Project description:We present here a transcriptional regulator, PA3898, which controls biofilm formation and multidrug efflux-pumps in P. aeruginosa. A mutant of this regulator significantly reduced the ability of P. aeruginosa to produce biofilm in vitro, and affected its in vivo fitness and pathogenesis in Drosophila melanogaster and BALB/c mouse lung infection models. Transcriptome analysis revealed that PA3898 modulates essential virulence genes/pathways, including multidrug efflux-pumps and phenazine biosynthesis.ChIP-seq identified its DNA binding sequences and confirmed PA3898 directly interacts with promoter regions of four genes/operons, two of which are mexAB-oprM and phz2.
Project description:We performed ChIP-seq analyses of RhlR to map the C4-homoserine lactone-dependent and PqsE-dependent RhlR binding sites in the P. aeruginosa genome.
Project description:Estrogen Receptor alpha (ERα) is a key driver of most breast cancers, and it is the target of endocrine therapies used in the clinic to treat women with ERα positive (ER+) breast cancer. The two methods ChIP-seq (chromatin immunoprecipitation coupled with deep sequencing) and RIME (Rapid Immunoprecipitation of Endogenous Proteins) have greatly improved our understanding of ERα function during breast cancer progression and in response to anti-estrogens. A critical component of both ChIP-seq and RIME protocols is the antibody that is used to pull down the bait protein. To date, most of the ChIP-seq and RIME experiments for the study of ERα have been performed using the sc-543 antibody from Santa Cruz Biotechnology. However, this antibody has been discontinued, thereby severely impacting the study of ERα in normal physiology as well as diseases such as breast cancer and ovarian cancer. Here, we compare the sc-543 antibody with other commercially available antibodies, and we show that 06-935 (EMD Millipore) and ab3575 (Abcam) antibodies can successfully replace the sc-543 antibody for ChIP-seq and RIME experiments.
Project description:The Pseudomonas aeruginosa quorum-sensing (QS) systems contribute to bacterial homeostasis and pathogenicity. Although many regulators have been characterized to control the production of virulence factors and QS signaling molecules, its detailed regulatory mechanisms still remain elusive. Here, we performed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) on 10 key QS regulators. The direct regulation of these genes by corresponding regulator has been confirmed by Electrophoretic mobility shift assays (EMSAs) and quantitative real-time polymerase chain reactions (qRT-PCR). Binding motifs are found by using MEME suite and verified by footprint assays in vitro. Collectively, this work provides new cues to better understand the detailed regulatory networks of QS systems. ChIP-seq of 10 QS regulators in Pseudomonas aeruginosa
Project description:Pseudomonas aeruginosa PA3973 encodes a putative TetR family transcriptional regulator, with a helix-turn-helix motif involved in DNA binding. We applied phenotype analyses, as well as transcriptome profiling (RNA-seq), and genome-wide identification of binding sites using ChIP-seq to unravel the biological role of PA3973. The ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome, among them 139 were located in intergenic regions. The 13 bp sequence was identified as the preferential binding site of PA3973. The PA3973 regulon encompasses genes involved in stress response, including the putative PA3973-PA3970 operon. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3973 showed changes in the mRNA level of 648 genes; among them, 374 were down-regulated. Concomitantly, ChIP-seq analysis identified more than 300 PA3973 binding sites in the P. aeruginosa genome, among them 139 were located in intergenic regions. The 13 bp sequence was identified as the preferential binding site of PA3973.
Project description:we performed ChIP-seq assays to identify in vivo targets of GltR. The plasmid mini-gltR-flag-lacz was constructed and the resultant plasmid was fused to P. aeruginosa strain PAO1, yielding PAO1/mini-gltR-flag-lacz. We investigated GltR-binding to the chromosome of PAO1 during growth with glucose by ChIP-Seq. Sequence reads obtained from three independent ChIP-Seq experiments using anti-flag antibody and mapped to the P. aeruginosa PAO1 genome.Using the MACS software,we identified 55 enriched loci (q-value < 0.05) harboring GltR-binding peaks, that were enriched > 3-fold, but were absent in control sample conducted without anti-flag antibody.