Project description:Dam identification (DamID) is a powerful technique to generate genome-wide maps of chromatin protein binding. Due to its high sensitivity it is particularly suited to study the genome interactions of chromatin proteins in small tissue samples in model organisms such as Drosophila. Here we report an intein-based approach to tune the expression level of Dam and Dam-fusion proteins in Drosophila by addition of a ligand to fly food. This helps to suppress toxic effects of Dam. In addition we describe a strategy for genetically controlled expression of Dam in a specific cell type in complex tissues. We demonstrate the utility of the latter by generating a glia-specific map of Polycomb in small samples of brain tissue. We determined DamID scores for Polycomb, normalized by Dam only control, for Drosophila larval central brain, larval fat bodies and repo+ glial cells of larval central brain. All samples were performed with 2 biological replicates. In case of Dam only control for larval central brain, each biological replicate was performed with 3 technical replicates.
Project description:The glucocorticoid receptor (GR) binds the human genome at >10,000 sites, but only regulates the expression of hundreds of genes. To determine the functional effect of each site, we measured the glucocorticoid (GC) responsive activity of nearly all GR binding sites (GBSs) captured using chromatin immunoprecipitation (ChIP) in A549 cells. 13% of GBSs assayed had GC-induced activity. The responsive sites were defined by direct GR binding via a GC response element (GRE) and exclusively increased reporter- gene expression. Meanwhile, most GBSs lacked GC-induced reporter activity. The non-responsive sites had epigenetic features of steady state enhancers and clustered around direct GBSs. Together, our data support a model in which clusters of GBSs observed with ChIP-seq reflect interactions between direct and tethered GBSs over tens of kilobases. We further show that those interactions can synergistically modulate the activity of direct GBSs, and may therefore play a major role in driving gene activation in response to GCs. Glucoroticoid receptor binding site chip-seq libraries were cloned into STARR-seq for massively parallel functioal analysis. The results were confirmed by ChIP-Exo performed on the GR in A549 cells treated with 100 nM dexamethasone for one hour. This dataset [6] contains the ChIP exo data from cells treated with 100 nM DEX.
Project description:Transcription rate analysis in Yeast by means of GRO, mRNA amount and ChIP-on-chip during the depletion of Spt16p. Keywords: Genomic run-on GRO ChIP-chip Transcription rate analysis by means of GRO and mRNA amount (RA) of three independent replicates during the depletion of Spt16p (Control and 5 & 7 hours after the depletion). Each time point replicate has been hybridized on a different macroarray (F14-F16). ChIP-on-chip analysis of Spt16 were done during exponential grow in YPD.
Project description:Recent evidence suggests that RNA interaction can regulate the activity and localization of DNA binding proteins, particularly chromatin-associated proteins. However, it is unknown if these observations are specialized instances for a few key RNAs and chromatin factors in specific contexts, or a general mechanism underlying the establishment of chromatin state and regulation of gene expression. Here, we introduce formaldehyde RNA ImmunoPrecipitation (fRIP-Seq), a sensitive method for cataloging RNA-protein interactions, to survey the RNA associated with a panel of 24 chromatin-associated and traditional RNA binding proteins. For each protein that reproducibly bound measurable quantities of bulk RNA (90% of the panel), we detected enrichment for hundreds to thousands of both noncoding and mRNA transcripts. We found that the enriched sets of RNA share biochemical, functional, and epigenetic properties. Thus, these data provide strong evidence that non-random RNA association is a common feature across diverse classes of chromatin modifying complexes. RNA associated with 24 different proteins using fRIP was compared to total RNA-seq
Project description:The goal of the microarray was to investigate the transcriptome changes induced by exogenous NAD+ in the wild-type Col-0 plants. Results showed that exogenous NAD+-induced dramatic transcriptional changes in Arabidopsis. Particularly, a large group of salicylic acid pathway genes including NPR1 and its traget genes were induced by NAD+, whereas the jasmonic acid/ethylene pathway defense marker gene PDF1.2 was inhibited by NAD+ treatment. In addition, a group of the pathogen-associated molecular pattern pathway genes were also induced by exogenous NAD+. These results indicate that exogenous NAD+ induces defense pathways against (hemi)biotrophic pathogens but suppresses defense against necrotrophs. Two to three replicates with leaves from 8-12 plants per sample were collected at 0, 4, and 24 hr after NAD+ treatment. Leaf tissues were collected as the control at 0 hr, and NAD+-treated leaf tissues were collected at 4 and 24 hr. After extraction, RNA concentration was determined on a NanoDrop Spectrophotometer (Thermofisher Scientific, Waltham, MA) and sample quality was assessed using the 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Equal amount of RNA from the biological replicates were used for microarray analysis.
Project description:Analysis of genome-wide differences of transcription using Genomic run-on (GRO), RNApol II ChIP-on-Chip, cDNA analysis and ChIP-on-Chip. This SuperSeries is composed of the following subset Series: GSE14060 RNA pol II ChIP on chip (RPCC) GSE14077 RPCC analysis of rap1-sil, tpk1 & tpk2 GSE14080 GRO analysis of rap1-sil, tpk1 & tpk2 GSE14082 Analysis of Spt16 depletion GSE1002 YPD to YPGal timecourse Refer to individual Series
Project description:Chronic lymphocytic leukemia (CLL) is characterized by substantial clinical heterogeneity, despite relatively few genetic alterations. To provide a basis for studying epigenome deregulation in CLL, we established genome-wide chromatin accessibility maps for 88 CLL samples from 55 patients using the ATAC-seq assay, and we also performed ChIPmentation and RNA-seq profiling for ten representative samples. Based on the resulting dataset, we devised and applied a bioinformatic method that links chromatin profiles to clinical annotations. Our analysis identified sample-specific variation on top of a shared core of CLL regulatory regions. IGHV mutation status â which distinguishes the two major subtypes of CLL â was accurately predicted by the chromatin profiles, and gene regulatory networks inferred for IGHV-mutated vs. IGHV-unmutated samples identified characteristic differences between these two disease subtypes. In summary, we found widespread heterogeneity in the CLL chromatin landscape, established a community resource for studying epigenome deregulation in leukemia, and demonstrated the feasibility of chromatin accessibility mapping in cancer cohorts and clinical research. Genome-wide profiling of chromatin states and gene expression levels in 88 CLL samples from 55 individuals gave rise to 88 ATAC-seq profiles, 40 ChIPmentation profiles (10 samples, each with 3 different antibodies and matched immunoglobulin control), and 10 RNA-seq profiles. Raw sequence data has been deposited at the EBI's European Genome-phenome Archive (EGA) under the accession number EGAS00001001821 (controlled access to protect patient privacy).
Project description:In an effort to begin to delineate the bicarbonate regulon, we used microarray analysis with cells grown to late exponential growth phase (3 hr) and then submitted to a 15 min exposure with 0.1 M NaHCO3. Our goal was to define the first set of genes affected by the presence of bicarbonate.
Project description:There is a large body of evidence supporting the beneficial role of adipose-derived stem cells (ASCs) in tissue repair and regeneration. These effects appear to be mainly mediated by paracrine signaling pathways and enhanced during hypoxia. Mass spectrometry (MS) represents a valuable tool for proteomic profiling of cultured ASCs, which may help revealing the identity of the factors secreted by the cells under different conditions. However, serum starvation essentially required to obtain samples compatible with secretome analysis by MS can have significant influence on ASCs. Here, we present a novel and optimized culturing approach based on the use of a clinically relevant serum-free formulation, which was used to assess the effect of hypoxia on the ASC proteomic profile.