Combined RNA-seq and ChIP-seq Identifies Gene Targets for Transcriptional Regulation in Vasopressin-Sensitive Collecting Duct Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Pablo Sandoval
PROVIDER: E-GEOD-79584 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:In mammals, the peptide hormone vasopressin controls renal water excretion, largely through its actions in the renal collecting duct to regulate the molecular water channel aquaporin-2 (AQP2). There are two modes of regulation: 1) short-term regulation by membrane trafficking; and 2) long-term regulation involving vasopressin-induced changes in the abundance of the aquaporin-2 protein. Defects in the long-term regulation have been implicated in multiple water balance disorders. With the objetive to identify genes that are transcriptionally regulated in response to vasopressin in cultured mouse collecting duct cells (mpkCCD), we carried out both RNA-seq to measure all transcripts (n=9) and ChIP-seq for RNA polymerase II (Polr2a) binding along the genome (n=3). Observations were made both after 24-hr treatment with the vasopressin analog dDAVP and with vehicle. Protocol:Confluent monolayers mpkCCD cells were washed in ice-cold 1X PBS (Ca+2/Mg+2-free) followed by cross-linking in 1.11% formaldehyde in Covaris Fixing Buffer (5.5 ml) for 5min at room temperature with gentle rocking (PN 520075, Covaris, Woburn, Massachusetts). Cross-linking was terminated by the addition of 0.3 ml of 1X Covaris Quenching Buffer with rocking. The buffer solution was aspirated and 1X ice-cold PBS was added to each plate. Cells were scraped into 15ml conical tubes, spun down (200 x g, 5 min), and washed twice with PBS. After the second wash, cell pellets were resuspended in 10 ml of 1X Covaris Lysis Buffer for 10 min at 4o C with rocking. Nuclei were pelleted by spinning at 1,700 x g for 5 min at 4o C. Nuclear pellets was resuspended in the 1X Covaris Wash Buffer and incubated for 10 mins at 4o C with rocking. Samples were spun at 1,700 x g for 5 min at 4o C. Nuclear pellets were washed twice with the Covaris Non-ionic Shearing Buffer and spun at 1,700 x g for 5 min at 4o C. Nuclei were resuspended in Covaris Non-ionic Shearing Buffer (Maximum of 1-3x 10^7cells per ml of buffer). Shearing. Two 20 ul aliquots of the resuspended pellets were removed prior to shearing to be used as subsequent unsheared controls for immunoblotting and agarose gel electrophoresis. The remaining samples were transferred to AFA tubes (TC12 x 12 mm) and volumes adjusted to 1 ml (V)). Samples were sheared using a Covaris S2 SonoLAB Single system for two minutes (Duty cycle: 5%; Intensity: 4; cycle: 200). Total nuclear protein concentration of each sample in mcg/ml (R) was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). To test shearing efficiency, a 20 ul aliquot from each tube was removed after shearing to test the efficacy of shearing. The aliquots, including the unsheared sample, were incubated with 100 ul 1X TE buffer, 10 ul 10% SDS, and RNAse A (10 ug/ul, Cell Signaling Technology, Danvers, Massachusetts) for 30 mins at 37o C. 1 ul proteinase K (20 ug/ul) was added and the samples were incubated overnight at 65o C. The DNA was purified using DNA Clean & Concentrator columns (Catalog #: D4013, Zymo Research, Irvine, CA) and ran on an E-gel EX 2% agarose (Invitrogen, Carlsbad, CA). Target fragment sizes for chromatin immunoprecipitation were in the range of 150-700 bp in length with average fragment size of 300 bp. Testing epitope integrity (immunoblotting). Total protein content was determined with the BCA Protein Assay Kit (Thermo Scientific, Rockford, IL). Samples were then diluted with sample buffer (5X Laemmli buffer) at 20% (v/v) of the entire sample volume. Proteins were resolved by SDS-PAGE (4-20% polyacrylamide gels, Criterion, Bio-Rad, Hercules, CA) and transferred electrophoretically onto nitrocellulose membranes as previously described (Hwang, 2010). Membranes were probed with anti-RNA polymerase II (Catalog #: MMS-126R, Covance). The antibody was used at a 1:500 dilution (anti-RNA polymerase II) (in Odyssey blocking buffer containing 0.1% Tween 20) overnight at 4°C. After 1-h incubation with secondary antibody (Li-Cor Biosciences 680 anti-rabbit immunoglobulin G or 800 anti-mouse immunoglobulin G; Lincoln, NE) at 1:5000 dilution, sites of antibody-antigen reaction were detected using an Odyssey infrared imager (Li-Cor). Chromatin immunoprecipitation. 10 ul of sample equivalent to 2% input was removed prior to IP. Chromatin Immunoprecipitation was performed using 30-90 ug of sheared chromatin in 500 uL of 1X Covaris Non-ionic Shearing Buffer with 10 μl anti-RNA polymerase II antibodies. Samples were incubated overnight at 4°C with rotation, followed by addition of ChIP Grade Protein G Magnet Beads (Catalog #: 9006, Cell Signaling Technology) and incubation for 2 h at 4o C with rotation. The beads were washed a total of four times, including three low salt washes followed by one high salt wash for 5 mins/wash at 4o C (SimpleChIP protocol #9003, Cell Signaling Technology). Immunoprecipitated chromatin was eluted from the beads with 150 ul of 1X ChIP Elution Buffer for 30 mins at 65o C in a thermomixer. To reverse crosslinks, 6 ul of 5M NaCl and 2 ul of 20 ug/ul Proteinase K were added to the chromatin followed by incubation at 65o C overnight. DNA was purified according to SimpleChIP protocol #9003, Cell Signaling Technology. q-PCR. To check the success of the chromatin immunoprecipitation, input and ChIPâ??ed DNA samples were amplified with SYBR Green PCR Master Mix (Applied Biosystems) on a 7900HT Fast Real-Time PCR System. Primer sets corresponding to Hox9a and actin genes were used as positive controls for both histone and RNA polymerase II IPâ??s. (see Supplementary Table X for primer sequences). Library construction, amplification and sequencing. Loaded fixed volume of total eluate. Library construction was performed on a NuGEN instrument using the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit. Libraries were then enriched via PCR. After amplification, DNA was measured. A fixed amount of the total measured DNA was used for sequencing in an Illumina Hiseq2000 instrument to obtain single read data. High-throughput sequencing and read mapping. Libraries for Illumina sequencing of the samples were prepared using the the protocol and reagents outlined in the Ovation SP Ultralow Library Systems kit , NuGen Mondrian SP+ (NuGEN, San Carlos, CA) . Samples were sequenced using an Illumina HiSeq2000, obtaining 1- by 50-bp reads. We combine RNA-seq and ChIP-seq to identify genes whose mRNA abundances change in tandem with changes in RNA polymerase II binding, as a mean to identify genes that are transcriptionally regulated in response to vasopressin. In addition, the pattern of RNA polymerase II binding along the gene provides important information about regulatory mechanisms. We identify two populations of genes regulated by vasopressin in mpkCCD cells, viz. 1) those with increases in transcript abundance and increases in RNA polymerase II binding throughout the gene body and; 2) genes that show increases in RNA polymerase II binding in the promotor proximal region but do not produce increases in transcript level. The former group is restricted to 35 genes and includes Aqp2. The latter group is made up of the majority of expressed genes and is presumably due to a widespread increase in transcriptional initiation without proportionate release of pausing.
2016-07-01 | E-GEOD-79573 | biostudies-arrayexpress
Project description:We sequenced mRNAs and mapping the binding of RNA polymerase 2 in collecting duct cells treat with Vasopressin or vehicle. Confluent mpkCCD cell monolayers were maintained in serum-free medium for 24 h prior to treatment with or without 0.1 nM dDAVP in serum-free medium for 24 h. followed RNA polymerase 2 Chip seq.
2016-07-01 | E-GEOD-79525 | biostudies-arrayexpress
Project description:Vasopressin is the major hormone that regulates renal water excretion. It does so by binding to a receptor in renal collecting duct cells, triggering signaling pathways that ultimately regulate the abundance, location, and activity of the water channel protein aquaporin 2. We took an advantage of quantitative large scale proteomic technologies and oligonucleotide microarrays to quantify steady state changes in protein and transcript abundances in response to vasopressin in a collecting duct cell line, mpkCCD clone 11 (Yu et al. PNAS 2009, 106:2441-2446). This cell line originally developed by Alan Vandewalle’s group recapitulates vasopressin-mediated AQP2 expression and phosphorylation as seen in native colleting duct cells. The mpkCCD cells were grown on membrane supports to permit polarization. Once transepithelial resistance reached 5kohm per centimeter square and higher, the cells were exposed to the vasopressin V2 receptor analog, dDAVP, at a physiological concentration, 0.1nM, for 5 days. Control experiments were done with cells exposed to vehicle alone. Total RNA was harvested and processed for transcript expression analysis using Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. Each experimental treatment, vehicle and dDAVP, was repeated 3 times.
2010-12-09 | E-GEOD-19938 | biostudies-arrayexpress
Project description:Protein Kinase A (PKA) is a widely studied protein that has been viewed by most investigators as a single entity, although its catalytic subunits are coded in the genome by two separate genes, PKA catalytic alpha (Gene symbol: Prkaca) and PKA catalytic beta Gene symbol: Prkacb). At an amino-acid level, the two are 91.5 percent identical and the catalytic domains are virtually identical. (Footnote: A third entity PKA catalytic gamma, is not widely expressed and will not be considered here.) We have recently succeeded in using CRISPR-Cas9 to create disruptive mutations in both PKA genes (PKA double KO, or PKA dKO) in vasopressin-responsive kidney epithelial cells (mpkCCD cells). Here we carry out mass spectrometry based quantitative proteomics and phosphoproteomics separately in PKA catalytic-alpha and PKA catalytic-beta single knockouts address the issue of function difference between these two PKA catalytic subunits.
2020-06-01 | PXD015050 | Pride
Project description:The mouse mpkCCD cell line is a continuous cultured epithelial cell line with characteristics of renal collecting duct principal cells. This line is widely used to study epithelial transport and its regulation. To provide a data resource useful for experimental design and interpretation in studies using mpkCCD cells, we have carried out 'deep' proteomic profiling of these cells using three levels of fractionation (differential centrifugation, SDS-PAGE, HPLC) followed by tandem mass spectrometry to identify and quantify proteins. The use of multiple levels of fractionation increases the ability to detect low abundance proteins. As a result, we identified 6766 proteins in mpkCCD cells at a high level of stringency. These proteins are expressed over 7 orders of magnitude of protein abundance. The data are provided to users as a public data base at https://helixweb.nih.gov/ESBL/Database/mpkFractions/. The MS data were mapped back to gel slices to generate 'virtual western blots' for each protein. For most of the 6766 proteins, the apparent molecular weight from SDS-PAGE agreed closely with the calculated molecular weight. However, a substantial fraction (>15%) of proteins were found to run aberrantly, either with much higher or much lower than predicted. These proteins were analyzed to identify mechanisms responsible for altered mobility on SDS-PAGE (high or low isoelectric point, high or low hydrophobicity, physiological cleavage, residence in the lysosome, post-translational modifications and expression of alternative isoforms due to alternative exon usage). Additionally, this analysis identified a previously unrecognized isoform of aquaporin-2 with apparent molecular weight <20 kD.
2015-10-06 | PXD002434 | Pride
Project description:The distal convoluted tubule (DCT) and the cortical collecting ducts (CCD) are portions of renal tubule that are partly responsible for maintaining the systemic concentrations of potassium, sodium, calcium and magnesium. Despite being structurally similar, DCT and CCD cells have different transport capabilities due to a variety of different membrane-associated transport proteins. However, DCT and CCD cells appear to be modulated via the same hormones. The objective of this study was assess the differential response of DCT and CCD cells to long-term exposure to the hormones vasopressin or angiotensin II, both of which modulate DCT and CCD cells differently. Mass spectrometry based quantitative proteomics was used to profile the differential proteome between DCT and CCD.
2017-12-15 | PXD005454 | Pride
Project description:The regeneration of the airway mucociliary epithelium involves several sequential events including migration, proliferation, polarization and final differentiation (i.e ciliogenesis). The airway mucociliary epithelium is consituted of three main cell types : ciliated cells, secretory cells and basal cells. We used microRNA microrrays to investigate the signature of microRNA during the four step of regeneration of the airway epithelium. Four time points (ALI-D0, ALI-D7, ALI-D14, ALI-D21) of regeneration of the airway epithelium for 3 donors.
2011-06-06 | E-GEOD-22144 | biostudies-arrayexpress
Project description:Human airway smooth muscle cells were co-cultured with BEAS-2B epithelial cells (or Control). Airway smooth muscle RNA was extracted and sent for Illumina HT-12 micro-array to examine gene expression.
2020-04-30 | E-MTAB-9004 | biostudies-arrayexpress
Project description:Vasopressin/cAMP/protein kinase A (PKA)/aquaporin-2 (AQP2) water channels signaling in the kidneys is a canonical pathway that determines AQP2 activity in response to body fluid balance. AQP2 phosphorylation by PKA increases water reabsorption from urine for the prevention of further water loss. We used several activators of cAMP/PKA signaling as screening tools to generate various phosphorylation patterns of PKA substrates and AQP2 in a mouse cortical collecting duct principal cell line (mpkCCDcl4). We next quantified phosphorylation levels of PKA substrates and AQP2 after western blot analysis using a phospho-PKA substrates antibody. Finally, we screen for PKA substrates whose phosphorylation levels were well correlated with those of AQP2. Immunoprecipitation with a phospho-PKA substrates antibody followed by mass spectrometry analysis revealed that the leading candidate in this assay proved to be a lipopolysaccharide-responsive and beige-like anchor protein (LRBA). Phosphorylation levels of LRBA was nearly perfectly correlated with those of AQP2.
2022-07-23 | PXD033852 | Pride
Project description:Caco-2 cells (a human gastrointestinal epithelial cell line) grown to form confluent cell layers on permeable membrane supports were treated with variously (A) iron depleted medium, (B) iron depleted medium with 10 uM ferric nitrilotiracetate added to the apical medium or (C) iron depleted medium with 30 uM human holo-transferrin added to the basolateral medium for 7 days (days 14-21 after cell seeding). RNA was extract from the cells on day 21 for transcription profiling by array.
2015-04-27 | E-MTAB-3442 | biostudies-arrayexpress