Nucleosome positioning in Yeast expressing active or inactive murine DNMTs
ABSTRACT: Effect of induced Methylation on Nucleosome positioning in yeast. Mnase digested DNA from a control strain, a strain expressing the 4 murine DNMTs or a strain expressing catalytically inactive murine DNMTs were extracted and sequenced on a hiseq 2000
Project description:Effect of induced Methylation on Nucleosome positioning in yeast. Mnase digested DNA from a control strain and a strain expressing the 4 murine DNMTs were extracted and sequenced on a hiseq 2000
Project description:Localisation of CpG methylation in yeast expressing murine DNMTS Genomic DNA was purified from a control strain and a strain expressing murine DNMTs, treated with Bisulfite and sequenced on a hiseq 2000
Project description:The oomycete pathogen Phytophthora sojae causes root rot of soybean. During infection, the pathogen is thought to deliver dozens, if not hundreds, of effector proteins into the host to manipulate intracellular systems. Although these pathogen proteins often exhibit similar N-terminal delivery domains, the remaining effector region is rarely homologous to known protein domains, making it difficult to predict its biochemical function during infection. As a complement to studies in the natural host, Saccharomyces cerevisiae has been successfully used as a model system to explore the biochemical function of individual pathogen effectors. The presumption is that many effectors target conserved eukaryotic pathways in the host and consequently the expression of effectors in yeast will confer corresponding phenotypes. Indeed, putative effector functions identified using yeast functional genomic approaches have subsequently been validated in the natural host. Over-expression of the Phytophthora sojae effector Avh172 (PsAvh172) inhibits the growth of Saccharomyces cerevisiae, suggesting that the effector targets a biological pathway conserved with plants. In this study, the transcriptomes of yeast expressing PsAvh172 or an empty vector were compared to examine the global transcriptional response, in hopes of discerning the effectors biochemical target.
Project description:ISWI-family chromatin remodelers organize nucleosome arrays, while SWI/SNF-family remodelers (RSC) disorganize and eject nucleosomes, implying an antagonism that is largely unexplored in vivo. Here, we describe two independent genetic screens for rsc suppressors that yielded mutations in the promoter-focused ISW1a complex, or mutations in the ‘basic patch’ of histone H4 (an epitope that regulates ISWI activity), strongly supporting RSC-ISW1a antagonism in vivo. RSC and ISW1a largely co-localize, and genomic nucleosome studies using rsc isw1 mutant combinations revealed opposing functions: promoters classified with a nucleosome-deficient region (NDR) gain nucleosome occupancy in rsc mutants, but this gain is attenuated in rsc isw1 double mutants. Furthermore, promoters lacking NDRs have the highest occupancy of both remodelers, consistent with regulation by nucleosome occupancy, and decreased transcription in rsc mutants. Taken together, we provide the first genetic and genomic evidence for RSC-ISW1a antagonism, and reveal different mechanisms at two different promoter architectures. Genome-wide nucleosome occupancy maps in RSC and rsc null strains were generated by paired-end sequencing of mononucleosomal DNA. Strains carrying the Sth1 degron allele and either pGal-UBR1 (YBC3386) or ubr1 null (YBC3387) represent RSC null and RSC wildtype, respectively.
Project description:We show that ligation-competent Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Using deep sequencing, we demonstrate that ligation junctions preferentially occur around nucleosome midpoints rather than in internucleosomal linker regions. Disrupting chromatin assembly or lagging strand polymerase processivity impacts both the size and the distribution of Okazaki fragments, suggesting a role for nascent chromatin, assembled immediately after the passage of the replication fork, in the termination of lagging strand synthesis. Our studies represent the first high-resolution analysis of eukaryotic Okazaki fragments in vivo, and establish a mechanistic link between the fundamental processes of DNA replication and chromatin assembly. 4 samples: replicate samples of wild-type and pol32 knockout
Project description:We studied the transcriptional profile in response to acute PtdIns-4,5P2 depletion induced by heterologous expression of a plasma membrane-directed version of mammalian PI3K catalytic subunit (p110α-CAAX). Three biological samples were analyzed for samples expressing for 4 hours p110α-CAAX (PI3K) versus the kinase dead mutant p110α-CAAX K802R (KD), and one microarray experiment was carried out for each sample.
Project description:The activity of the epigenetic writers DNA methyltransferases (Dnmts) after olfactory reward conditioning is important for both stimulus-specific long-term memory (LTM) formation and extinction. It, however, remains unknown which components of memory formation Dnmts regulate (e.g., associative vs. non-associative) and in what context (e.g., varying training conditions). Here, we address these aspects in order to clarify the role of Dnmt-mediated DNA methylation in memory formation. We used a pharmacological Dnmt inhibitor and classical appetitive conditioning in the honeybee Apis mellifera, a well characterized model for classical conditioning. We quantified the effect of DNA methylation on naïve odor and sugar responses, and on responses following olfactory reward conditioning. We show that (1) Dnmts do not influence naïve odor or sugar responses, (2) Dnmts do not affect the learning of new stimuli, but (3) Dnmts influence odor-coding, i.e., 'correct' (stimulus-specific) LTM formation. Particularly, Dnmts reduce memory specificity when experience is low (one-trial training), and increase memory specificity when experience is high (multiple-trial training), generating an ecologically more useful response to learning. (4) In reversal learning conditions, Dnmts are involved in regulating both excitatory (re-acquisition) and inhibitory (forgetting) processes.