Project description:Meiotic recombination is initiated by the Spo11-dependent programmed DNA double-strand breaks (DSBs) that are preferentially concentrated within genomic regions known as hotspots, but the factor(s) which specify the positions for meiotic DSB hotspots remain unclear. Here, we show that the frequency and distribution of R-loops, a type of functional chromatin structure comprising single-stranded DNA and a DNA:RNA hybrid, change dramatically throughout meiosis. We detected the formation of multiple de novo R-loops in the pachytene stage, and found they co-localize with meiotic DSB hotspots. We further show that transcription-replication head-on collisions could promote R-loop formation during meiosis, and apparently direct the initiation of meiotic DSB formation. Furthermore, the hotspots can be eliminated by reverse the direction of either transcription or replication, and reconstituted by reverse both of their direction. Our study reveals that R-loops may play dual roles in meiotic recombination. In addition to participation in meiotic DSB processing, some meiotic DSB hotspots may be originated from the transcription-replication head-on collisions during meiotic DNA replication.
Project description:We used CRISPR/Cas9 to delete the DBP1 ORF in SK1 budding yeast cells. We then used ribosome profiling and mRNA-seq to observe gene expression profiles of wild-type and dbp1∆ cells during meiosis.
Project description:The R-loop is a common chromatin feature presented from prokaryotic to eukaryotic genomes and has been revealed to be involved in multiple cellular processes. Here, we developed a novel R-loop profiling technique, ULI-ssDRIP-seq, to decte global R-loops from a limited number of cells. Based on this method, we profiled the R-loop landscapes during parental-to-zygotic transition and early development regulatory in zebrafish, and revealed a series of important characters of R-loops.
Project description:Dynamic proteome in diploid yeast cells undergoing mitotic growth and meiotic development. The proteome of growing (YPA) and differentiating yeast cells at 6 hours (SPII 6, middle meiosis) and 8 hours (SPII8, late meiosis) were compared using the TOP3 GeLC-MS/MS method.
Project description:Fission yeast cells undergo meiosis and sporulation under conditions of nutritional stress, most frequently nitrogen starvation. This is a complex developmental process, which results in the formation of four spores that are highly resistant to environmental stress. We have carried out time course experiments with diploid fission yeast cells undergoing meiosis and sporulation. In this experiment we have used wild type diploids, and induced meiosis by removal of nitrogen from the medium. As a reference we used a pool consisting of equal amount of RNA from all time points of the experiments. This experiment is part of a series, with accession numbers E-SNGR-2 to E-SNGR-7.
Project description:Genome haploidization at meiosis depends on two consecutive nuclear divisions, which are controlled by an oscillatory system consisting of Cdk1-cyclin B and the APC/C bound to the Cdc20 activator. How the oscillator generates exactly two divisions has been unclear. We have studied this question in yeast where exit from meiosis involves accumulation of the APC/C activator Ama1 at meiosis II. We show that inactivation of the meiosis I-specific protein Spo13/MEIKIN results in a single-division meiosis due to premature activation of APC/CAma1. In the wild-type, Spo13 bound to the polo-like kinase Cdc5 prevents Ama1 synthesis at meiosis I by stabilizing the translational repressor Rim4. In addition, Cdc5-Spo13 inhibits the activity of Ama1 by converting the B-type cyclin Clb1 from a substrate to an inhibitor of Ama1. Cdc20-dependent degradation of Spo13 at anaphase I unleashes a feedback loop that increases Ama1’s synthesis and activity, leading to irreversible exit from meiosis at the second division. Thus, by repressing the exit machinery at meiosis I, Cdc5-Spo13 ensures that cells undergo two divisions to produce haploid gametes.
Project description:Transcriptional profile of fission yeast meiosis and sporulation. Fission yeast cells undergo meiosis and sporulation under conditions of nutritional stress, most frequently nitrogen starvation. This is a complex developmental process, which results in the formation of four spores that are highly resistant to environmental stress. We have carried out time courses of diploid fission yeast cells undergoing meiosis and sporulation. To achieve the best possible synchrony we have used thermosensitive mutants in the meiotic inhibitor pat1. We first pre-synchronized the cells in G1 by removal of nitrogen at the permissive temperature for pat1, and then induced meiosis by inactivating pat1 with a temperature shift. pat1-induced meiosis is a standard technique, resulting in a highly synchronous meiosis that is very similar to the normal process. The use of pat1 mutants and a temperature-shift may cause some artifacts, so these results should be viewed together with temperature shift controls and especially with the results of the time course done with wild type diploids, available under accession numbers E-SNGR-3 to E-SNGR-7. In all cases we used as reference a pool consisting of equal amounts of RNA from all time points of the experiment. This experiment describes the first biological replicate for pat1 using array design A-SNGR-2. Note that there are three other biological replicates, which use array design A-SNGR-1 in experiment E-SNGR-2.