Project description:ChIP-exo/nexus experiments present modifications on the commonly used ChIP-seq protocol for high resolution mapping of transcription factor binding sites. Although many aspects of the ChIP-exo data analysis are similar to those of ChIP-seq, these high throughput experiments pose a number of unique quality control and analysis challenges. We develop a statistical quality control pipeline and accompanying R package, ChIPexoQual, to enable exploration and analysis of ChIP-exo and related experiments. ChIPexoQual evaluates a number of key issues including strand imbalance, library complexity, and signal enrichment of data. Assessment of these features are facilitated through diagnostic plots and summary statistics calculated over regions of the genome with varying levels of coverage. We evaluated our QC pipeline with both large collections of public ChIP-exo/nexus data and multiple, new ChIP-exo datasets from E. coli. ChIPexoQual analysis of these datasets resulted in guidelines for using these QC metrics across a wide range of sequencing depths and provided further insights for modelling ChIP-exo data. Finally, although ChIP-exo experiments have been compared to ChIP-seq experiments with single-end (SE) sequencing, we provide, for the first time, comparisons with paired-end (PE) ChIP-seq experiments. We illustrate that, at fixed sequencing depths, ChIP-exo provides higher sensitivity, specificity, and spatial resolution than PE ChIP-seq and both significantly outperform their SE ChIP-seq counterpart.
Project description:<p>Gene expression is a biological process regulated at different molecular levels, including chromatin accessibility, transcription, and RNA maturation and transport. In addition, these regulatory mechanisms have strong links with cellular metabolism. Here we present a multi-omics dataset that captures different aspects of this multi-layered process in yeast. We obtained RNA-seq, metabolomics, and H4K12Ac ChIP-seq data for wild-type and mip6delta strains during a heat-shock time course. Mip6 is an RNA-binding protein that contributes to RNA export during environmental stress and is informative of the contribution of post-transcriptional regulation to control cellular adaptations to environmental changes. The experiment was performed in quadruplicate, and the different omics measurements were obtained from the same biological samples, which facilitates the integration and analysis of data using covariance-based methods. We validate our dataset by showing that ChIP-seq, RNA-seq and metabolomics signals recapitulate existing knowledge about the response of ribosomal genes and the contribution of trehalose metabolism to heat stress.</p>
Project description:Identfification of MEF2A target genes using ChIP-exo in skeletla muscle and primary cardiomyocytes. Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells. MEF2A target genes were identified in 48 hr DM C2C12 myoblasts cells and primary cardiomyocytes using ChIP-exo. Binding profiles on MEF2A in each cell type were compared. Cross sectional-analysis between ChIP-exo identified targets and RNA-seq analysis of MEF2A deplted myoblasts was also done.
Project description:Recently, a number of advances have been implemented into the core ChIP-seq (chromatin immunoprecipitation coupled with next-generation sequencing) methodology to streamline the process, reduce costs or improve data resolution. Several of these emerging ChIP-based methods perform additional chemical steps on bead-bound immunoprecipitated chromatin, posing a challenge for generating similarly treated input controls required for artifact removal during bioinformatics analyses. Here we present a versatile method for producing technique-specific input controls for ChIP-based methods that utilize additional bead-bound processing steps. This reported method, termed protein attached chromatin capture (PAtCh-Cap), relies on the non-specific capture of chromatin-bound proteins via their carboxylate groups, leaving the DNA accessible for subsequent chemical treatments in parallel with chromatin separately immunoprecipitated for the target protein. Application of this input strategy not only significantly enhanced artifact removal from ChIP-exo data, increasing confidence in peak identification and allowing for de novo motif searching, but also afforded discovery of a novel CTCF binding motif.
Project description:Several recently emerging ChIP-seq (chromatin immunoprecipitation followed by sequencing) based methods perform chemical steps on bead-bound immunoprecipitated chromatin, posing a challenge for generating similarly treated input controls required for bioinformatics and data quality analyses. Here we present a versatile method for producing technique-specific input controls for ChIP-based methods that utilize additional bead-bound processing steps. Application of this method allowed for discovery of a novel CTCF binding motif from ChIP-exo data.
Project description:Identfification of MEF2A target genes using ChIP-exo in skeletla muscle and primary cardiomyocytes. Identfification of MEF2A target genes using ChIP-exo and RNA-seq in skeletal muscle and primary cardiomyocytes. MEF2 plays a profound role in the regulation of transcription in cardiac and skeletal muscle lineages. To define the overlapping and unique MEF2A genomic targets, we utilized ChIP-exo analysis of cardiomyocytes and skeletal myoblasts. Of the 2783 and 1648 MEF2A binding peaks in skeletal myoblasts and cardiomyocytes, respectively, 294 common binding sites were identified. Genomic targets were compared to differentially expressed genes in RNA-seq analysis of MEF2A depleted myogenic cells.
Project description:RUVBL2 is most important AAA+ ATPase for RNA polymerase II assembly and transcription regulation, through DNA remodeling or by directly interaction with PIC,this study will comprehensively to study the promiscuous functions of this proteins through the ChIP-MS, ChIP-seq, RNA-seq and nascent RNA seq and biochemistry analysis. Our study would provide more systematic and novel responsibility of this molecule, especially for the development and carcinomas.
Project description:The ferric uptake regulator (Fur) plays a critical role in the transcriptional regulation of iron metabolism in many bacteria. However, the full regulatory potential of Fur beyond iron metabolism remains undefined. Here, we comprehensively reconstructed the Fur transcriptional regulatory network in Escherichia coli K-12 MG1655 in response to iron availability using genome-wide measurements (ChIP-exo and RNA-seq). Polyomic data analysis revealed that a total of 81 genes in 42 transcription units (TUs) are directly regulated by three different modes of Fur regulation, including apo- and holo-Fur activation as well as holo-Fur repression. We showed that Fur connects iron transport and utilization enzymes with negative-feedback loop pairs for iron homeostasis. In addition, direct involvement of Fur in the regulation of DNA synthesis, energy metabolism, and biofilm development was found. These results indicate that Fur exhibits a comprehensive regulatory role affecting many fundamental cellular processes linked to iron metabolism in order to coordinate E. coli responses to the availability of iron. [ChIP-exo]: A total of twelve samples were analyzed. WT and Fur-8-myc tagged cells were cultured in the presense and absence of iron with biological duplicates. To analyze static RNAP binding, rifampicin was also added to the media with biological duplicates. DPD = iron chelator.