Chip-seq to identify σE dependent genes in EtOH stressed and unstressed Streptomyces coelicolor A3(2)
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ABSTRACT: Chip-seq experiment used to identify the binding sites of alternative σ factor σE in Streptomyces coelicolor. We access the binding sites with and without EtOH stress condition (see methods). To capture the binding of σE, the σE gene was tagged on genome by HA (Human Influenza hemagglutinin derived) epitope (TAC CCA TAC GAC GTC CCA GAC TAC GCT) on its C-terminus, wild type strain without tagged σE served as negative control.
Project description:Chip-seq to identify the HrdB (SCO5820) binding sites and regulated genes in Streptomyces coelicolor. The HrdB gene was tagged on genome by HA (Human Influenza hemagglutinin derived) epitope (TAC CCG TAC GAT GTG CCG GAT TAC GCG). We have 3 replicates with HrdB tagged by HA and 2 replicates with wild-type strain as controls. Chip-seq experiment was conducted using antibody against HA tag in vegetative growth phase (see protocols).
Project description:Plant-beneficial fungi from the genus Trichoderma (Hypocreales, Ascomycota) can control oomyceteous plant-pathogenic Pythium myriotylum (Peronosporales, Oomycota) and thus serve as bioeffectors for the eco-friendly products of crop protection. However, the underlying mechanisms of microbe-microbe interactions have yet to be fully understood. In this study, we focused on the role of the Trichoderma secretome induced by P. myriotylum mycelia. For this purpose, we selected strains showing strong (T. asperellum, T. atroviride, T. virens), moderate (T. cf. guizhouense, T. reesei), and weak (T. parepimyces) activities, respectively, and cultured with the sterilized P. myriotylum mycelia. Secreted proteins were analyzed using label-free LC-MS/MS, bioinformatic localization prediction, gene ontology (GO) annotation, and ortholog analysis. The exoproteomic analysis quantified proteins in the six Trichoderma spp., suggesting unequal antagonistic mechanisms among the strong and weak strains, respectively, with different proportions of putative cellulases, proteases, redox enzymes, and extracellular proteins of unknown function. Notably, proteolysis-related proteins were abundant, while the abundant proteases tended not to be conserved across the species (i.e., non-orthologous). Putative cellobiohydrolases were detected abundantly in all Trichoderma species except for the weak antagonist T. parepimyces, even though its genome encodes for these proteins. Notably, secretomes of the most potent anti-Pythium bioeffectors tended to have higher endo-cellulase activity. Cellulose and other glucans are major components of the oomycete cell wall, which was partly reflected in the cellulases produced by the Trichoderma species. The varying abundances of orthologous proteins suggested the evolution of differing transcription regulation mechanisms across the Trichoderma genus in response to the ubiquitous presence of Oomycota.
Project description:Firstly our study demonstrated that TSA can inhibits cell proliferation, induces cell apoptosis and cell cycle arrest in CCA cell lines in vitro. To identify the target transcripts of TSA to suppress CCA tumorigenesis, mRNA expression profiles were determined by microarray analysis. We chose TFK-1 cells treated by TSA in indicated concentration (IC50) 48 hours for the microarray. Then we found out TACC3 was downregulated, and demonstrated that TACC3 was high expression and may played a role as an target of TSA in inhibiting CCA cells proliferation and migration via in vitro and vivo experiments. We chose TFK-1 cells treated by TSA in indicated concentration (IC50) 48 hours as the experiment sample and without TSA as the control sample to do this microarray analysis for three times. Then regulated genes would be tested to investigate the potential roles in CCA tumorigenesis,
Project description:Histone modifications affect DNA-templated processes ranging from transcription to genomic replication. In this study, we examine the cell cycle dynamics of the trimethylated form of histone H3 lysine 4 (H3K4me3), a mark of active chromatin that is viewed as â??long-livedâ?? [1], and that is involved in memory during cell state inheritance in metazoans [2]. We synchronized yeast using two different protocols, then followed H3K4me3 patterns as yeast passed through subsequent cell cycles. While most H3K4me3 patterns were conserved from one generation to the next, we found that methylation patterns induced by alpha factor or high temperature were erased within one cell cycle, during S phase. Early-replicating regions were erased before late-replicating regions, implicating replication in H3K4me3 loss. However, incomplete H3K4me3 erasure occurred at the majority of loci even when replication was prevented, suggesting that most erasure results from an active process. Indeed, deletion of the demethylase Jhd2 slowed erasure at most loci. Together, these results indicate overlapping roles for passive dilution and active enzymatic demethylation in erasing ancestral histone methylation states in yeast. References: [1] Ng HH, Robert F, Young RA, Struhl K (2003) Targeted recruitment of Set1 histone methylase by elongating Pol II provides a localized mark and memory of recent transcriptional activity. Mol Cell 11: 709-719. [2] Ringrose L, Paro R (2004) Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu Rev Genet 38: 413-443. The overall design of the experiment consists of two cell cycle experiments, each consisting of the following subsets: gene expression, ChIP with anti-H3K4Me3 antibody, and ChIP input. The experiments are as follows: CCA - BY4741 bar1- cells synchronized by alpha factor arrest; CCTS - BY4741 cdc28(ts) cells synchronized by arrest at non-permissive temperature. The individual time courses are enumerated as follows: CCA gene expression (array title "Synchronized cells, xxx min, cell cycle CCA"), 18 arrays; CCA ChIP for anti-H3K4Me3 (array title "H3K4Me3 ChIP cell cycle CCA, xxx min"), 17 arrays, 1 replicate; CCA ChIP input (array title "ChIP Input cell cycle CCA, xxx min"), 18 arrays, 1 replicate; CCTS gene expression (appears in a different dataset as biological replicate "I"; array title "Synchronized cells, xxx min, biological replicate I"), 18 arrays; CCTS ChIP for anti-H3K4Me3 (array title "H3K4Me3 cell cycle CCTS, xxx min"), 2 technical replicates, 18 arrays per replicate; CCTS ChIP input (array title "ChIP Input cell cycle CCTS, xxx min"), 2 technical replicates, 18 arrays per replicate. Gene expression arrays were run against a reference of unsynchronized cells, ChIP-chip anti-H3K4Me3 samples were run against an IP (of the same epitope) of unsynchronized cells, and ChIP input samples were run against either a pool of all the time points in the time course (CCTS) or against sonicated DNA isolated from unsynchronized cells (CCA). Four additional experiments have been performed. They are referred to as series X in the title. Series 1: anti H3K4me3 CHIP time course of BY4741 bar1 delete cells (wt) synchronized with alpha factor and released in the cell cycle, 9 arrays, 1 replicate. Series 2: anti H3K4me3 CHIP time course of BY4741 bar1 jhd2 delete cells (jhd2 delete) synchronized with alpha factor and released in the cell cycle, 7 arrays, 1 replicate. Series 3: anti H3K4me3 CHIP time course of CYM36 cells (cdc7ts) synchronized with alpha factor and released in the cell cycle at the permissive 24C or at the restrictive 37C temperature, 22 arrays, 2 replicates. Series 4: anti H3K4me3 CHIP time course of BY4741 bar1 delete cells (wt) grown in galactose and synchronized with alpha factor and either released in the cell cycle in glucose media or alpha factor arrested and switched to glucose media, 6 arrays, 1 replicate.
Project description:Lymph node metastasis is one of the main causes for the low survival rate of gastric cancer patients. Exploring key proteins players in the progression of gastric adenocarcinoma (GAC) may provide new prognostic parkers and therapeutic strategies. we applied proteomic analysis to compare tumor tissues from GAC patients with or without lymph node metastasis, and captured unique molecular features of GAC patients with LNM. Analysis of the phosphoproteome provided a snapshot of abnormal phosphorylation signaling pathways and abnormal kinases activities. Furthermore, we found that TNXB and SPON1, two ECM proteins are associated with LNM status in GAC patients. Thus, our study suggests a number of proteins and kinases that has the potential to serve as prognosis markers to predict patient outcome.
Project description:We identified SMYD5 (SET and MYND Domain-Containing Protein 5) physiologically methylates core ribosomal protein L40 at lysine 22 (rpL40K22me3).In mouse GAC model, we observed rpL40K22me3 stoichiometry is saturating in both normal and GAC tissue.
Project description:The GacS/GacA two component regulatory system globally activates the production of secondary metabolites including phenazines in Pseudomonas chlororaphis 30-84. To better understand the regulatory role of the Gac system, we conducted RNA-seq analyses to determine the regulon of the response regulator GacA. Transcriptome analyses identified over 700 genes differentially regulated by GacA. Consistent with our previous findings, phenazine biosynthetic genes were significantly down-regulated in a gacA mutant. The expression levels of phenazine regulatory genes such as phzI, phzR, iopA, iopB, rpoS and pip were also decreased. Moreover, the expression of three none-coding RNAs (ncRNAs) including rsmX, rsmY and rsmZ was significantly decreased by gacA mutation consistent with the presence of GacA binding sites in their promoters. Our results also demonstrated that over-expression of rsmZ from a non-gac regulated promoter resulted in the restoration of AHL and phenazine production as well as the expression of other secondary metabolites in gac mutants. The role of RsmA and RsmE in phenazine production was also investigated. Over-expression of rsmE, but not rsmA, resulted in decreased AHL production and phenazine gene expression in P. chlororaphis. Consistently, a mutation in rsmE bypassed the requirement of GacA in phenazine gene expression. On the contrary, constitutive expression of the phzI/phzR quorum sensing system was not able to rescue phenazine production in the gacA mutant indicating the direct impact of Gac system on the transcript stability of phenazine biosynthetic genes. Together, these results indicate that the Gac system regulates phenazine production at multiple levels and exerts its positive effect on AHL and phenazine biosynthesis via RsmZ and RsmE. A model is proposed to illustrate the GacA regulon in P. chlororaphis 30-84. A total of 6 samples were analyzed in AB medium + 2% casamino acids, Pseudomonas chlororaphis wild type strain (3 replicates); Pseudomonas chlororaphis gacA mutant (3 replicates).
Project description:Genetic abnormalities of cholangiocarcinoma (CCA) have been widely studied; however, epigenomic changes related to cholangiocarcinogenesis have been less well characterized. We have profiled the DNA methylomes of 28 primary CCA and six matched adjacent normal tissues using Infinium’s HumanMethylation27 BeadChips with the aim of identifying gene sets aberrantly epigenetically regulated in CCA. Using a linear model for microarray data we identified 1610 differentially methylated autosomal CpG sites with 809 CpG sites (representing 603 genes) being hypermethylated and 801 CpG sites (representing 712 genes) being hypomethylated in CCA versus adjacent normal (false discovery rate ? 0.05). Gene Ontology and Gene Set Enrichment analyses identified gene sets significantly associated with hypermethylation at linked CpG sites in CCA including homeobox genes and target genes of PRC2, EED, SUZ12 and Histone H3 trimethylation at lysine 27. We confirmed frequent hypermethylation at the homeobox genes HOXA9 and HOXD9 by bisulfite pyrosequencing in a larger cohort of CCA (n = 103). Our findings indicate a key role for hypermethylation of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in CCA. These data have implications for CCA carcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. Methods: Thirty-two primary CCA, 6 matched adjacent normal, 5 CCA cell lines and an immortal biliary cell line were analyzed for DNA methylation profiles using the Infinium HumanMethylation27 BeadChip. Differential methylation was statistically analyzed using LIMMA. Specific functional terms and Gene Set Enrichment Analysis were performed to identify specific function of aberrantly methylated genes. Results: We identified 711 and 590 autosomal CpG sites as being hypermethylated and hypomethylated representing 527 and 521 genes, respectively, in CCA compared to adjacent normal (false discovery rate 0.05). A number of gene sets were significantly associated with hypermethylation at linked CpG sites in CCA including homeobox genes and the target genes of PRC2, EED, SUZ12, and H3K27me3, whereas target genes of NOS and OCT4 were hypomethylated at linked CpG sites in tumors. Frequent aberrant DNA methylation at HOXA9 and HOXD9 was confirmed by bisulfite pyrosequencing in a larger cohort of CCA (n = 107). Conclusions: This is the first report of a DNA methylation profile of multiple CpG sites at genes associated with a stem cell-like phenotype as a common molecular aberration in CCA. These data have implications for CCA carcinogenesis, as well as possible novel treatment options using histone methyltransferase inhibitors. Array-based methylation profiling was performed using the Infinium HumanMethylation27 BeadChip in 28 primary cholangiocarcinomas and six matched adjacent normal tissues. The reproducibility of the Infinium HumanMethylation27 BeadChips was evaluated using five technical replicates of the ovarian cancer cell line PEO1. Differential methylation cutoff was estimated from five technical replicates by bootstrap resampling and set at ?? ? 0.15 corresponding to a FDR ? 0.05.
Project description:To test if CDK8 acts directly at HIF1A target genes, we performed ChIP-seq experiments in HCT116 cells under normoxic and hypoxic conditions. ChIP-seq for CDK8 versus Input under normoxia and 24hrs hypoxia (1% O2).
Project description:Strep A are human-exclusive bacterial pathogens killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved main surface carbohydrate, the Group A Carbohydrate (GAC). GAC contains a rhamnose polysaccharide (RhaPS) backbone, which is a validated universal vaccine candidate. RhaPS vaccine candidate production is currently limited to chemical routes. We engineered the GAC synthesis route to enable the industry standard recombinant production route to couple RhaPS to selected carrier proteins. The integrity of the correct chemical structure of the glycoconjugate vaccines was confirmed by NMR spectroscopy and mass spectrometry. Purified IdeS-RhaPS and NanA-RhaPS produce carbohydrate specific antibodies in mice and rabbits confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates. We provide a promising route to the production of tailor-made low-cost glycoconjugate vaccines against Strep A and other bacterial pathogens.