R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE97072 | GEO | 2017/10/12
SECONDARY ACCESSION(S): PRJNA380626
REPOSITORIES: GEO
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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.
2013-07-09 | E-GEOD-45530 | biostudies-arrayexpress
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.
2013-07-09 | GSE45530 | GEO
Project description:We report the genome localization of R-loops in early and late-stage embryos relative to Drosophila cultured cells. We demonstrate that proper absolute R-loop levels chage during embryogenesis and that resolution of R-loops is critical for embryonic development. R-loop mapping by strand-specific DRIP-seq revealed that R-loop localization is plastic across development, both in the genes which form R-loops and where they localize relative to gene bodies. Importantly, these changes are not driven by changes in the transcriptional program. Rather, negative GC skew and absolute changes in AT skew are associated with R-loop formation in Drosophila. Furthermore, we demonstrate that while some chromatin binding proteins and histone modification such as H3K27me3 are associated with R-loops throughout development, other chromatin factors associated with R-loops in a developmental specific manner.
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Project description:R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflicts (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.
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Project description:R-loops are three-stranded nucleic acid structures that have important regulatory roles in cells. Deregulation of R-loop dynamics, however, can lead to DNA damage and genome instability, which results from the action of endonucleases such as XPG on R-loops. The mechanism by which this R-loop processing occurs and the impact on the cell remain unclear. Here, we show that cells with R-loops induced by deregulation of the RNA-DNA helicase Senataxin (SETX), the breast cancer gene BRCA1, or RNA splicing, accumulate cytoplasmic RNA-DNA hybrids in an XPG-dependent manner. Sequencing of cytoplasmic hybrids upon loss of SETX shows that they are derived from a subset of nuclear RNA-DNA hybrids that have longer lifetimes, signal on both sense and antisense strands and contain regions with switches in nucleotide skew. We further show that induced cytoplasmic hybrids bind the pattern recognition receptors cGAS and TLR3, activating IRF3 and inducing apoptosis. Ataxia oculomotor apraxia type 2 (AOA2) patient cells which harbor a mutated SETX also exhibit XPG-dependent activation of the innate immune response. These findings identify RNA-DNA hybrids as novel, immunogenic nucleic acid species which aberrantly accumulate in the cytoplasm of cells upon R-loop processing, and can ultimately induce cell apoptosis. We anticipate that aberrant R-loop processing and subsequent innate immune activation may be a pathological process that contributes to many disease states, such as neurodegeneration and cancer.
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