ChIP-Seq of the Sinorhizobium meliloti CRP-like protein Clr in combination with either cAMP or cGMP under different growth conditions
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ABSTRACT:
PROVIDER: PRJEB53364 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR9823490_1.fastq.gz | Fastqsanger.gz | |||
| ERR9823490_2.fastq.gz | Fastqsanger.gz | |||
| ERR9823491_1.fastq.gz | Fastqsanger.gz | |||
| ERR9823491_2.fastq.gz | Fastqsanger.gz | |||
| ERR9823492_1.fastq.gz | Fastqsanger.gz |
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Project description:In bacteria, Crp-Fnr superfamily transcription factors mediate 3',5'-cyclic adenosine monophosphate (cAMP) and 3',5'-cyclic guanosine monophosphate (cGMP) signaling. The CRP-like protein Clr of the soil-dwelling and plant-symbiotic α-proteobacterium Sinorhizobium meliloti was previously shown to activate target promoters in both its cAMP- and cGMP-bound states (Krol et al., Microbiology 62:1840–1856, 2016). In order to further characterize the overall regulon of Clr in S. meliloti Chromatin Immuno Precipitation DNA-Sequencing (ChIP-Seq) experiments were performed with C-terminally FLAG tagged Clr under four different growth conditions, namely growth in TY complex medium and MOPS minimal medium, each supplemented with either cAMP or cGMP. For each condition, the respective immunoprecipitated (IP) and non-immunoprecipitated (control) samples were analyzed and compared to locate genomic positions in which Clr-DNA binding occurs. In combining ChIP-Seq with Electrophoretic Mobility Shift Assays and promoter-probe assays we expanded the list of known Clr-regulated target promoters and showed that virtually all of these promoters containing a palindromic Clr binding site (CBS) motif are activated both by Clr•cAMP and Clr•cGMP.
2023-03-23 | E-MTAB-11788 | biostudies-arrayexpress
Project description:Fungal degradation of lignocellulosic biomass requires various (hemi-)cellulases and plays key roles in biological carbon cycle. Although cellulases induction recently described in some saprobic filamentous fungi, regulation of cellulase transcription has not been studied thoroughly. Here, we identified and characterized the novel cellulase regulation factors clr-4 in Neurospora crassa and its ortholog Mtclr-4 in Myceliophthora thermophila. Deletion of clr-4 and Mtclr-4 displayed similarly defective phenotypes in cellulolytic enzymes production and activities. Transcriptomics analysis of Δclr-4/ΔMtclr-4 revealed down-regulation of not only encoding genes of (hemi-)cellulases and pivotal regulators (clr-1, clr-2 and xyr-1), but also the key genes of cAMP signaling pathway such as adenylate cyclase cr-1. Consistently, the significant decreased levels of intracellular cAMP were observed in Δclr-4/ΔMtclr-4 compared to wild-type during cellulose utilization. Electrophoretic mobility shift assays (EMSA) verified that CLR-4 could directly bind to the promoter regions of adenylyl cyclase (Nccr-1) and cellulose regulator clr-1, while MtCLR-4 bind to upstream regions of adenylyl cyclase Mtcr-1 and biomass deconstruction regulators Mtclr-2 and Mtxyr-1. Concluded, the novel cellulase expression regulators (CLR-4/MtCLR-4) findings here significantly enrich our understanding of the regulatory network of cellulose degradation and provide new targets for industrial fungi strain engineering for plant biomass deconstruction in biorefinery.
2021-03-18 | GSE111986 | GEO
Project description:There is growing appreciation that the feeling of well-being, alterations in mood and susceptibility to a variety of medical disorders depend on the proper expression of the master circadian clock and the synchrony among the other oscillators found in many peripheral tissues. To improve our understanding of the role of peripheral oscillators, an improved understanding of the molecular machinery sub-serving the circadian variation in gene expression is essential. Our long-term goal is to understand the molecular mechanisms that initiate circadian gene expression following external stimulation in the mammalian pineal gland. Are all or just some of the "circadian clock" genes induced by NE, cAMP, or cGMP? In this project we compare a series of gene expression patterns using DNA microarray in response to cAMP, cGMP and NE stimulation to understand why NE does not initiate circadian rhythms in the rat pineal gland. As a preliminary experiment we will compare gene expression 0, 1, and 4 h after start of chemical stimulation. A failure of NE to initiate circadian rhythms is due to failure of activation of certain "circadian clock" genes. We found that circadian rhythms are initiated by stimulation of cAMP or cGMP analogue in the rat pineal gland, while norepinephrine (NE) stimulation only moderately induce Period1 mRNA (one of "circadian clock" genes) 24 h after start of stimulation. From these results we hypothesize that external stimulation activates some "circadian clock" genes simultaneously, and that failure of circadian rhythm initiation following NE stimulation is due to insufficient "circadian clock" genes activations. Male rats of wistar strain are kept in 12h-12h light dark cycles at least for one week before start of experiments. Pineal glands are removed from animals and placed in the culture dish. Rat pineal glands are cultured for 3 days before chemical stimulation. On the day of experiment, the pineal cultures are stimulated using one of NE, cAMP and cGMP analogues for 1 or 4 h before harvest. The samples are immediately frozen and total RNA are extracted from each group which are from a pool of 8 pineal glands using Trizol reagent (Invitrogen). Concentrations of total RNA are determined using spectrophotometer, and six microgram of total RNA is aliquoted in each sample tube for further analysis. Since we already have Affymetrix Rat Genome U34A array GeneChips, we would like to send Chips as well as our total RNA samples. Experiment Overall Design: as above
2008-06-12 | E-GEOD-2865 | biostudies-arrayexpress
Project description:cAMP receptor protein (CRP, also known as the catabolite activator protein [CAP]) is arguably the best-studied of the global transcription factors of E coli. CRP alone is responsible for regulating at least 283 operons. Upon binding cAMP, the CRP dimer binds DNA and directly interacts with RNA polymerase (RNAP). At Class II promoters, CRP binds near position -41,5 relative to the transcription start site and contacts the amino-terminal domain of the RNAP α subunit (RNAPα-NTD). This interaction requires AR2, a patch of primarily positively charged residues (H19, H21, E96, and K101) that interact with negatively charged residues on RNAPα-NTD. Acetylome analyses consistently detect lysine 100 (K100) of CRP as acetylated. Since K100 is adjacent to the positively charged AR2, we hypothesized that the K100 positive charge may also play a role in CRP function. We further hypothesized that acetylation of K100 would neutralize this positive charge, leading to a potential regulatory mechanism
2017-03-24 | GSE96955 | GEO
Project description:We observed the expression profile of the total mRNA in crp (TTHA1437) deletion mutant strain of Thermus thermophilus HB8 during infection of bacteriophage ϕYS40. Keywords: time course, bacteriophage, infection, CRP, cAMP receptor protein, deletion mutant
2009-11-03 | GSE16955 | GEO
Project description:The C-type lectin-related protein, Clr-f, encoded by Clec2h in the mouse NK gene complex (NKC), is a member of a family of immune regulatory lectins that guide immune responses at distinct tissues of the body. Clr-f is highly expressed in the kidney; however, its activity in this organ is unknown. To assess the requirement for Clr-f in kidney health and function, we generated a Clr-f-deficient mouse (Clr-f−/−) by targeted deletions in the Clec2h gene. Mice lacking Clr-f exhibited glomerular and tubular lesions, immunoglobulin and C3 complement protein renal deposits, and significant abdominal and ectopic lipid accumulation. Whole kidney transcriptional profile analysis of Clr-f−/− mice at 7, 13, and 24 weeks of age revealed a dynamic dysregulation in lipid metabolic processes, stress responses, and inflammatory mediators. Examination of the immune contribution to the pathologies of Clr-f−/− mouse kidneys identified elevated IL-12 and IFNγ in cells of the tubulointerstitium, and an infiltrating population of neutrophils and T and B lymphocytes. The presence of these insults in a Rag1−/−Clr-f−/− background reveals that Clr-f−/− mice are susceptible to a T and B lymphocyte-independent renal pathogenesis. Our data reveal a role for Clr-f in the maintenance of kidney immune and metabolic homeostasis.
2022-03-19 | GSE198814 | GEO
Project description:ChIP-seq analysis of the cAMP binding protein (CRP) in Enteroaggregative Escherichia coli 042
| PRJEB84381 | ENA
Project description:In bacteria, the most prevalent receptor proteins of 3',5'-cyclic AMP (cAMP) and 3',5'-cyclic GMP (cGMP) are found among transcription factors of the Crp-Fnr superfamily. The prototypic Escherichia coli catabolite activator protein (CAP) represents the main Crp cluster of this superfamily and is known to bind cAMP and cGMP but to mediate transcription activation only in its cAMP-bound state. In contrast, both cyclic nucleotides mediate transcription activation by Sinorhizobium meliloti Clr, mapping to cluster G of Crp-like proteins. We present crystal structures of Clr-cAMP and Clr-cGMP bound to the core motif of the palindromic Clr DNA binding site (CBS). We show that both cyclic nucleotides shift ternary Clr-cNMP-CBS-DNA complexes (where cNMP is cyclic nucleotide monophosphate) to almost identical active conformations, unlike the situation known for the E. coli CAP-cNMP complex. Isothermal titration calorimetry measured similar affinities of cAMP and cGMP binding to Clr in the presence of CBS core motif DNA (equilibrium dissociation constant for cNMP (KDcNMP], ~7 to 11 μM). However, different affinities were determined in the absence of this DNA (KDcGMP, ~24 μM; KDcAMP, ~6 μM). Sequencing of Clr-coimmunoprecipitated DNA as well as electrophoretic mobility shift and promoter-probe assays expanded the list of experimentally proven Clr-regulated promoters and CBS. This comprehensive set of CBS features conserved nucleobases that are consistent with the sequence readout through interactions of Clr amino acid residues with these nucleobases, as revealed by the Clr-cNMP-CBS-DNA crystal structures. IMPORTANCE Cyclic 3',5'-AMP (cAMP) and cyclic 3',5'-GMP (cGMP) are both long known as important nucleotide secondary messengers in eukaryotes. This is also the case for cAMP in prokaryotes, whereas a signaling role for cGMP in this domain of life has been recognized only recently. Catabolite repressor proteins (CRPs) are the most ubiquitous bacterial cAMP receptor proteins. Escherichia coli CAP, the prototypic transcription regulator of the main Crp cluster, binds both cyclic mononucleotides, but only the CAP-cAMP complex promotes transcription activation. In contrast, Crp cluster G proteins studied so far are activated by cGMP or by both cAMP and cGMP. Here, we report a structural analysis of the cAMP- and cGMP-activatable cluster G member Clr from Sinorhizobium meliloti, how binding of cAMP and cGMP shifts Clr to its active conformation, and the structural basis of its DNA binding site specificity.
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Project description:Alpha-rhizobia are soil-dwelling alphaproteobacteria existing either in free-living states or in symbiosis with a leguminous plant host. cyaJ is a putative adenylate/guanylate cyclase transmembrane protein. Overexpression of cyaJ leads to increased > levels of cAMP. To investigate the effect of increased cAMP on the transcriptome of Sinorhizobium meliloti we performed microarray analysis of > Sinorhizobium meliloti Rm2011 carrying an empty vector pWBT or cyaJ overexpression construct pWBT-cyaJ.
2016-08-17 | E-MTAB-4932 | biostudies-arrayexpress