A genome-wide analysis of PhoP~P binding to chromosomal DNA reveals several novel features of the PhoPR-mediated phosphate limitation response in Bacillus subtilis
ABSTRACT: Identification of the specific PhoP binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the PhoPR regulon in Bacillus subtilis and its role in adaptation to phosphate-limiting conditions and cell wall metabolism, as well as implying a role in several other cellular processes. For each sample analyzed in this study three biological replicates were performed. Three different samples were taken from a strain expressing the PhoP-SPA protein as well as from wild-type (168) without a tagged PhoP. Samples were taken from exponentially growing cells in low phosphate medium (LPDM) as well as from phosphate-limited cells (T2). Each sample compares ChIP DNA vs. Total DNA from the same cells.
Project description:Identification of the specific WalR (YycF) binding regions on the B. subtilis chromosome during exponential and phosphate starvation growth phases. The data serves to extend the WalRK regulon in Bacillus subtilis and its role in cell wall metabolism, as well as implying a role in several other cellular processes. For each sample analyzed in this study three biological replicates were performed. Three different samples were taken from a strain expressing the WalR-SPA protein as well as from wild-type (168) without a tagged WalR. Samples were taken from exponentially growing cells in low phosphate medium (LPDM) as well as from phosphate-limited cells (T2). Each sample compares ChIP DNA vs. Total DNA from the same cells.
Project description:S. coelicolor was grown in low-phosphate concentration R5 liquid medium which facilitated the transition to a phosphate starvation induced stationary phase and the subsequent induction of the PhoP regulon. The genome wide binding locations of PhoP during phosphate starvation were determined by chromatin-immunopreciptiation followed by hybridisation to high density S. coelicolor DNA microarrays. The use of both wild-type S. coelicolor and phoP-null mutant strains allowed the identification of specific PhoP binding locations.
Project description:S. coelicolor was grown in low-phosphate concentration R5 liquid medium which facilitated the transition to a phosphate starvation induced stationary phase and the subsequent induction of the PhoP regulon. Global gene expression profiling of a wild-type and a phoP-null mutant was performed using samples isolated, respectively, at 24, 30, 35 and 45 h of growth. RNA was extracted and the resulting cDNA was hybridised to S. coelicolor high density DNA microarrays.
Project description:The human liver contains multiple cell types whose epigenetic patterns are undetermined. We examined the promoter methylome of purified and uncultured hepatic stellate cells (HSCs), hepatocytes (HEPs) and liver sinusoidal endothelial cells (LSECs), by methylated DNA immunoprecipitation (MeDIP) and array hybridization. Uncultured HSCs, LSECs and Heps show ~7000-8000 methylated promoters, with 60-70% similarity between all cell types. GO analysis for commonly methylated genes reveals involvement in germ cell development, segregating germ-line from somatic lineage methylation. HSCs, LSECs and HEPs also contain ~500-1000 uniquely methylated promoters; these are implicated in signaling and biosynthetic processes (HSCs), lipid transport and metabolism (LSECs), and chromatin assembly (HEPs). The promoter methylome of culture-activated HSCs deviates from that of their uncultured (quiescent) counterparts. HSC culture-induced activation also enhances methylation differences between individual donors; however this does not necessarily relate to changes in gene expression. HSc activation results in a net gain of promoter DNA methylation, despite the demethylation and de novo methylation of thousands of promoters. Our data provide to our knowledge the first genome-wide maps of promoter DNA methylation in human purified and uncultured liver cell types. Although methylation profiles are largely similar between HSCs, LSECs and hepatocytes, the detection of cell type-specific methylation patterns suggests a differential epigenetic programming of these cell types in the liver. Determine the promoter DNA methylation pattern of 3 uncultured, reshly isolated, human healthy liver cell types (hepatocytes (HEPs), liver sinusoidal endothelial cells (LSECs) and haptic stellate cells (HSCs), and of HSCs after a 24-h culture-induced activation.
Project description:The PhoPR two-component system is essential for virulence in Mycobacterium tuberculosis where it controls expression of approximately 2% of the genes, including those for the ESX-1 secretion apparatus, a major virulence determinant. Mutations in phoP lead to compromised production of pathogen-specific cell wall components and attenuation both ex vivo and in vivo. Using antibodies against the native protein in ChIP-seq experiments (chromatin immunoprecipitation followed by high-throughput sequencing) we demonstrated that PhoP binds to at least 35 loci on the M. tuberculosis genome. The PhoP regulon comprises several transcriptional regulators as well as genes for polyketide synthases and PE/PPE proteins. Integration of ChIP-seq results with high-resolution transcriptomic analysis (RNA-seq) revealed that PhoP controls 30 genes directly, whilst regulatory cascades are responsible for signal amplification and downstream effects. The most prominent site of PhoP regulation was located in the intergenic region between rv2395 and PE_PGRS41, where the mcr7 gene codes for a small non-coding RNA (ncRNA). Northern blot experiments confirmed the absence of Mcr7 in the M. tuberculosis phoP mutant as well as low-level expression of the ncRNA in M. tuberculosis complex members other than M. tuberculosis. By means of genetic and proteomic analyses we demonstrated that Mcr7 modulates translation of the tatC mRNA thereby impacting the activity of the Twin Arginine Translocation (Tat) protein secretion apparatus. As a result, secretion of the immunodominant Ag85 complex and the beta-lactamase BlaC is affected, among others. Mcr7, the first ncRNA of M. tuberculosis whose function has been established, therefore represents a missing link between the PhoPR two-component system and the downstream functions necessary for successful infection of the host. RNA-Seq: M. tuberculosis H37Rv (GC1237) cultures grown in vitro to exponential phase. One wild type sample plus one isogenic phoP mutant sample
Project description:We have employed short-capped RNA sequencing (sc-RNA-seq) in order to identify genes whose expression is regulated by promoter proximal pausing of RNA Polymerase II (RNAPI) in response to stress stimulation. We used serum-deprived mouse Swiss 3T3 fibroblasts, either untreated (control) or treated with anisomycin to induce the p38/MAP kinase pathway. Serum starved (72 h 0.2% FCS) mouse 3T3 cells were treated with anisomycin (188.5 nM) for 1 h (in duplicates). Untreated, serum-starved cells were used as a control. We isolated nuclear RNA, performed size fractionation followed by isolation of short-capped RNAs (scRNA). scRNAs were subsequently converted into DNA library and sequenced.
Project description:We have generated single-nucleotide resolution, nascent transcription profiles from HeLa cells by developing Native Elongation Transcript sequencing technology for mammalian chromatin (mNET-seq). Our extensive data sets provide a substantial resource to study mammalian nascent transcript profiles. We reveal unanticipated phosphorylation states for RNA polymerase II C-terminal domain (Pol II CTD) at both gene ends. We also observe that following 5’ splice site cleavage by the spliceosome, upstream exon transcripts are tethered to Pol II CTD phosphorylated on the serine 5 position (S5P) which is accumulated over downstream exons. We further show that depletion of termination factors substantially reduces Pol II pausing at gene ends leading to termination defects. Remarkably termination factors play an additional promoter role by restricting non-productive RNA synthesis and redistributing Pol II CTD S2P to promoters. These data demonstrate that CTD phosphorylation is more dynamic and variably distributed across mammalian transcription units than previously envisaged. To monitor nascent RNA within the mammalian Pol II complex, and its association with different CTD phosphorylation states, we employed mNET-seq methodology on HeLa cells, complemented with direct sequencing of chromatin-bound RNA (ChrRNA-seq). mNET-seq was preformed using the antibodies 8WG16, CMA602, CMA603 and CMA601, which are specific for unphosphorylated CTD, Ser2 phosphorylation, Ser5 phosphorylation and all CTD isoforms, respectively. In another experiment, to evaluate the effect of transcription termination factors in nascent RNA production by Pol II, mNET-seq and complemented with ChrRNA-seq was preformed on HeLa cells transfected with siRNA against PTBP1, CPSF73, CstF64+CstF64tau or Xrn2, and the gene profiles were compared with profiles from HeLa transfected with siRNA for Luciferase generated by the same protocol.
Project description:Microarrays were used to identify Y. pestis genes that are differentially regulated under conditions of phoP overexpression. RNA samples were isolated from cultures of two isogenic Y. pestis strains, one overexpressing phoP (KIM10+phoP(delta)/PhoP) and the other not (KIM10+phoP(delta)/MMB67EH), and their gene expression profiles were compared.
Project description:Bacillus subtilis responds to phosphate starvation stress by inducing the PhoP and SigB regulons. While the PhoP regulon provides a specific response to phosphate starvation stress, maximizing the acquisition of phosphate (Pi) from the environment and reducing the cellular requirement for this essential nutrient, the SigB regulon provide non-specific resistance to stress by protecting essential cellular components such as DNA and membranes. We have characterized the phosphate starvation stress response of B. subtilis at a genome-wide level using DNA macroarrays. A combination of outlier and cluster analyses identified putatively new members of the PhoP regulon, namely yfkN (2',3'-cyclic-nucleotide 2'-phosphodiesterase), yurI (ribonuclease), yjdB (unknown) and vpr (extracellular serine protease). YurI is thought to be responsible for the non-specific degradation of RNA, whilst the activity of YfkN on various nucleotide phosphates suggests that it could act on substrates liberated by YurI which produces 3` or 5` phosphoribonucleotides. The putative new PhoP regulon members are either known or predicted to be secreted and are likely to be important for the recovery of inorganic phosphate from a variety of organic sources of phosphate in the environment. Keywords: other
Project description:We used ChIP-chip analysis to identify where the nucleoid associated protein StpA binds on the Salmonella Typhimurium genome. We identified 285 chromosomal regions bound by StpA, ranging in size from 400 to 3500 base pairs and containing 572 genes. Comparison of the ChIP data revealed that StpA displayed an identical binding profile to H-NS; all genomic regions associated with StpA were also bound by H-NS. Of the 88 StpA-dependent genes that are de-repressed in the absence of StpA, only 25 were bound by StpA, suggesting that StpA generally regulates gene expression indirectly. To identify the direct StpA targets, we co-immunoprecipitated DNA from a strain expressing a 3xFLAG-tagged StpA-protein using an anti-3xFLAG antibody. As a negative control, we used the same antibody (anti-3xFLAG) to co-immunoprecipitate DNA from an isogenic strain expressing the non-tagged StpA-protein. Three independent biological replicates were generated. For comparative purposes we also performed the co-immunoprecipitation on wild type Salmonella using a monoclonal anti-H-NS antibody. In that case, the negative control was obtained by performing the co-immunoprecipitation protocol on wild type Salmonella without adding antibody.