Genome-wide maps of R-loops in Drosophila embryogenesis and cultured cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE185403 | GEO | 2022/06/21
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.
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Project description:R-loops serve as key modulators in various biological processes in humans and plants.Current high-throughput methodologies for R-loop profiling have their own intrinsic pros and cons. Therefore, any robust, sensitive, and amenable methods, complementary and/or advantageous to existing methodologies, are much needed for advancing R-loop biology. We here present an RNase H based cleavage combined with DNA polymerase-mediated in situ biotin-dATP/dCTP labeling of newly synthesized DNA for sequencing, termed ISRH-seq, an antibody-independent approach for strand-specific mapping of R-loops. After conducting a parallel comparison with DRIP-seq, we found that ISRH-seq can identify more R-loop peaks with the same sequencing depth. Moreover, ISRH- and DRIP-specific R-loops had distinct genomic and epigenetic features, exhibited distinct relationships with expression of overlapping genes, and functional divergence. Importantly, we found that ISRH-seq can be scalable down to 250-100ng DNA input, indicative of a robust and sensitive high-throughput methodology. In addition, it can be applied for R-loop profiling under stress conditions. Thus, our study provides a valuable orthogonal methodology for comprehensive profiling of R-loops in plants. It could be easily adapted to other non-plant systems, especially for these biologically important tissues or cell types with limited cell numbers, thereby advancing understanding of R-loop biology.
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Project description:R-loops are involved in many biological processes in cells, yet the regulatory principles for R-loops in vivo and their impact on development remain to be explored. Here, we modified the CUT&Tag strategy to profile R-loops in Drosophila at multiple developmental stages. While high GC content promotes R-loop formation in mammalian cells, it is not required in Drosophila. In contrast, RNAPII abundance on chromatin appears to be a universal inducing factor for R-loop formation in the Drosophila genome, including active promoters and enhancers, and H3K27me3 decorated repressive regions and intergenic repeat sequences. Importantly, such a regulatory relationship is dynamically maintained throughout development and many development-related transcription factors that may regulate RNAPII activation and R-loop dynamics were uncovered. By ablating the elongation factor Spt6, we further showed the global R-loop induction coupled with RNAPII pausing, genome instability, and defects in larvae survival and cuticle development. Overexpression of rnh1 largely reversed the upregulation or downregulation of gene expression depending on gene length, DNA damage, and defects in survival and cuticle development. Altogether, our findings indicate that dynamic R-loop regulation is dictated by RNAP pausing and transcription activity, and plays a feedback role in gene regulation, genome stability maintenance, and Drosophila development.
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Project description:Cold is a major naturally occurring stress that usually causes complex symptoms and severe yield loss in plants. R-loops can function in a series of biological processes, including normal development and stress responses, in plants. However, how R-loops function in cold responses is still largely unknown in plants, especially in cold sensitive rice. Herein, we conducted DRIP-seq in combination with other newly generated omics data (RNA-seq, DNase-seq, ChIP-seq and MNase-seq) in rice with or without cold treatment. We found that cold treatment caused R-loop dynamics across the genome, indicating that R-loops are cold sensitive non-B DNA structures. Moreover, cold biased R-loops had high GC content and novel motifs the binding of distinct TFs as compared to CK biased R-loops, indicative of occurrence of differential biological implications of R-loop dynamics in response to cold stress. More importantly, we for the first time to provide evidence showing that, in addition to the direct involvement of R-loops in the regulation of cold-responsive gene transcription, R-loops can indirectly modulate transcription of subsets of cold-responsive genes through R-loop overlapping TF-centered or cis-regulatory element related regulatory networks and lncRNAs in rice. Thus, our study for the first time provides evidence showing functions of R-loop dynamics in rice cold responses. It also provides some potential R-loop loci for genetic and epigenetic manipulation towards breeding of enhanced cold tolerance rice varieties.
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Project description:To plot the R-loop landscape of LUAD cells, we conducted DRIP-seq using S9.6, an anti-DNA/RNA hybrid antibody.We next tested the effect of FANCI deficiency on R-loop distribution and found that FANCI knockout clearly altered the R-loop landscape, showing a strong loss trend.Our analysis revealed that changes in R-loop distribution mediated by FANCI deficiency blocks the activity of the Ras signaling pathway, thereby suppressing tumor-cell proliferation and dissemination. Importantly, our study highlights the causal role of FANCI-mediated changes in R-loop distribution that leads to LUAD development
2023-11-23 | GSE248308 | GEO
Project description:R-loop, a three-stranded RNA/DNA structure, has been linked to induced genome instability and regulated gene expression. To enable precision analysis of R-loops in vivo, we develop an RNase H-based approach, revealing predominant R-loop formation near gene promoters with strong G/C skew and propensity to form G-quadruplex in non-template DNA, corroborating with all biochemically established properties of R-loops. Transcription perturbation experiments further indicate that R-loop induction correlates to transcriptional pausing. Interestingly, we note that most mapped R-loops are each linked to a nearby free RNA end, and by using a ribozyme to co-transcriptionally cleave nascent RNA, we demonstrate that such free RNA end coupled with a G/C-skewed sequence is necessary and sufficient to induce R-loop. These findings provide a topological solution for RNA invasion into duplex DNA and suggest an order for R-loop initiation and elongation in an opposite direction to that previously proposed.
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Project description:3D chromatin structure, enhancers, and gene transcription function together to regulate cellular differentiation, establish cellular identity, and respond to external stimuli; however, the functional interplay between these regulatory elements remains incompletely understood. To infer potential causal relationships, we mapped 3D chromatin architecture, histone H3 K27 acetylation, chromatin accessibility, and gene expression across eight narrowly-spaced time points of macrophage activation. Enhancers and genes that were connected by a loop exhibited stronger correlation between histone H3K27 acetylation and expression than can be explained by distance or proximity alone, suggesting that the presence of a chromatin loop may facilitate functional regulatory connections via methods beyond simply increasing their frequency of physical proximity. Changes in enhancer acetylation preceded changes in gene expression by 30-60 minutes further supporting a causal relationship. Changes in gene expression exhibit a directional bias at differential loop anchors. The anchors of gained enhancer-promoter loops are associated with increased gene expression of genes oriented away from the center of the loop. Surprisingly, lost enhancer-promoter loops were also associated with increased gene expression, particularly within the bounds of the loop. Analysis of absolute levels of transcription revealed that lost loops were characterized by particularly high levels of internal transcription. Taken together, these results are consistent with a reciprocal relationship between looping and transcription. In this model, loops can enhance transcription by facilitating enhancer-promoter contacts, but also high levels of transcription can negatively impact loop strength by antagonizing loop extrusion mechanisms.
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Project description:3D chromatin structure, enhancers, and gene transcription function together to regulate cellular differentiation, establish cellular identity, and respond to external stimuli; however, the functional interplay between these regulatory elements remains incompletely understood. To infer potential causal relationships, we mapped 3D chromatin architecture, histone H3 K27 acetylation, chromatin accessibility, and gene expression across eight narrowly-spaced time points of macrophage activation. Enhancers and genes that were connected by a loop exhibited stronger correlation between histone H3K27 acetylation and expression than can be explained by distance or proximity alone, suggesting that the presence of a chromatin loop may facilitate functional regulatory connections via methods beyond simply increasing their frequency of physical proximity. Changes in enhancer acetylation preceded changes in gene expression by 30-60 minutes further supporting a causal relationship. Changes in gene expression exhibit a directional bias at differential loop anchors. The anchors of gained enhancer-promoter loops are associated with increased gene expression of genes oriented away from the center of the loop. Surprisingly, lost enhancer-promoter loops were also associated with increased gene expression, particularly within the bounds of the loop. Analysis of absolute levels of transcription revealed that lost loops were characterized by particularly high levels of internal transcription. Taken together, these results are consistent with a reciprocal relationship between looping and transcription. In this model, loops can enhance transcription by facilitating enhancer-promoter contacts, but also high levels of transcription can negatively impact loop strength by antagonizing loop extrusion mechanisms.
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Project description:3D chromatin structure, enhancers, and gene transcription function together to regulate cellular differentiation, establish cellular identity, and respond to external stimuli; however, the functional interplay between these regulatory elements remains incompletely understood. To infer potential causal relationships, we mapped 3D chromatin architecture, histone H3 K27 acetylation, chromatin accessibility, and gene expression across eight narrowly-spaced time points of macrophage activation. Enhancers and genes that were connected by a loop exhibited stronger correlation between histone H3K27 acetylation and expression than can be explained by distance or proximity alone, suggesting that the presence of a chromatin loop may facilitate functional regulatory connections via methods beyond simply increasing their frequency of physical proximity. Changes in enhancer acetylation preceded changes in gene expression by 30-60 minutes further supporting a causal relationship. Changes in gene expression exhibit a directional bias at differential loop anchors. The anchors of gained enhancer-promoter loops are associated with increased gene expression of genes oriented away from the center of the loop. Surprisingly, lost enhancer-promoter loops were also associated with increased gene expression, particularly within the bounds of the loop. Analysis of absolute levels of transcription revealed that lost loops were characterized by particularly high levels of internal transcription. Taken together, these results are consistent with a reciprocal relationship between looping and transcription. In this model, loops can enhance transcription by facilitating enhancer-promoter contacts, but also high levels of transcription can negatively impact loop strength by antagonizing loop extrusion mechanisms.
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