Directional RNA sequencing of mRNA from Sinorhizobium meliloti cells grown with limiting phosphate and with excess phosphate.
Ontology highlight
ABSTRACT: Analysis of the RNA-seq reads confirmed that transcription of many of the previously identified Pho-regulon genes of Sinorhizobium meliloti was regulated by the availability of inorganic phosphate in the media. The transcriptional start sites of many of the Pho-regulon genes as determined by RNA-seq reads were found to correspond to those determined from primer extension analysis. mRNA from S. meliloti RmP110 grown in MOPS + 2 mM Pi and MOPS + 20 M-BM-5M Pi M-bM-^@M-^S directional RNA-seq via Illumina sequencing
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.
Project description:Cadmium is toxic and intricate pathways linked to metallothioneins (MT) drive the detoxification process. Whist the mechanisms are well understood in mammalian systems, detailed knowledge is still elusive in invertebrates (which notably differ to mammalian systems). The model nematode Caenorhabditis elegans is ideally suited for assessing metallothionein mediates detoxification in invertebrates as is relatively short-lived, can be easily exposed and its genome is fully sequenced and widely annotated. The aim of the experiment was to identify new MT mediated target genes involved in Cd toxicosis. The global transcriptome was compared in wild type and a MT double knockout strain raised in the presence or absence of 30 µM Cd.
Project description:The sense of hearing depends on the faithful transmission of sound information from the ear to the brain by spiral ganglion (SG) neurons. However, how SG neurons develop the connections and properties that underlie auditory processing is largely unknown. We catalogued gene expression in mouse SG neurons at six developmental stages, ranging from embryonic day 12 (E12), when SG neurons first extend projections, up until postnatal day 15 (P15), after the onset of hearing. For comparison, we also analyzed the closely-related vestibular ganglion (VG) at the same time points. To identify genes involved in SG axon guidance and branching, target selection, synaptogenesis, synaptic refinement, and synaptic function, we collected SG at E12 and E13, E16, P0, P6, and P15. We also collected VG at the same time points. For E12 and E13 time points, SG and VG were microdissected from Rnx-cre; Z/EG embryos, which express GFP in the VG. E16-P15 VG was also isolated by microdissection from Rnx-cre; Z/EG animals. E16-P15 SG neurons were isolated by FACS sorting dissociated cochlea from Mafb-GFP animals.
Project description:Despite correlations between histone methyltransferase (HMT) activity and gene regulation, direct evidence that HMT activity is responsible for gene activation is sparse. We address the role of the HMT activity for MLL1, a histone H3 lysine 4 (H3K4) methyltransferase critical for maintaining hematopoietic stem cells (HSCs). Here we show that the SET domain and thus HMT activity of MLL1 is dispensable for maintaining HSCs and for supporting leukemogenesis driven by the MLL-AF9 fusion oncoprotein. Upon Mll1 deletion, histone H4 lysine 16 (H4K16) acetylation was selectively depleted at MLL1 target genes in conjunction with reduced transcription. Surprisingly, inhibition of SIRT1 was sufficient to prevent the loss of H4K16 acetylation and the reduction in MLL1 target gene expression. Thus, recruited MOF activity, and not the intrinsic HMT activity of MLL1, is central for the maintenance of HSC target genes. In addition, this work reveals a role for SIRT1 in opposing MLL1 function. 11 Samples, 5 controls and 5 KOs with antibodies H3K4me1, H3K4me3, and H3K27Ac. One input Sample.
Project description:Background: The overproduction of the main auxin indole-3-acetic acid (IAA) in Sinorhizobium meliloti RD64 leads to beneficial effects both in free-living rhizobium and in host plant during symbiosis. These effects could be related to alteration in gene expression induced by IAA. Preliminary studies of these alterations induced in free-living cells have already started. In this work the transcriptional profiles of free-living S. meliloti wild type 1021 and RD64 strains obtained by RNA-seq analysis were used to better investigate this issue. Results: Transcriptome analysis by next-generation sequencing (RNA-seq) allowed the identification of the genes differentially expressed in RD64 cells, grown in a rich medium under aerobic conditions. Most of the up-regulated genes can be classified into two main categories. The first class comprised the main regulator fixJ and the two intermediate regulators fixK and nifA, which control the expression of genes involved in nitrogen-fixation. For these two latter genes the highest expression levels were observed. Several other genes, known to be FixJ targets, such as those involved in denitrification process and proline metabolism, were also identified. The second class of up-regulated genes comprised the gene coding for the sigma factor RpoH1 and other genes regulated in RpoH1-dependent manner in S. meliloti and involved in stress response. The main category of genes negatively affected can be classified in transport of sugars and other small molecules, chemotaxis and cell envelope functions. Under microaerobic condition, quantitative real-time PCR analysis of genes coding for the two-component regulatory system FixJL and for the regulator NifA showed that for all three genes the induction levels measured in RD64 cells were even higher when compared to aerobic conditions. In particular, for the fixJ gene the expression level increased more than tenfold. The up-regulation of nifA gene was also observed under aerobic conditions both when nitrogen level in the medium was limiting and when it was high. Conclusion: This work provided evidence that the overexpression of the auxin IAA in S. meliloti free-living cells triggers the induction of many genes involved in bacterial response to different abiotic stresses and allows the expression of key genes involved in nitrogen fixation. mRNA profiles of S. meliloti wild type 1021 and its derivative IAA-overproducing RD64 strain were generated in triplicate by using HiSeq2000 sequencer
Project description:Argonaute proteins lie at the heart of the RNA-Induced Silencing Complex (RISC), wherein they use small RNA guides to recognize targets. Some Argonaute proteins can directly cleave their targets, whereas others recruit co-factors to repress independently of “slicing.” Prior studies established the architecture of Argonaute proteins; however, we have not yet had a detailed picture of an Argonaute whose biochemical and biological functions were well established. Here we describe the crystal structure of human Argonaute-2 bound to miR-20a at 2.2 Å resolution. Overall architecture and domain organization is shared with its prokaryotic counterparts, though Ago2 is somewhat more open, with its PAZ domain further removed from the other domains. The miRNA is anchored at both ends by the Mid and PAZ domains but makes several kinks and turns along the binding groove. The RNA confers remarkable stability on the Argonaute protein, locking this otherwise flexible enzyme into a stable conformation. total small RNAs (19-29nt) and small RNAs associated with purified hArgonaute2 purified from SF9 cells. These were processed and sequenced on Illumina GA-II platform.
Project description:Polycomb group (PcG) proteins maintain the silenced state of key developmental genes, but how these proteins are recruited to specific regions of the genome is still not completely understood. In Drosophila, PcG proteins are recruited to Polycomb response elements (PREs) comprised of a flexible array of sites for sequence-specific DNA binding proteins, “PcG recruiters”, including Pho, Spps, Cg, GAF and many others. Pho is thought to play a central role in PcG recruitment. Early data showed that mutation of Pho binding sites in PREs in transgenes abrogated the ability of those PREs to repress gene expression. In contrast, genome-wide experiments in pho mutants or by Pho knockdown showed that PcG proteins can bind to PREs in the absence of Pho. Here we directly addressed the importance of Pho binding sites in two engrailed (en) PREs at the endogenous locus and in transgenes. Our results show that Pho binding sites are required for PRE activity in transgenes with a single PRE. In a transgene, two PREs together lead to stronger, more stable repression and confer some resistance to the loss of Pho binding sites. Making the same mutation in Pho binding sites has little effect on PcG-protein binding at the endogenous en gene. Overall, our data support the model that Pho is important for PcG binding but emphasize how multiple PREs and chromatin environment increase the ability of PREs to function in the absence of Pho.
Project description:Two samples from a larger study of the effect of Botrytis cinerea infection on gene expression in Arabidopsis thaliana. These two samples also form part of an investigation of the sequence dependancy of DNA and RNA fragmentation within ChIP-seq and RNA-seq experiments Two technical replicates from the 24 time point of a time series
Project description:Inorganic phosphate is an essential nutrient required by organisms for growth. During phosphate starvation, Saccharomyces cerevisiae activates the phosphate signal transduction (PHO) pathway leading to the expression of the secreted acid phosphatase, PHO5. The fission yeast, Schizosaccharomyces pombe, regulates expression of the ScPHO5 homolog (pho1+) via a non-orthologous PHO pathway. The genes induced by phosphate limitation and the molecular mechanism by which the genetically identified positive (pho7+) and negative (csk1+) regulators function are not known. Here we use a combination of molecular biology, expression microarrays, and chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to characterize the role of pho7+ and csk1+ in the PHO response. We define the set of genes that comprise the initial response to phosphate starvation in S. pombe. We identify a conserved PHO response for the ScPHO5 (pho1+), ScPHO84 (spbc8e4.01c+), and ScGIT1 (spbc1271.09+) orthologs. We use ChIP-Seq to identify members of the Pho7 regulon and characterize Pho7 binding in response to phosphate-limitation and Csk1 activity. We demonstrate that activation of pho1+ requires Pho7 binding to a UAS in the pho1+ promoter and that Csk1 repression does not regulate Pho7 enrichment. Further, we find that Pho7-dependent activation is not limited to phosphate-starvation, as additional environmental stress response pathways require pho7+ for maximal induction. We provide a global analysis of the PHO pathway in S. pombe. Our results elucidate the conserved core regulon required for responding to phosphate starvation between distantly related ascomycetes and a better understanding of flexibility in environmental stress response networks. ChIP Sequencing of the Schizosaccharomyces pombe 972h- transcription factor Pho7-TAP in high-Pi, no-Pi, and csk1M-NM-^T conditions