Gene expression profiling of Eed knockout and Ezh2 knockout small intestinal crypts [ChIP-seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Martijn Koppens
PROVIDER: E-GEOD-81577 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Small Cell Lung Cancer (SCLC) is the most aggressive type of lung cancer with early metastatic dissemination and invariable development of resistant disease for which no effective treatment is available to date. Mouse models of SCLC based on inactivation of Rb1 and Trp53 developed earlier showed frequent amplifications of two transcription factor genes: Nfib and Mycl. Overexpression of Nfib but not Mycl in SCLC mouse results in an enhanced and altered metastatic profile, and appears to be associated with genomic instability. NFIB promotes tumor heterogeneity with the concomitant expansive growth of poorly differentiated, highly proliferative, and invasive tumor cell populations. Consistent with the mouse data, NFIB expression in high-grade human neuroendocrine carcinomas correlates with advanced stage III/IV disease warranting its further assessment as a potentially valuable progression marker in a clinical setting. Genomic DNA from mouse small cell lung tumor samples was analyzed by mate pair sequencing and low coverage sequencing. And RNA from Nfib overexpressing mouse small cell lung cancer cell lines was further analyzed for high quality RNA profiles using Illumina Hiseq2500. This series contains only RNA-seq data.
2016-07-29 | E-GEOD-82005 | biostudies-arrayexpress
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 have completed the high quality reference genome for domestic sheep (Oar v3.1). Early-stage Illumina GA sequence platform sequenced less reads in high GC content regions than in other regions. To read through higher GC content regions, we generated 2 Gb MeDIP-seq data for filling gaps in sheep reference genome assembly.
2014-06-06 | E-GEOD-56644 | biostudies-arrayexpress
Project description:In this study we performed MeRIP-Seq to study N6-methyl adenosine (m6A) and and N6,2′ -O-dimethyladenosine (m6Am) modification of mRNA. We investigated the effect of the microbiota on the transcriptome and epitranscriptomic modifications in murine liver and cecum. We compared m6A/m modification profiles in cecum of conventionally raised (CONV) and germ-free (GF) mice. We additionally included GF mice colonised with the flora of CONV mice for four weeks (ex-GF), for which show that they exhibit similar patterns of the most abundant genera of gut bacteria as CONV mice. We added mice treated with several antibiotics to deplete the gut flora (abx)and vancomycin treated mice in which the genera Akkermansia, Escherichia/Shigella and Lactobacillus were enriched. Furthermore, we included GF mice colonised with the commensal bacterium Akkermansia muciniphila (Am), Lactobacillus plantarum (Lp) and Escherichia coli Nissle (Ec) and analysed their m6A/m modification profiles. In addition, we analysed changes in m6A/m- modified liver RNA for CONV, GF, and Am, Lp and Ec mice.
2020-02-12 | E-MTAB-6560 | biostudies-arrayexpress
Project description:Synthetic DNA-binding proteins have found broad application in gene therapies and as tools for interrogating biology. Engineered proteins based on the CRISPR/Cas9 and TALE systems have been used to alter genomic DNA sequences, control transcription of endogenous genes, and modify epigenetic states. Although the activity of these proteins at their intended genomic target sites have been assessed, the genome-wide effects of their action have not been extensively characterized. Additionally, the role of chromatin structure in determining the binding of CRISPR/Cas9 and TALE proteins to their target sites and the regulation of nearby genes is poorly understood. Characterization of the activity these proteins using modern high-throughput genomic methods would provide valuable insight into the specificity and off-target effects of CRISPR- and TALE-based genome engineering tools. We have analyzed the genome-wide effects of TALE- and CRISPR-based transcriptional activators targeted to the promoters of two different endogenous human genes in HEK293T cells using a variety of high-throughput DNA sequencing methods. In particular, we assayed the DNA-binding specificity of these proteins and their effects on the epigenome. DNA-binding specificity was evaluated by ChIP-seq and RNA-seq was used to measure the specificity of these activators in perturbing the transcriptome. Additionally, DNase-seq was used to identify the chromatin state at target sites of the synthetic transcriptional activators and the genome-wide chromatin remodeling that occurs as a result of their action. Our results show that these genome engineering technologies are highly specific in both binding to their promoter target sites and inducing expression of downstream genes when multiple activators bind to a single promoter. Moreover, we show that these synthetic activators are able to induce the expression of silent genes in heterochromatic regions of the genome by opening regions of closed chromatin and decreasing DNA methylation. Interestingly, the transcriptional activation domain was not necessary for DNA-binding or chromatin remodeling in these regions, but was critical to inducing gene expression. This study shows that these CRISPR- and TALE-based transcriptional activators are exceptionally specific. Although we detected limited binding of off-target sites in the genome and changes to genome structure, these off-target event did not lead to any detectable changes in gene regulation. Collectively, these results underscore the potential for these technologies to make precise changes to gene expression for gene and cell therapies or fundamental studies of gene function. HEK293T cells were transfected in triplicate with plasmids expressing synthetic transcription factors. The synthetic TFs were either (a) dCas9-VP64 fusion protein and a targeting guide RNA (gRNA), or (b) a TALE-VP64 fusion protein engineered to bind to a specific target site in the genome. As a control, cells were transfected with plasmids expressing GFP. After transfection, RNA-seq was used to identify both on-target and off-target binding sites for the synthetic TFs. The data in this submission were generated using the TALE transfection experiments.
2015-05-27 | E-GEOD-68341 | biostudies-arrayexpress
Project description:To test the hypothesis that defects in the termination of inflammatory signaling led to skin inflammation that results in the “Itchy” phenotype, we isolated RNA from the lesional skin of Itch-/- mice and from the skin of wild-type mice and performed genome-wide mRNA expression profiling by RNA sequencing. We ranked genes based on the fold change in their expression (increased or decreased) in Itch-/- skin relative to that in wild-type skin. The expression of several TNF–induced genes were increased in Itch-/- skin, including, IL-1?, IL-6, IL-11, IL-19, IL-1RL1, CCL4, CXCL3, CXCL2, CCL3, and CD14. mRNA profiles comparison between wild type (WT) skin and Itch-/- mice lesional skin
2015-03-04 | E-GEOD-65686 | biostudies-arrayexpress
Project description:Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling. Despite the success of recently approved therapies targeting AR signaling, such as abiraterone and second-generation anti-androgens MDV3100 (enzalutamide), durable responses are limited, presumably due to acquired resistance. Recently, JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit antiproliferative effects in a range of malignancies. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ1. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer. Examination of ASH2L genome-wide binding in prostate cancer cells after AR stimulation.
2015-03-02 | E-GEOD-60841 | biostudies-arrayexpress
Project description:Men who develop metastatic castration-resistant prostate cancer (CRPC) invariably succumb to the disease. The development and progression to CRPC following androgen ablation therapy is predominantly driven by unregulated androgen receptor (AR) signaling1-3. Despite the success of recently approved therapies targeting AR signaling such as abiraterone4-6 and second generation anti-androgens MDV3100 (enzalutamide)7,8, durable responses are limited, presumably due to acquired resistance. Recently JQ1 and I-BET, two selective small molecule inhibitors that target the amino-terminal bromodomains of BRD4, have been shown to exhibit antiproliferative effects in a range of malignancies9-12. Here we show that AR signaling-competent CRPC cell lines are preferentially sensitive to BET bromodomain inhibition. BRD4 physically interacts with the N-terminal domain of AR and can be disrupted by JQ111,13. Like the direct AR antagonist, MDV3100, JQ1 disrupted AR recruitment to target gene loci. In contrast to MDV3100, JQ1 functions downstream of AR, and more potently abrogated BRD4 localization to AR target loci and AR mediated gene transcription including induction of TMPRSS2-ERG and its oncogenic activity. In vivo, BET bromodomain inhibition was more efficacious than direct AR antagonism in CRPC xenograft models. Taken together, these studies provide a novel epigenetic approach for the concerted blockade of oncogenic drivers in advanced prostate cancer. Examination of AR, BRD2, BRD3, BRD4, ERG, RNA Pol II and H3K27ac in prostate cancer cells with respect to BET inhibitors
2014-04-25 | E-GEOD-55062 | biostudies-arrayexpress
Project description:We use a spatial transcriptomic technique (TOMO-seq) to build a 3D map of gene expression patterns in the mouse olfactory mucosa, which can be used to start addressing many unanswered questions, about olfaction and the importance of gene expression zones in the olfactory mucosa related to it, and also beyond olfaction (eg, mechanisms of formation of spatial patterns of gene expression)
2022-02-17 | E-MTAB-10211 | biostudies-arrayexpress
Project description:DNA Double-Strand Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the tridimensional genome organization around DSBs and its role in DSB repair foci formation. We performed Hi-C experiments before and after DSB induction and upon ctrl or SCC1 depletion, 4C-seq experiments before and after DSB induction and upon cohesin (SCC1) depletion or ATM inhibition. We also performed ChIP-seq of pATM(S1981), CTCF, P-SMC3(S1083), MDC1 and a calibrated ChIP-seq of SCC1 with or without damage. ChIP-chip of SMC3 (S1083) and SMC1 (S966) were used to show the recruitment of these marks on DNA repair foci. ChIP-chip of gammaH2AX was realized upon SCC1 depletion and ChIP-seq of gammaH2AX was realized upon SCC1 or WAPL to show the role of the cohesin complex in gammaH2AX foci formation. ChIP-seq of gammaH2AX was realized upon ATM or ATR inhibition to show that ATM is the major kinase that phosphorylates H2AX at clean breaks in human cells.
2021-01-29 | E-MTAB-8851 | biostudies-arrayexpress