K. lactis Ste12-myc ChIP-Seq in pheromone-responding a cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Kluyveromyces lactis
SUBMITTER: Trevor Sorrells
PROVIDER: E-GEOD-65792 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:The purpose of this experiment was to determine the genes directly regulated by Ste12 in K. lactis. The experiment was performed in a cells to determine if the a-specific genes were bound by Ste12. Ste12 was tagged with c-myc and was immunoprecipitated with a c-myc antibody. Cells were starved in SD media lacking phosphate for 2 hours, then treated with 10µg/mL K. lactis alpha factor for 2 hours before harvesting. A control untagged strain was processed in parallel. The experiment was repeated 3 times, and sequenced on an Illumina HiSeq 2500.
2015-07-06 | GSE65792 | GEO
Project description:Most available knowledge on fungal arginine metabolism is derived from studies on Saccharomyces cerevisiae, in which arginine catabolism is initiated by releasing urea via the arginase reaction. Orthologs of the S. cerevisiae genes encoding the first three enzymes in the arginase pathway were cloned from Kluyveromyces lactis and shown to functionally complement the corresponding deletion in S. cerevisiae. Surprisingly, deletion of the single K. lactis arginase gene KlCAR1 did not completely abolish growth on arginine as nitrogen source. Growth rate of mutant strongly increased during serial transfer in shake-flask cultures. A combination of RNAseq-based transcriptome analysis and 13C-15N-based flux analysis was used to elucidate the arginase-independent pathway. Isotopic 13C15N-enrichment in ?-aminobutyrate revealed succinate as the entry point in the TCA cycle of the alternative pathway. Transcript analysis combined with enzyme activity measurements indicated increased expression in the Klcar1? mutant of a guanidinobutyrase (EC.3.5.3.7), an enzyme not previously demonstrated in fungi. Expression of the K. lactis KLLA0F27995g (renamed KlGBU1) encoding guanidinobutyrase enabled S. cerevisiae to use guanidinobutyrate as sole nitrogen source and its deletion in K. lactis almost completely abolish growth on this nitrogen source. Phylogenetic analysis suggests that this enzyme activity is widespread in fungi. The goal of the present study was to characterize arginine catabolism in K. lactis. To this end, CAR1, CAR2 and PRO3 orthologs in K. lactis were identified and functionally analysed by deletion, expression in S. cerevisiae and enzyme activity assays. Since deletion of the arginase gene in K. lactis was found not to completely abolish growth on arginine as a sole nitrogen source, the alternative pathway for arginine catabolism operating in this yeast was studied by a combination of transcriptome analysis, 13C and 15N isotope-based flux analysis and enzyme activity assays in cell extracts. To investigate arginine metabolism in the arginase-negative K. lactis strain, strains GG1632 (Klku80? KlCAR1 reference strain) and IMS0367 (Klcar1? Arg+) were grown in aerobic bioreactor batch cultures on glucose chemically defined medium with arginine as sole nitrogen source. RNA sequencing of samples taken during the exponential phase of growth on glucose-arginine media of the reference strain G1631 and the arginase less strain IMS0367 were compared resulting in the characterization of a new function.
2014-07-16 | E-GEOD-56060 | biostudies-arrayexpress
Project description:Snt2 is a yeast chromatin-interacting protein whose function has not been well characterized, that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we show that in response to H2O2, Snt2 and Ecm5 colocalize to promoters of genes involved in various aspects of the environmental stress response. By integrating these ChIP-seq results with expression analysis, we identify a key set of target genes that require Snt2 for proper expression after H2O2 stress. Finally, by mapping Snt2 and Ecm5 localization before and after rapamycin treatment, we identify a subset of H2O2-specific Snt2 and Ecm5 target promoters that are also targeted in response to rapamycin. Our results establish a function for Snt2 in regulating transcriptional changes in response to oxidative stress, and suggest Snt2 may have a role in additional stress pathways. Crosslinking ChIP analysis to identify sites of Snt2 or Ecm5 genomic localization before, 0.5 hours after, or 4 hours after treatment with H2O2 (final concentration 0.4 mM). Snt2 and Ecm5 were genomically tagged with a 13Myc tag at their C termini. ChIPs were performed using a Myc antibody on either Snt2-Myc or Ecm5-Myc strains, or on an untagged wildtype strain (BY4741) as a control. Inputs and ChIPs from untagged strain were sequenced as controls.
2013-07-24 | E-GEOD-42971 | biostudies-arrayexpress
Project description:Snt2 is a yeast chromatin-interacting protein whose function has not been well characterized, that was recently shown to associate with Ecm5 and the Rpd3 deacetylase. Using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq), we show that in response to H2O2, Snt2 and Ecm5 colocalize to promoters of genes involved in various aspects of the environmental stress response. By integrating these ChIP-seq results with expression analysis, we identify a key set of target genes that require Snt2 for proper expression after H2O2 stress. Finally, by mapping Snt2 and Ecm5 localization before and after rapamycin treatment, we identify a subset of H2O2-specific Snt2 and Ecm5 target promoters that are also targeted in response to rapamycin. Our results establish a function for Snt2 in regulating transcriptional changes in response to oxidative stress, and suggest Snt2 may have a role in additional stress pathways. Crosslinking ChIP analysis to identify sites of Snt2 or Ecm5 genomic localization before, 0.5 hours after, or 4 hours after treatment with rapamycin (final concentration 5 nM) or with DMSO as a control. Snt2 and Ecm5 were genomically tagged with a 13Myc tag at their C termini. ChIPs were performed using a Myc antibody on either Snt2-Myc or Ecm5-Myc strains, or on untagged wildtype strain (BY4741) as a control. Inputs and ChIPs from untagged strain were sequenced as controls.
2013-07-24 | E-GEOD-43001 | biostudies-arrayexpress
Project description:Lcn972 is a non-modified bacteriocin that targets exclusively Lactococcus sp. Addition of Lcn972 inhibits cell wall biosynthesis at the septum by binding to the cell wall precursor lipid II. Resistance to Lcn972 develops upon selection with subinhibitory concentrations. The transcriptome of the highly resistant L. lactis D1 derived from the susceptible strain L. lactis MG1614 was compared. Fourteen genes were significantly up-regulated and 29 were down-regulated (expression change > 2-fold, p<0.001). Down-regulation was mostly found in sugar catabolic genes. Up-regulated genes included members of the cell envelope stress (CesR) regulon, the penicillin-binding protein pbpX and llmg2447, which may encode a putative extracytoplasmic function (ECF) anti-sigma factor located downstream of a non-functional ECF-sigX. Up-regulation of llmg2447 was linked to integration of IS981 that exchanged the -35 promoter region of llmg2447. Over-expression of llmg2447 resulted in highly Lcn972-resistant L. lactis transformants. The transcriptomes of Lactococcus lactis MG1614 susceptible to Lcn972 and L. lactis D1 resistant to Lcn972, grown under laboratory conditions, were compared using four biological replicates.
2012-12-06 | E-GEOD-38349 | biostudies-arrayexpress
Project description:pBL1 is a Lactococcus lactis theta-replicating 10.9-kbp plasmid that encodes the synthetic machinery of the bacteriocin Lcn972. In this work, the transcriptomes of exponentially growing L. lactis with and without pBL1 were compared. A discrete response was observed with a total of ten genes showing significantly changed expression. Up-regulation of the lactococcal oligopeptide uptake system (opp) was observed, likely linked to a higher nitrogen demand required for Lcn972 biosynthesis. Striking, celB coding for the membrane porter IIC of the cellobiose-PTS and the upstream gene llmg0186 were down-regulated. Growth profiles for L. lactis strains MG1363, MG1363/pBL1 and MG1363ΔcelB grown in CDM-cellobiose confirmed slower growth of pBL1 and ΔcelB while no differences were scored on glucose. The presence of pBL1 shifted the fermentation products towards a mixed acid profile and promoted substantial changes in intracellular pool sizes for glycolytic intermediates in cellobiose-growing cells as determined by HPLC and NMR. Overall, these data support the genetic evidence of a constriction in cellobiose uptake. Notably, several cell wall precursors accumulated, while other UDP-activated sugars pools were lower, which could reflect rerouting of precursors towards the production of structural or storage polysaccharides. Moreover, slow cellobiose-growing cells and those lacking celB were more tolerant to Lcn972 than cellobiose adapted cells. Thus, down-regulation of celB could help to build-up a response against the antimicrobial activity of Lcn972 enhancing self-immunity of the producer cells. The transcriptomes of Lactococcus lactis MG1614 with and without the bacteriocinogenic plasmid pBL1, grown under laboratory conditions, were compared using three biological replicates.
2011-09-14 | E-GEOD-30625 | biostudies-arrayexpress
Project description:Cellular processes are subject to variability, or noise, yet mechanisms that promote cell-to-cell uniformity are poorly understood. We have identified such a role for Dig1, a redundant (with Dig2) MAPK-responsive inhibitor of the S. cerevisiae mating pathway transcription factor Ste12. Cells lacking Dig1, but not Dig2, exhibited increased variability in outputs of the mating pathway. dig1∆ mutants also displayed a Ste12-dependent defect in growth in the absence of mating pheromone and a quantitative defect in the process of mating, itself. In cells expressing two reporter genes driven by the same promoter, both intrinsic/uncorrelated and extrinsic/correlated noise were found to increase. Remarkably, the extrinsic noise phenotype in cells lacking Dig1 correlates with the aggregation of target genes: we observed subnuclear foci of Ste12 specifically in dig1∆ cells and, using a newly-developed method to immunoprecipitate a single locus from crosslinked chromatin, we found that Dig1 inhibits long-range interactions between Ste12 target genes in vivo. Dig1 may shield binding surfaces on Ste12 whose unmasking leads to inappropriate gene associations and increased gene expression noise. These studies reveal how investigations of variability modulation mechanisms can yield unexpected biological insights.
2010-12-15 | GSE17583 | GEO
Project description:K. lactis Ste12-myc ChIP-Seq in pheromone-responding a cells
| PRJNA275069 | ENA
Project description:ChIP-chip to determine the regulation of the K. lactis hsgs (ChIP of MATa1, MATalpha2, and RME1). The MATa1 and MATalpha2 ChIPs were performed in an a/alpha cell using N-terminally HA-tagged proteins and the RME1 ChIPs were perfomed in an a cell using C-terminally myc-tagged protein. For the RME1 ChIPs, the cells grown with out phosphate. For the MATa1 andMATalpha2 ChIPs the cells were grown in YEPD. Epitope tagged strains were compared to untagged control strains. Two biological replicates were preformed for the RME1 ChIP. For the MATa1 and MATalpha2 ChIP, peaks were considered indicative of binding if both MATa1 and MATalpha2 showed enrichment above the untagged control.
2010-12-01 | E-GEOD-25209 | biostudies-arrayexpress
Project description:Most eukaryotic genes express mRNAs with alternative polyadenylation sites at their 3’ ends. Here we show that polyadenylated 3’ termini in three yeast species (S. cerevisiae, K. lactis, D. hansenii) are remarkably heterogeneous. Instead of a few discrete 3’ ends, the average yeast gene has an “end zone”, a >200 bp window with >60 distinct poly(A) sites, the most utilized of which represents only 20% of the mRNA molecules. The pattern of polyadenylation within this zone varies across species, with D. hansenii possessing a higher focus on a single dominant point closer to the ORF terminus. Some polyadenylation occurs within mRNA coding regions with a strong bias towards the promoter. The polyadenylation pattern is determined by a highly degenerate sequence over a broad region and by a local sequence that relies on A residues after the cleavage point. Many dominant poly(A) sites are predicted to adopt a common secondary structure that may be recognized by the cleavage/polyadenylation machinery. We suggest that the end zone reflects a region permissive for polyadenylation, within which cleavage occurs preferentially at the A-rich sequence. In S. cerevisiae strains, D. hansenii genes adopt the S. cerevisiae polyadenylation profile, indicating that the polyadenylation pattern is mediated primarily by species-specific factors. Four sequencing lanes containing direct RNA sequence from S. cerevisiae (strains JGY2000 and two replicates of AB1380), K. lactis strain CLIB209, D. hansenii strain NCYC2572, and S. cerevisiae strains JYAC06 and JYAC07, each harboring D. hansenii sequences on a YAC. AB1380 is the non-YAC-containing S. cerevisiae parental strain for JYAC06 and JYAC07.
2013-07-03 | E-GEOD-47661 | biostudies-arrayexpress