ChIP-chip analysis of CspC binding in Salmonella enterica serovar Typhimurium strain SL1344
ABSTRACT: 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 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: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: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: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: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:We performed transcriptome abundance analysis of Salmonella Typhimurium strain SL1344 swap which has been genetically engineered to express the hns open-reading frame from the stpA promoter and the stpA open reading frame from the hns promoter. This strain is designated SL1344(swap). Transcript abundance was compared with that of wild-type SL1344. This comparison was performed to determine the effect of chromosome location of the expression of two related global regulators and how alterations to their expression patterns would impact on their regulons. Three independent RNA samples were harvested from wild-type SL1344 and SL1344(swap) grown to exponential phase (OD600nm = 0.3) and hybridized to a microarray containing multiple probes for each of the SL1344 open reading frames. Please note that the original raw data file for the 'SL1344_wildtype_BR2' sample is unavailable, however, the 'wildtype SL1344 BR2.gpr' contains the raw data missing only the header information.
Project description:Genetic loss-of-function studies in development, cancer and somatic cell reprogramming suggested that the group of macroH2A histone variants could function through stabilizing the differentiated state by a yet unknown mechanism. Here, we now present results demonstrating that macroH2A variants have a major function in maintaining nuclear organization and heterochromatin architecture. Specifically, we find that a substantial amount of macroH2A is associated with heterochromatic repeat sequences in an H3K9me3-dependent manner. We further identify macroH2A on sites of interstitial heterochromatin. Loss of macroH2A leads to major defects in nuclear organization including loss of nuclear circularity, disaggregation of nucleoli and a global loss of dense heterochromatin. Domains formed by repeat sequences when depleted of macroH2A are disorganized, expanded and fragmented, and partially re-expressed. This can be explained by our finding that macroH2A tethers these repeats to the nucleostructural protein Lamin B1. ChIPseq to determine the distribution of histone variants macroH2A1 and macroH2A2 were realized in the HepG2 cell line
Project description:DNA Double Strands Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the role of senataxin, a RNA:DNA helicase involved in the regulation of transcription and the maintenance of genome integrity, at sites of DNA Double Strand Breaks. We performed ChIP-Seq mapping of senataxin before and after damage, genome-wide DNA:RNA hybrids (DRIP) mapping before and after damage. We also performed RNA-Seq and RNA pol II mapping (total and phosphorylated on serine 2 of the CTD) by ChIP-Seq in undamaged cells to discriminate between damage in active versus inactive regions.
Project description:SepH was identified having a potential role in sporulation following ChIP-seq analysis that showed the sepH gene is under the control of the WhiA and WhiB transcription factors (Bush et al., 2013; Bush et al., 2016). Since SepH has a helix-turn-helix motif, this ChIP-Seq experiment was carried out to determine whether SepH specifically binds DNA and if so, where.