Project description:This study profiles RNA:DNA hybrid formation in human and mouse cell lines. DRIPc-seq (strand-specific R-loop mapping) was performed on human NT2 cells and mouse 3T3 cells. DRIP-seq (R-loop mapping) was performed on human NT2 and K562 and mouse E14 and 3T3 cell lines. MethylC-seq and RNA-seq were performed on NT2.
Project description:DNA topoisomerase I is an essential enzyme in higher eukaryotes that regulates DNA torsional tension during fundamental processes, such as replication and transcription. During its catalytic activity, a transient Topoisomerase I-DNA cleavage complex, called Top1cc, forms to allow strand rotation and duplex relaxation. However, the stabilization of Top1cc can lead to increased DNA:RNA hybrid duplexes, DNA double-strand cuts (DSBs) and genome instability as shown by formation of micronuclei, extranuclear chromatin enveloped by a nuclear membrane. To better comprehend the underlying mechanisms, we have established genomic maps of Top1cc-mediated R-loop changes and DSBs at short times upon Top1cc induction. Our findings show that Top1ccs dynamically changed the genomic distribution of R-loops, with regions of stable hybrid gains. DSBs were at specific gene loci, strongly associated with stable anterior R-loops at highly-transcribed genes and close to backtracked RNA polymerase II. Moreover, DSBs and stable anterior R-loops were highly enriched at early replication origins, thus revealing the location of replication conflicts with backtracked RNA polymerases.
Project description:Strand asymmetry in the distribution of guanines and cytosines, measured by GC skew, predisposes DNA sequences towards R-loop formation upon transcription. Previous work revealed that GC skew and R-loop formation associate with a core set of unmethylated CpG island (CGI) promoters in the human genome. Here, we show that GC skew can distinguish four classes of promoters, including three types of CGI promoters, each associated with unique epigenetic and gene ontology signatures. In particular, we identify a strong and a weak class of CGI promoters and show that these loci are enriched in distinct chromosomal territories reflecting the intrinsic strength of their protection against DNA methylation. Interestingly, we show that strong CGI promoters are depleted from the X chromosome while weak CGIs are enriched, a property consistent with the acquisition of DNA methylation during dosage compensation. Furthermore, we identify a third class of CGI promoters based on its unique GC skew profile and show that this gene set is enriched for Polycomb group targets. Lastly, we show that nearly 2,000 genes harbor GC skew at their 3’ ends and that these genes are preferentially located in gene-dense regions and tend to be closely arranged. Genomic profiling of R-loops accordingly showed that a large proportion of genes with terminal GC skew form R-loops at their 3’-ends, consistent with a role for these structures in permitting efficient transcription termination. Altogether, we show that GC skew and R-loop formation offer significant insights into the epigenetic regulation, genomic organization, and function of human genes. DRIP-seq was performed on genomic DNA extracted from human pluripotent Ntera2 cells. The DNA was either fragmented using HindIII, EcoRI, BsrGI, XbaI and SspI (DRIP-seq 1) or BamHI, NcoI, ApaLI, NheI and PvuII (DRIP-seq 2, two technical replicates). Input DNA was also fragmented with each restriction enzyme cocktail and sequenced alongside.
Project description:BackgroundR-loops are three-stranded nucleic acid structures that usually form during transcription and that may lead to gene regulation or genome instability. DRIP (DNA:RNA Immunoprecipitation)-seq techniques are widely used to map R-loops genome-wide providing insights into R-loop biology. However, annotation of DRIP-seq peaks to genes can be a tricky step, due to the lack of strand information when using the common basic DRIP technique.ResultsHere, we introduce DRIP-seq Optimized Peak Annotator (DROPA), a new tool for gene annotation of R-loop peaks based on gene expression information. DROPA allows a full customization of annotation options, ranging from the choice of reference datasets to gene feature definitions. DROPA allows to assign R-loop peaks to the DNA template strand in gene body with a false positive rate of less than 7%. A comparison of DROPA performance with three widely used annotation tools show that it identifies less false positive annotations than the others.ConclusionsDROPA is a fully customizable peak-annotation tool optimized for co-transcriptional DRIP-seq peaks, which allows a finest gene annotation based on gene expression information. Its output can easily be integrated into pipelines to perform downstream analyses, while useful and informative summary plots and statistical enrichment tests can be produced.
Project description:m6A RNA methylation of major satellite repeat transcripts facilitates chromatin association and RNA:DNA hybrid formation in mouse heterochromatin
Project description:Experiment to obtain the genome-wide distribution of DNA:RNA hybrid prone loci in Saccharomyces cerevisiae by DNA:RNA immunoprecipitation and tiling microarray (DRIP-chip). Samples: wild type, Rnase H deletion mutant, hpr1 deletion mutant, sen1-1 temperature sensitive mutant.
Project description:Strand asymmetry in the distribution of guanines and cytosines, measured by GC skew, predisposes DNA sequences towards R-loop formation upon transcription. Previous work revealed that GC skew and R-loop formation associate with a core set of unmethylated CpG island (CGI) promoters in the human genome. Here, we show that GC skew can distinguish four classes of promoters, including three types of CGI promoters, each associated with unique epigenetic and gene ontology signatures. In particular, we identify a strong and a weak class of CGI promoters and show that these loci are enriched in distinct chromosomal territories reflecting the intrinsic strength of their protection against DNA methylation. Interestingly, we show that strong CGI promoters are depleted from the X chromosome while weak CGIs are enriched, a property consistent with the acquisition of DNA methylation during dosage compensation. Furthermore, we identify a third class of CGI promoters based on its unique GC skew profile and show that this gene set is enriched for Polycomb group targets. Lastly, we show that nearly 2,000 genes harbor GC skew at their 3’ ends and that these genes are preferentially located in gene-dense regions and tend to be closely arranged. Genomic profiling of R-loops accordingly showed that a large proportion of genes with terminal GC skew form R-loops at their 3’-ends, consistent with a role for these structures in permitting efficient transcription termination. Altogether, we show that GC skew and R-loop formation offer significant insights into the epigenetic regulation, genomic organization, and function of human genes.
Project description:<p>RNA sequencing was performed on human DRGs and relative gene abundances were calculated.</p> <p>Various analyses were performed:</p> <p> <ol> <li>Human DRG gene expression profiles were contrasted with a panel of gene expression profiles of relevant tissues in human and mouse ( integrating, among other sources, datasets from ENCODE and GTex ) in order to identify.</li> <ol type="a"> <li>DRG-enriched gene expression, co-expression modules of DRG-expressed genes, and key transcriptional regulators in humans.</li> <li>Contrasting the human and mouse DRG transcriptomes to identify DRG-enriched gene expression patterns that were conserved between human and mouse, identifying putative cell types of expression of these genes, and potential known drugs that might target the corresponding gene products.</li> <li>Characterization of non-coding RNA profile of human and mouse DRGs.</li> <li>Characterization of DRG-enriched alternative splicing and alternative transcription start site usage based transcript variants in humans and mouse, and the overlap between these two species.</li> <li>Contrasting of human DRG and GTex human tibial nerve samples to identify putative axonally transported mRNAs in sensory neurons.</li> </ol> <li>Human DRG transcriptomes from donors suffering from neuropathic and/or chronic pain were contrasted with controls to identify.</li> <ol type="a"> <li>Differentially expressed genes, pathways and regulators path play a potential role in neuronal plasticity, electrophysiological activity, immune signaling and response.</li> <li>Predictive models (Random Forests) were built to jointly predict the sex and pain state of samples based on information contained solely in autosomal gene expression profile.</li> <li>Gene co-expression modules were identified and gene set enrichment analysis performed.to identify sample - pathway associations, and to broadly characterize plasticity in human DRG cell types.</li> </ol> </ol> </p>