Identification of Sfh and H-NS binding sites in the Salmonella Typhimurium genome
ABSTRACT: The global regulator H-NS represses transcription in gram negative bacteria. Sfh is a homologue of H-NS and is encoded by plasmid pSfR27. Sfh provides a 'stealth' function that allows pSfR27 to be transmitted to a new host without disrupting the competitive fitness of the new host We used ChIP-on-chip to profile Sfh (3xFLAG-tagged) and H-NS binding sites in Salmonella Typhimurium strain SL1344 and found that Sfh provides its 'stealth' function by targeting a sub-set of H-NS bound genes that display reduced levels of H-NS occupancy with the SL1344 chromosome upon acquisition of plasmid pSfR27 Identification of Sfh binding sites in strains SL1344 (pSfR27) and SL1344 hns (pSfR27) as well as identification of H-NS binding sites in strains SL1344, SL1344 (pSfR27), and SL1344 (pSfR27) sfh
Project description:We performed Chromatin Immunoprecipitation (ChIP) and microarray hybridization analysis of CspC binding in Salmonella Typhimurium strain SL1344 which has been genetically engineered to express a 3xFLAG tagged CspC protein. Chromatin samples were prepared from SL1344 CspC 3xFLAG grown to exponential phase (OD600nm = 0.2). CspC FLAG ChIP and mock normal mouse IgG ChIP reactions were carried out. The purified ChIP DNA samples were hybridized to SL1344 tiling microarrays.
Project description:The LysR family transcription factor LeuO is believed to antagonize the global repressor H-NS. ChIP-on-chip analysis of LeuO, H-NS and RNAP binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between LeuO and H-NS regulated genes. Furthermore LeuO binding was associated with RNA polymerase recruitment, indicating a role for LeuO in activating transcription. Analysis of LeuO, H-NS and RNA Polymerase binding in Low-phosphate media (LPM)
Project description:ChIP-on-chip analysis of RNAP and RpoD binding to the Salmonella enterica serovar Typhimurium chromosome demonstrated a high degree of overlap between RNAP and RpoD binding and provided us with important insights into the global distribution of these factors. Furthermore this data was correlated with information on the location of 1873 transcription start sites identified by RNA-Seq technology, thereby providing a detailed transcriptional map of Salmonella Typhimurium. Analysis of RNAP, RNAP-Rifampicin and and RpoD binding in Luria Broth (LB)
Project description:OmpR is a DNA binding protein belonging to the OmpR/EnvZ two component system. This system is known to sense changes in osmolarity in Escherichia coli. Recently, OmpR in Salmonella enterica serovar Typhimurium was found to be activated by acidic pH and DNA relaxation. In this study, ChIP-on-chip was employed to ascertain the genome-wide distribution of OmpR in Salmonella Typhimurium and Escherichia coli in acidic and neutral pH. In addition we investigated the affect of DNA relaxation on OmpR binding in Salmonella Typhimurium. Analysis of OmpR binding at pH 7 and pH 4.5 in E-minimal medium in both SL1344 and CSH50. Three independent biological replicates at each pH 7 and pH 4.5 was performed. This was done in each strain background. DNA was immunopreciptated using an anti-FLAG anitbody which binds to flag-tagged OmpR in both strains. Two control ‘mock’ experiments were performed under the same culture condtions in each strain background; DNA was immunopreciptated using normal mouse IgG antibody. To analyse the effect of DNA relaxation onOmpR binding SL1344 was maintained in the exponential growth phase in LB broth with and without treatment with the drug novobiocin (25 μg/ml) for 40 min. This was peformed on two independent occasions. In all cases the experimental immunoprecipitated DNA was hybridised against the input DNA.
Project description:Mixed Lineage Leukemia (MLL) and its metazoan Trithorax orthologs have been linked with the epigenetic maintenance of transcriptional activity. To identify mechanisms by which MLL perpetuates active transcription in dividing cells, we investigated its role during M-phase of the cell cycle. Unlike other chromatin modifying enzymes examined, we found that MLL associates with gene promoters packaged within condensed mitotic chromosomes. Genome-wide location analysis identified a globally rearranged pattern of MLL occupancy during mitosis in a manner favoring genes that were highly transcribed during interphase. Knockdown experiments revealed that MLL retention at gene promoters during mitosis accelerates transcription reactivation following mitotic exit. MLL tethers Menin, RbBP5, and ASH2L to its occupied sites during mitosis, but is dispensable for preserving histone H3K4 methylation. These findings implicate mitotic bookmarking as a component of Trithorax-based gene regulation which may facilitate inheritance of active gene expression states during cell division. anti-MLL ChIP (antibody 456) and anti-pol2 chip (sc-899) in chromatin prepared from interphase and mitotic HeLa cells
Project description:The global regulator H-NS represses transcription in gram negative bacteria. Sfh is a homologue of H-NS and is encoded by plasmid pSfR27. Sfh provides a 'stealth' function that allows pSfR27 to be transmitted to a new host without disrupting the competitive fitness of the new host We used ChIP-on-chip to profile Sfh (3xFLAG-tagged) and H-NS binding sites in Salmonella Typhimurium strain SL1344 and found that Sfh provides its 'stealth' function by targeting a sub-set of H-NS bound genes that display reduced levels of H-NS occupancy with the SL1344 chromosome upon acquisition of plasmid pSfR27 Overall design: Identification of Sfh binding sites in strains SL1344 (pSfR27) and SL1344 hns (pSfR27) as well as identification of H-NS binding sites in strains SL1344, SL1344 (pSfR27), and SL1344 (pSfR27) sfh
Project description:Despite the prevalence of antisense transcripts in bacterial transcriptomes, little is known about how their synthesis is controlled. We report that a major function of the Escherichia coli termination factor Rho and its co-factor NusG is suppression of ubiquitous antisense transcription genome-wide. Rho binds C-rich unstructured nascent RNA (high C/G ratio) prior to its ATP-dependent dissociation of transcription complexes. NusG is required for efficient termination at minority subsets (~20%) of both antisense and sense Rho-dependent terminators with lower C/G ratio sequences. In contrast, a widely studied nusA deletion proposed to compromise Rho-dependent termination had no effect on antisense or sense Rho-dependent terminators in vivo. Global co-localization of the nucleoid-associated protein H-NS with Rho-dependent terminators and genetic interactions between hns and rho suggest that H-NS aids Rho in suppression of antisense transcription. The combined actions of Rho, NusG, and H-NS appear to be analogous to the Sen1-Nrd1-Nab3 and nucleosome systems that suppress antisense transcription in eukaryotes. Chromatin immunoprecipitation (ChIP) experiments were performed using antibodies against RNA polymerase (RNAP; Beta subunit) in wild-type cells or cells deleted for hns, nusG, or a partial deletion of nusA. Differentially labeled ChIP DNA and genomic DNA were competitively hybridized to an E. coli K-12 MG1655 tiling array with overlapping probes at ~12bp spacing across the entire genome. The series contains 12 datasets.
Project description:Transcriptional profiling of Salmonella Typhimurium strains SL1344 (pSfR27) and SL1344 (pSfR27) delta sfh to identify 'Sfh-dependent' transcripts Two condition experiment in which the transcriptomes of wild-type SL1344 (pSfR27) and SL1344 (pSfR27) delta sfh were compared to identify 'Sfh-dependent' transcripts
Project description:Among the most important regulators of gene expression in bacteria are 'nucleoid-associated proteins'. These proteins alter the topology of the bound DNA by bending, wrapping or bridging it, thus having multiple effects, including transcriptional regulation, on the bacterial cell. Among the best-studied nucleoid proteins are H-NS and Fis, which bind to specific sequences on the DNA. H-NS is a global repressor of gene expression. Fis alters the global conformation of the DNA by introducing branched structures in it; but its effect on gene expression on a genomic scale remains largely unclear.<br><br>Several bacterial transcriptional regulators including H-NS and Fis have been studied using ChIP-chip. However, the higher resolution and dynamic range offered by ChIP-Seq have not been exploited for any bacterial species. By performing ChIP-Seq of these two proteins, we present the first such study in a bacterium. In addition to providing a proof-of-principle for the use of this technology for bacteria, we perform our study at multiple time-points during growth in rich medium, thus generating new insights into how these proteins function under different cellular conditions. Further, by analysing our data in conjunction with newly-generated gene expression and RNA polymerase-chromosome interaction data we provide new interpretation of the genome-scale patterns of the interactions of these proteins to the DNA.