Project description:To understand the proteome and phosphoproteome level responses to meiotic DNA breaks, we have compared protein phosphorylation and protein levels in cells spo11-YF (cannot form meiotic DNA breaks) cells to SPO11 (can form meiotic breaks) cells.
Project description:Methamphetamine (METH) addiction is associated with a plethora of neuropsychiatric symptoms. In rodents, administration of the drug is accompanied by complex changes in gene expression in the dorsal striatum. Very little is known about the effects of the drug on gene expression and epigenetic modifications in the nucleus accumbens (NAc). The present study was conducted in order to investigate the effects of a single injection of METH on gene expression in that structure. We also queried whether changes in transcript levels were accompanied by alterations in histone acetylation as well as in the expression of the histone acetyltransferase (HAT), ATF2, and of the histone deacetylases (HDACs), HDAC1 and HDAC2. Microarray analyses revealed that METH caused significant time-dependent increases in the expression of several genes that had previously been implicated in the acute and longterm effects of psychostimulants in the brain. These include several immediate early genes (c-fos, Egrs, c-jun, and Nurr1) and corticotropin-releasing factor (Crf). There were also increases in the levels of neuromedin U, Tnf-alpha, and Kcnk18, among others. In contrast, the METH injection caused decreases in the expression of many genes including Npas4 and cholecystokin (Cck). Pathway analyses showed that differentially affected genes participate in behavioral performance, cell-to-cell signaling and interactions, and regulation of gene expression. Other differentially affected genes are known to be involved in cellular compromise and death pathways. PCR analyses were used to confirm the changes in the expression of c-fos, fosB, c-jun, junB, Crf, NmU, Cck, and Npas4 transcripts. Western blot analyses also identified METH-mediated decreases in the acetylation of histone H3 at lysine 9 (H3K9) and lysine 18 (H3K18) as well as in histone 4 at lysine 16 (H4K16). In contrast, the METH injection caused time-dependent increases in the acetylation of H4K8 and H4K12. The changes in histone acetylation were also accompanied by decreased expression of HDAC1 but increased expression of ATF2 and of HDAC2. These results suggest that METH-induced alterations in global gene expression might be due, in part, to diverse effects of METH-induced histone acetylation secondary to changes in HAT and HDAC expression. At the indicated time after the METH or saline injections, rats (n = 5 per group) were euthanized and the NAc was dissected and immediately put on dry ice. The tissues were kept at -70 oC until RNA extraction. Total RNA was isolated from the nucleus accumbens according to the manufacturer’s manual using Qiagen RNeasy mini kit (Qiagen, Valencia, CA, USA). RNA integrity was detected using an Agilent 2100 Bioanalyzer (Agilent, Palo Alto, CA, USA).
Project description:To understand the proteome and phosphoproteome level responses to meiotic DNA breaks, we have compared protein phosphorylation and protein levels in cells spo11-YF (cannot form meiotic DNA breaks) cells to SPO11 (can form meiotic breaks) cells.
Project description:We conducted a genome-wide expression analysis of wild-type males using three cell populations isolated from mitotic, meiotic and post-meiotic phases of spermatogenesis in Drosophila melanogaster. Our approach was to directly isolate testis regions enriched with RNAs from each of the three specific germline phases. We used microarrays to detail the global gene expression profile in spermatogenesis and identified up- and down-regulated genes between two different spermatogenic phases in pairwise comparisons Experiment Overall Design: Cell types present at various stages of spermatogenesis are generally located in a gradient along the proximal-distal axis of the testis, however most are not exclusively restricted to any one geographic region. Cells enriched for mitotic, meiotic and post-meiotic phases were obtained by dissection of apical, proximal and distal regions of the testis, respectively
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.
Project description:Sumoylation is emerging as a post-translation modification important for chromosome duplication and stability. The origin recognition complex (ORC), which directs DNA replication initiation by loading the MCM replicative helicases onto origins, is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are largely unclear. Here we report the effects of hyper- and hypo-sumoylation of yeast ORC using multiple approaches. We show that ORC hyper-sumoylation preferentially reduces the activity of a subset of early origins, while Orc2 hypo-sumoylation has an opposing effect. Mechanistically, ORC hyper-sumoylation leads to reduced MCM loading in vitro and diminished MCM chromatin association in vivo. The importance of an appropriate level of ORC sumoylation is suggested by the data that either hyper- or hypo-sumoylation of ORC results in genome instability and a dependence on other genome maintenance factors for cell fitness. Thus, yeast ORC sumoylation status needs to be fine-tuned to achieve optimal origin activity control and genome stability.
Project description:Progression through meiosis in Schizosaccharomyces pombe is regulated by stage-specific gene expression and translation, changes in RNA stability, expression of anti-sense transcripts and also requires stage-specific, targetted proteolysis of regulatory proteins. We have used SILAC labelling to examine the relative levels of proteins in S. pombe as diploid cells undergo meiosis. We found that the relative level of 880 proteins changes at least two-fold at some stage of meiosis. Most of these proteins either increase or decrease in level during the meiotic divisions, while some show transient peaks of expression. The most notable changes are in the proteostasis network, which shows a significant increase in components involved in protein turnover concomitant to a decrease in proteins involved in ribosome biogenesis. There was also an increase in ESCRT III protein levels; biological analysis reveals a role for some ESCRT III components in chromosome segregation and spore formation. Correlation with previous studies of gene expression and ribosome occupancy through meiosis reveals that changes in steady state protein levels are mainly regulated post-transcriptionally.
Project description:DNA double-strand breaks (DSBs) initiate meiotic recombination. Past DSB-mapping studies have used rad50S or sae2? mutants, which are defective in break processing, to accumulate DSBs, and report large (= 50 kb) “DSB-hot” regions that are separated by “DSB-cold” domains of similar size. Substantial recombination occurs in some DSB-cold regions, suggesting that DSB patterns are not normal in rad50S or sae2? mutants. We therefore developed novel methods that detect DSBs using ssDNA enrichment and microarray hybridization, and that use background-based normalization to allow cross-comparison between array datasets, to map genome-wide the DSBs that accumulate in processing-capable, repair-defective dmc1î and dmc1î rad51î mutants. DSBs were observed at known hotspots, but also in most previously-identified “DSB-cold” regions, including near centromeres and telomeres. While about 40% of the genome is DSB-cold in rad50S mutants, analysis of meiotic ssDNA from dmc1? shows that most of these regions have significant DSB activity. Thus, DSBs are distributed much more uniformly than was previously believed. Southern-blot assays of DSBs in selected regions in dmc1?, rad50S and wild-type cells confirm these findings. Comparisons of DSB signals in dmc1, dmc1 rad51, and dmc1 spo11 mutant strains identify Dmc1 as the primary strand transfer activity genome-wide, and Spo11-induced lesions as initiating all meiotic recombination. Keywords: DSB mapping, ChIP-chip, single strand DNA , BND cellulose We use two different strategies to map the genome-wide distribution of meiotic DSBs in the yeast Saccharomyces cerevisiae. The first is a chromatin immunoprecipitation (ChIP) based approach that targets the Spo11p protein, which remains covalently attached to DSB ends in the rad50S mutant background. The second approach involves BND cellulose enrichment of the single strand DNA (ssDNA) recombination intermediate formed by end-resection at DSB sites following Spo11p removal. We use dmc1 and dmc1 rad51 mutants that accumulates meiotic single strand DNA intermediates
Project description:Crossing over between homologs is critical for the stable segregation of chromosomes during the first meiotic division. S. cerevisiae Mer3 (HFM1 in mammals) is a SF2 helicase and member of the ZMM group of proteins, that facilitates the formation of the majority of crossovers during meiosis. Here we describe the structural organisation of Mer3 and using AlphaFold modelling and XL-MS we further characterise the previously described interaction with Mlh1-Mlh2. We find that Mer3 also forms a previously undescribed complex with the recombination regulating factors Top3 and Rmi1 and that this interaction is competitive with Sgs1BLM helicase in a phospho-dependent manner. Using in vitro reconstituted D-loop assays we show that Mer3 inhibits the anti-recombination activity of Sgs1 helicase, but only in the presence of Dmc1. Thus we provide a mechanism whereby Mer3 interacts with a network of proteins to protect Dmc1 derived D-loops from dissolution.
Project description:Protein, Ubiquitylation, and Phosphorylation changes were quantified after 1 and 3 hours of rapamycin treatment in budding yeast. Approximately 3,600 proteins, 9,000 phosphorylation sites, and 2,500 ubiquitylation sites were quantified.