Development of GRO-cap (GRO-seq followed by 5'-cap enrichment) to map transcription start sites in C. elegans
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ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
SUBMITTER: Barbara Meyer
PROVIDER: E-GEOD-43085 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series
2013-06-18 | E-GEOD-43087 | biostudies-arrayexpress
Project description:Dosage Compensation is required to correct for uneven gene dose between the sexes. We utilized global run-on sequencing (GRO-seq) to examine how Caenorhabditis elegans dosage compensation reduces transcription of X-linked genes. To facilitate these experiments, we required accurate 5M-bM-^@M-^Y-ends of genes that have been missing due to a co-transcriptional trans-splicing event common in nematodes. We developed a modified GRO-seq protocol to identify TSSs that are supported by transcription, and determined that TSSs lie more than 1 kb upstream of the previously annotated TSS for nearly one-quarter of all genes. We then investigated the changes that occur in transcriptionally engaged RNA Polymerase when dosage compensation is disrupted, and find that dosage compensation controls recruitment of RNA Polymerase to X-linked genes. GRO-seq experiments (two biological replicates) were performed in nuclei from many wild-type states and a dosage compensation mutant
2013-06-18 | E-GEOD-43084 | biostudies-arrayexpress
Project description:Production of mRNA depends critically on the rate of RNA polymerase II (Pol II) elongation. To dissect Pol II dynamics in mouse ES cells, we inhibited Pol II transcription at either initiation or promoter-proximal pause escape with Triptolide or Flavopiridol, and tracked Pol II kinetically using GRO-seq. Both inhibitors block transcription of more than 95% of genes, showing that pause escape, like initiation, is a ubiquitous and crucial step within the transcription cycle. Moreover, paused Pol II is relatively stable, as evidenced from half-life measurements at ~3200 genes. Finally, tracking the progression of Pol II after drug treatment establishes Pol II elongation rates at over 1,000 genes. Notably, Pol II accelerates dramatically while transcribing through genes, but slows at exons. Furthermore, intergenic variance in elongation rates is substantial, and is influenced by a positive effect of H3K79me2 and negative effects of exon density and CG content within genes. We isolated replicates of nuclei of untreated mESCs and cells treated for 2, 5, 12.5, 25 and 50 min with 300nM flavopiridol, as well as nuclei treated for 12.5, 25, and 50 min with 500nM triptolide and performed GRO-seq with these.
2014-03-21 | E-GEOD-48895 | biostudies-arrayexpress
Project description:Study of the POU-homeodomain transcription factor, has revealed that, binding of Pit1-occupied enhancers to a nuclear matrin-3-rich network/architecture is a key event in effective activation of the Pit1-regulated enhancer / coding gene transcriptional program. All ChIP-seq(s) were designed to understand the unique features, associated molecular mechanisms and functions of Matrin3 in the Homeodomain transcription study. Gro-seq Samples are used to detect gene expression between knockdown control and knockdown two key factors in our study.
2014-08-03 | E-GEOD-58009 | biostudies-arrayexpress
Project description:Inhibition of transcriptional elongation plays an important role in gene regulation in metazoans, including C. elegans, which lacks Negative Elongation Factor homologs. Here we combine genomic and biochemical approaches to dissect a novel role of C. elegans AF10 homolog, ZFP-1, in transcriptional control. We show that ZFP-1 and its interacting partner DOT-1.1 have a global role in negatively modulating the level of Pol II transcription on essential widely expressed genes. Moreover,the ZFP-1/DOT-1.1 complex contributes to progressive Pol II stalling on essential genes during development and to rapid Pol II stalling during stress response. The slowing down of Pol II transcription by ZFP-1/DOT-1.1 is associated with an increase in H3K79 methylation and a decrease in H2B monoubiquitination, which promotes transcription. We propose a model where recruitment of ZFP-1/DOT-1.1 and deposition of H3K79 methylation at highly expressed genes initiates a negative feedback mechanism for modulation of their expression. GRO-seq (Global Run-On sequening) for nascent transcript detectiong on WT and zfp-1(ok554) mutant nematode (C. elegans) larvae in L3 stage, performed in duplicate per condition (4 samples total).
2013-07-05 | E-GEOD-47132 | biostudies-arrayexpress
Project description:Argonaute proteins and their small RNA co-factors short interfering RNAs (siRNAs) are known to inhibit gene expression at the transcriptional and post-transcriptional levels. In Caenorhabditis elegans, the Argonaute CSR-1 binds thousands of endogenous siRNAs (endo-siRNAs) antisense to germline transcripts and associates with chromatin in a siRNA-dependent manner. However, its role in gene expression regulation remains controversial. Here, we used a genome-wide profiling of nascent RNA transcripts to demonstrate that the CSR-1 RNAi pathway promotes sense-oriented Pol II transcription. Moreover, a loss of CSR-1 function resulted in global increase in antisense transcription and ectopic transcription of silent chromatin domains, which led to reduced chromatin incorporation of centromere-specific histone H3. Based on these findings, we propose that the CSR-1 pathway has a role in maintaining the directionality of active transcription thereby propagating the distinction between transcriptionally active and silent genomic regions. GRO-seq (Global Run-On sequencing) for detection of nascent transcripts. Two biological replicates were generated for csr-1 hypomorphic mutant and the corresponding WT samples using ~300,000 worms for each experiment, and two biological replicates were prepared for drh-3 (ne4253) mutant and the corresponding WT samples using ~100,000 worms for each experiment. The GRO-seq were performed in late L3/early L4 stage worms. (8 samples total)
2014-02-28 | E-GEOD-49946 | biostudies-arrayexpress
Project description:Densely sampled time course GRO-seq analysis was performed to determine global transcriptional activities at both enhancers and promoters upon Sendai Virus infection.
2018-10-07 | E-MTAB-6047 | biostudies-arrayexpress
Project description:Effluents from sewage treatment plants contain a mixture of micropollutants with the potential of harming aquatic organisms. Thus, addition of advanced treatment techniques to complement existing conventional methods has been proposed. Some of the advanced techniques could, however, potentially produce additional compounds affecting exposed organisms by unknown modes of action. In the present study the aim was to improve our understanding of how exposure to different sewage effluents affects fish. This was achieved by explorative microarray and quantitative PCR analyses of hepatic gene expression, as well as relative organ sizes of rainbow trout exposed to different sewage effluents (conventionally treated, granular activated carbon, ozonation (5 or 15 mg/L), 5 mg/L ozone plus a moving bed biofilm reactor, or UV-light treatment in combination with hydrogen peroxide). Exposure to the conventionally treated effluent caused a significant increase in liver and heart somatic indexes, an effect removed by all other treatments. Genes connected to xenobiotic metabolism, including cytochrome p450 1A, were differentially expressed in the fish exposed to the conventionally treated effluents, though only effluent treatment with granular activated carbon or ozone at 15 mg/L completely removed this response. The mRNA expression of heat shock protein 70 kDa was induced in all three groups exposed to ozone-treated effluents, suggesting some form of added stress in these fish. The induction of estrogen-responsive genes in the fish exposed to the conventionally treated effluent was effectively reduced by all investigated advanced treatment technologies, although the moving bed biofilm reactor was least efficient. Taken together, granular activated carbon showed the highest potential of reducing responses in fish induced by exposure to sewage effluents. The fish exposure was carried out in March 3-17, 2008 at Henriksdal STP (Stockholm, Sweden) using a large scale pilot plant with parallel treatment lines under ethic permits approved by the local animal committee in Gothenburg (permission no. 36-2007). A thorough description of the different treatment steps (both conventional and advanced) and the experimental setup during the exposure (Fig. 1) has been previously described elsewhere (Samuelsson et al., 2011). In brief, the raw influent underwent conventional activated sludge treatment to generate a reference effluent (Conventional Activated Sludge; CAS). This includes mechanical treatment (removal of large objects using grids with 3 mm column width); primary sedimentation; biological treatment (activated sludge aged 20 days); secondary sedimentation; sand filter. The additional advanced treatment processes were granular activated carbon (GAC), ozonation at 5 mg/L (OZ5) or 15 mg/L (OZ15), ozonation 5 mg/L followed by a moving bed biofilm reactor (MBBR) and irradiation by ultraviolet radiation (0.75 Wh/L) plus hydrogen peroxide (10 mg/L; UH). Two aquaria were used for positive controls. As the tap water available is not as well accepted by the fish, and to better reflect the organic content and overall chemistry of surface water, the control aquaria (TW1 and TW2, together TW) contained carbon-filtered tap water supplemented by 2% of conventionally treated reference effluent. We consider evaluation of implementation and running costs to be outside the scope of this paper. Juvenile rainbow trout (Oncorhynchus mykiss; 113M-BM-123 g; mean bodyweight M-BM-1 standard deviation) of both sexes were obtained from Antens Laxodling AB (AlingsM-CM-%s, Sweden). Rainbow trout is a commonly used species for this type of studies and our lab have ample experience using this species. Furthermore, rainbow trout of sufficient size for metabolomic studies (Samuelsson et al., 2011) can easily be obtained. Finally, the use of trout provides the opportunity for comparisons with other studies (e.g. Gunnarsson et al., 2009 and Albertsson et al., 2010). During an acclimatization period of four days on site, the fish were kept in tanks containing carbon-filtered tap water + 2% reference effluent. On the onset of the experiment, the fish were randomly distributed among eight 100 L aquaria (n=25/aquarium), supplied with treated effluent from the different parallel treatment lines. Aquaria duplicates were used for the controls. During both the acclimatization period as well as the 14 day exposure, the aquaria were aerated and the fish were not fed to reduce variability due to differences in food intake. It should be stressed that trout easily cope with food deprivations for several weeks (Kullgren et al., 2010). Measurements of temperature (13.5M-BM-10.5M-BM-0C), oxygen saturation (97M-BM-13%), pH (7.4M-BM-10.3), conductivity and flow rate were monitored 5 d per week throughout the exposure. In addition, to evaluate differences in removal efficiency between the treatments, total organic carbon (TOC), 7-day biochemical oxygen demand (BOD7), suspended solids (SS), total nitrogen (Tot-N) and total phosphorus (Tot-P) were measured in all effluents (Samuelsson et al., 2011). The sampling took place during two consecutive days (day 14 and 15) due to the large number of fish. For each day, an equal number of fish from each treatment was sampled in random order and killed by a blow to the head. The fish were weighed, their fork length was measured and their sex was determined by macroscopical observation of their gonads. Liver samples were collected for several different studies (hence sample size limitations), frozen on dry ice and stored at -70M-BM-0C until analysis. Homogenization of the frozen liver tissue was carried out using Tissuelyser (Qiagen, Hilden, Germany) and hepatic total RNA was extracted and purified using QIAcube and RNeasyM-BM-. Plus Mini Kit (Qiagen). The RNA quantity and quality were assessed by spectrophotometric measurements with the Nanodrop 1000 (NanoDrop Technologies, Wilmington, DE). To ensure that no degradation had occurred, the isolated RNA was analyzed using Experion automated electrophoresis (Bio-Rad, Hercules, CA). A 15k rainbow trout gene expression microarray was designed for the RT analyzer platform (febit, Heidelberg, Germany) by using The Institute for Genomic Research (TIGR) Rainbow Trout Gene Index (RTGI) database version 7.0 (http://compbio.dfci.harvard.edu/tgi/). Details on the probe design strategy, but for eelpout (Zoarces viviparus), and transcript selection strategy are described elsewhere (Kristiansson et al., 2009; Cuklev et al., 2011). However, in the present study, singletons and non-annotated expressed sequence tags (ESTs) were replaced by newly well-annotated ESTs in rainbow trout. When available, transcripts at GenBank (http://www.ncbi.nlm.nih.gov/nucleotide) were used. In our lab, results from similar microarrays using the same platform have shown good correlation with quantitative polymerase chain reaction (qPCR) data (Gunnarsson et al., 2009b; Kristiansson et al., 2009; Cuklev et al., 2011; Lennquist et al., 2011). To reduce variation in estrogen-responsive genes, only males were used for the subsequent gene expression analyses. Biotinylated antisense RNA (aRNA) was synthesized using MessageAmpM-bM-^DM-" II-Biotin Enhanced Single Round aRNA Amplification Kit (AmbionM-BM-.). The aRNA samples (20 M-BM-5g) were vacuum dried in a vacuum centrifuge, dissolved in 10 M-BM-5l water and fragmented according to the manufacturerM-bM-^@M-^Ys protocol. The following steps described in this subchapter were all performed by febit. Oligonucleotide arrays were synthesized by photo-controlled in situ synthesis using the Geniom One system (febit). Each biochip consists of eight individually accessible micro channels, each of which is referred to as a microarray in this manuscript. Eight individual samples from each aquarium were included in the analysis. In total, 64 microarrays were analyzed. A customized protocol, described in detail elsewhere (Cuklev et al., 2011), was used for prehybridization and hybridization. All samples were randomly placed on the biochips, with one sample from each exposure on each biochip. Signals were detected using the internal CCD-camera system of the RT analyzer instrument (febit) and quantified using the Geniom Wizard software. Integration times were between 156 and 570 ms, determined automatically by the instrument software. All microarray data processing and statistical calculations were performed in R-2.12.2 (www.r-project.org; R Development Core Team, 2010). The quality of pre- and post normalized arrays was verified with box- and MA plots. The data analysis was performed in the R-package LIMMA (Smyth, 2005). Data were normalized using the M-bM-^@M-^XquantileM-bM-^@M-^Y method. Moderated t-statistics and adjusted p-values of differential expression were calculated using the empirical Bayes model.
2012-05-09 | E-GEOD-37574 | biostudies-arrayexpress
Project description:Distal enhancers characterized by H3K4me1 mark play critical roles in developmental and transcriptional programs. However, potential roles of specific distal regulatory elements in regulating RNA Polymerase II (Pol II) promoter-proximal pause release remain poorly investigated. Here we report that a unique cohort of jumonji C domain-containing protein 6 (JMJD6) and bromodomain-containing protein 4 (Brd4) co-bound distal enhancers, termed anti-pause enhancers (A-PEs), regulate promoter-proximal pause release of a large subset of transcription units via long-range interactions. Brd4-dependent JMJD6 recruitment on A-PEs mediates erasure of H4R3me2(s), which is directly read by 7SK snRNA, and decapping/demethylation of 7SK snRNA, ensuring the dismissal of the 7SKsnRNA/HEXIM inhibitory complex. The interactions of both JMJD6 and Brd4 with the P-TEFb complex permit its activation and pause release of regulated coding genes. The functions of JMJD6/ Brd4-associated dual histone and RNA demethylase activity on anti-pause enhancers have intriguing implications for these proteins in development, homeostasis and disease. All Gro-seq(s) were designed to reveal the transcriptional targets of JMJD6 and Brd4, and assess the role of JMJD6 and Brd4 in Pol II promoter-proximal pause release. All ChIP-seq(s) were designed to understand the unique features, associated molecular mechanisms and functions of the anti-pause enhancers (A-PEs) discovered in the current study.
2014-01-14 | E-GEOD-51633 | biostudies-arrayexpress
Project description:Substantial evidence supports the hypothesis that enhancers are critical regulators of cell type determination, orchestrating both positive and negative transcriptional programs; however, the basic mechanisms by which enhancers orchestrate interactions with cognate promoters during activation and repression events remain incompletely understood. Here we report the required actions of the LIM domain binding protein, LDB1/CLIM2/NLI, interacting with the enhancer binding protein, ASCL1, to mediate looping to target gene promoters and target gene regulation in corticotrope cells. LDB1-mediated enhancer:promoter looping appears to be required for both activation and repression of these target target gene promoter genes. While LDB1-dependend activated genes are regulated at the level of transcriptional initiation, the LDB1-dependent repressed transcription units appear to be regulated primarily at the level of promoter pausing, with LDB1 regulating recruitment of MTA2, a component of the NuRD complex, on these negative enhancers, required for the repressive enhancer function. These results indicate that LDB1-dependent looping events can deliver repressive cargo to cognate promoters to mediate promoter pausing events in a pituitary cell type. Global Run On (GRO) assay followed by high throughput sequencing (GRO-seq)
2015-02-03 | E-GEOD-64515 | biostudies-arrayexpress