-^HM-^Fpgm M-bM-^HM-^FluxS mutant was called control. Six independent RNA samples from R88 were paired with six independent RNA samples from M-bM-^HM-^Fpgm M-bM-^HM-^FluxS mutant cultures for hybridization to six two-color microarrays. A dye-swap design was used to remove the Cy5 and Cy3 dye bias.
Project description:Transcription is a major obstacle for replication fork progression and a cause of genome instability. Such instability increases in mutants with a suboptimal assembly of the nascent messenger ribonucleo-protein particle (mRNP), as THO/TREX and the NPC-associated THSC/TREX-2 complex. Here we show that yeast sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells accumulate genome-wide replication obstacles as determined by the distribution of the Rrm3 helicase. Such obstacles preferentially occur at long and highly expressed genes, to which Sac3 and its interacting partner Thp1 are preferentially bound in wild-type cells. ChIP-chip studies were perfomed with antibodies against Flag-tagged Thp1 and Sac3 proteins in wild-type cells of the yeast S. Cerevisiae, as well as Flag-tagged Rrm3 protein in sac3M-bM-^HM-^F and thp1M-bM-^HM-^F cells that were compared with Rrm3 in wild-type cells from Santos-Pereira et al., 2013 (accession number GSE50185).
Project description:Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in five types of Alzheimer's disease (AD) mouse models (5xFAD, APP-tg, PS1-tg, PS2-tg and APP-KI) and four types of frontotemporal lobar degeneration (FTLD) mouse models(CHMP2B-KI, PGRN-KI, VCP-KI and TDP43-KI) at multiple time points (1, 3 and 6 months).
Project description:The strain bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H] improved salt resistance of bdf1M-bM-^HM-^F. To gain further insight into the mechanism of BDF1 in suppressing bdf1M-bM-^HM-^F salt sensitivity, DNA microarray analysis was performed to determine the reason for the salt sensitivity of bdf1M-bM-^HM-^F cells and the process of how coexpression of SIR2 and BDF2 improves salt resistance. Transcriptomic analysis under salt treatment (0.6 mol.L-1 NaCl for 45 min) was performed using three different strains: bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H], bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242] and bdf1M-bM-^HM-^F[pRS316][pYX242]. The transcription of 3244 genes were significantly changed( > 2-fold) in bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H] compared with bdf1M-bM-^HM-^F[pRS316][pYX242] upon NaCl stress (0.6 mol.L-1 NaCl for 45 min). Only 281 genes were significantly changed ( > 2-fold) in bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242] compared with bdf1M-bM-^HM-^F[pRS316][pYX242] upon NaCl stress (0.6 molM-bM-^HM-^YL-1 NaCl for 45 min). BF2:bdf1M-bM-^HM-^F[pRS316][pYX242]. BF2B: bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][pYX242]. BF2S:bdf1M-bM-^HM-^Fbdf2M-bM-^HM-^F[BDF2 L][SIR2 H]. BF2B vs BF2 under salt treatment (0.6 mol.L-1 NaCl for 45 min); BF2S vs BF2 under salt treatment (0.6 mol.L-1 NaCl for 45 min).
Project description:Yersinia pestis, the etiological agent of plague, is able to sense cell density by quorum sensing. The function of quorum sensing in Y. pestis is not clear. Here, the process of autoinducer-2 (AI-2) quorum sensing was investigated by comparing transcript profiles when AI-2 quorum-sensing signal is added in. The strain M-bM-^HM-^Fpgm(pigmentation-negative) mutant R88 was used as wild type. The control consisted of cells grown and treated under the same conditions without added signals. Six independent RNA samples from Y. pestis CO92 M-bM-^HM-^Fpgm cultures were paired with six independent RNA samples from AI-2 signal added cultures for hybridization to six two-color microarrays. A dye-swap design was used to remove the Cy5 and Cy3 dye bias.