Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Two Distinct Types of RNA:DNA Hybrids Revealed by Evaluating Divergent “R-loop” Profiles Detected with Different Mapping Strategies

ABSTRACT: R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.

ORGANISM(S): Homo sapiens

PROVIDER: GSE147886 | GEO | 2022/05/22

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Similar Datasets

Improved mapping and characterization of R-loop forming loci in fission yeast.

Project description:R-loops both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loop forming loci in the fission yeast, we used the S9.6-based DRIPc-seq approach to obtain improved strand-specific maps of R-loop forming loci at near nucleotide resolution. We show that the weak affinity of the S9.6 antibody for double-stranded RNA (dsRNA) is sufficient to confound the mapping of genuine R-loops by this approach. dsRNA elimination allowed the identification of two distinct classes of R-loops that differ by their sensitivity to endogenous RNase H activity. Both classes associate with common chromatin features, which are similar to those associated with R-loop formation in human. We used RNA-seq to identify transcripts whose steady-state levels were affected by genome-wide manipulation of R-loop levels. gdh2 is such a transcript and we show that extra RNase H1 stimulates the cis-acting transcription interference between an upstream non-coding RNA and gdh2. Surprisingly, we found that most transcripts whose levels were altered by in vivo manipulation of R-loop levels did not form R-loops. Conversely, the abundance of only few R-loop forming transcripts was impacted by genome-wide variation of R-loop levels. We conclude that prolonged manipulation of R-loop levels imparts indirect effects on the transcriptome that could complicate the use of this strategy to understand R-loop functions.

2018-06-01 | GSE101086 | GEO

R-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instabilityR-loop proximity proteomics identifies a role of DDX41 in transcription-associated genomic instability

Project description:Transcription can pose a threat to genomic stability through the formation of R-loops that obstruct the progression of replication forks. R-loops are three-stranded nucleic acid structures formed by an RNA-DNA hybrid with a displaced non-template DNA strand. We developed RDProx to identify proteins that regulate R-loops in human cells. RDProx relies on the expression of the hybrid-binding domain (HBD) of Ribonuclease H1 (RNaseH1) fused to the ascorbate peroxidase (APEX2), which permits mapping of the R-loop proximal proteome using quantitative mass spectrometry. We associated R-loop regulation with different cellular proteins and identified a role of the tumor suppressor DEAD box protein 41 (DDX41) in opposing R-loop-dependent genomic instability. Depletion of DDX41 resulted in replication stress, double strand breaks and increased inflammatory signaling. Furthermore, DDX41 opposes accumulation of R-loops at gene promoters and its loss leads to upregulated expression of TGFβ and NOTCH signaling genes. Germline loss-of-function mutations in DDX41 lead to predisposition to acute myeloid leukemia (AML) in adulthood. We propose that accumulation of co-transcriptional R-loops, associated gene expression changes and inflammatory response contribute to the development of familial AML with mutated DDX41.

2021-11-25 | PXD024517 | Pride

RPA and RAD51 ChIP-seq from B6 and B6xCAST F1 PRDM9-Humanized/CAST mouse testes

Project description:Meiotic recombination is initiated by programmed DNA double-strand breaks (DSBs) and proceeds via binding of RPA, RAD51 and DMC1 to single-stranded DNA (ssDNA) substrates created after the formation of DSBs. Here, we report high-resolution in vivo maps of RPA and RAD51 binding in meiosis, mapping their binding locations and lifespans in a B6 and a genetically modified B6xCAST F1 mouse. We ascribe signals separately to the individual homologous chromosomes in the hybrid mouse, thereby separating the signal of binding to the chromosome where DSBs occurred and the chromosome that was used as template for repair. Together with super-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: whereas DMC1 binds near the break-site, RAD51 binds away from it. We characterize the D-loop, a critical intermediate bound by RPA, in vivo. These data show that DMC1, not RAD51, performs strand exchange in mammalian meiosis. We find that the localisation of D-loop intermediates is similar in crossover and non-crossover pathways, with a longer lifespan for crossover-destined intermediates. These findings answer long-standing questions about the molecular intermediates of recombination.

2020-06-30 | GSE143582 | GEO

Proximity labeling identifies a repertoire of site-specific R-loop modulators

Project description:R-loops are three stranded nucleic acid structures that accumulate on chromatin in neurological diseases and cancers and that contribute to genome instability. Using a proximity-dependent labeling system, we identified distinct classes of proteins that regulate R-loops in vivo through different mechanisms. We show that ATRX suppresses R-loops by interacting with RNAs and preventing R-loop formation. Our proteomics screen also discovered an unexpected enrichment for proteins containing zinc fingers and homeodomains at R-loops. One of the most consistently enriched proteins at R-loops was activity-dependent neuroprotective protein (ADNP), which is frequently mutated in ASD and causal in ADNP syndrome. We find that ADNP is necessary to suppress R-loops in vivo at its genomic targets and that it resolves R-loops in vitro. Furthermore, deletion of the homeodomain severely diminishes R-loop resolution activity in vitro, results in R-loop accumulation at ADNP targets and compromises neuronal differentiation. Notably, patient derived human induced pluripotent stem cells that contain an ADNP syndrome-causing mutation exhibit R-loop and CTCF accumulation at ADNP targets. Our findings point to a specific role for ADNP-mediated R-loop resolution in physiological and pathological neuronal function and, more broadly, to a previously unappreciated role for zinc finger and homeodomain proteins in R-loop regulation, with important implications for developmental disorders and cancers.

2021-06-03 | MSV000087568 | MassIVE

ChIP-Seq in mouse embryonic stem cells of endogenous HA-tagged Tet1

Project description:R-loops are prevalent three-stranded nucleic acid structures, composed of a DNA:RNA hybrid and a displaced single-stranded DNA loop. Although aberrant R-loop formation is a threat to genome stability, R-loops are enriched at CpG islands (CGI) and are implicated in maintaining CGI promoters unmethylated. Mapping of genomic regions where Tet1 binds in an R-loop dependent manner we here identify almost exclusively CGIs. The results suggest that R-loops are genomic address labels for TET1.

2018-10-16 | GSE104067 | GEO

Genomic Profiling of Native R-Loops with a DNA-RNA Hybrid Recognition Sensor Protein

Project description:R-Loops are unique RNA-containing chromatin structures, which participate in various key biological processes and associate with multiple human diseases. Accurately and comprehensively profiling R-Loops in the genome is crucial to study their functions under the physiological and pathological conditions. However, the existing methodologies have produced broad discrepancies in R-Loop profiling and an independent strategy is urgently needed. Here, we constructed an artificial DNA-RNA hybrid recognition sensor protein GST-His6-2XHBD with the hybrid-binding domain of RNase H1, and found GST-His6-2XHBD could specifically interact with DNA-RNA hybrids and behaves similarly to the anti-DNA-RNA hybrid S9.6 antibody in DRIPc-seq. Furthermore, we established a convenient method, R-loop CUT&Tag, by combination of GST-His6-2XHBD with a Tn5-based cleavage under targets and tagmentation approach. R-Loop CUT&Tag generates highly specific signals for native R-Loops, and can sensitively detect the R-Loop signals at the promoter, genebody and enhancer. R-Loop CUT&Tag also provides possibilities to genome-widely map the native R-Loops with limited materials, and may benefit the resolving of the broad discrepancies between multiple R-Loop mapping methods.

2021-02-22 | GSE156400 | GEO

R-ChIP Using Inactive RNase H Reveals Dynamic Coupling of R-loops with Transcriptional Pausing at Gene Promoters

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.

2017-10-12 | GSE97072 | GEO

The extruded non-template strand determines the architecture of R-loops

Project description:We show that these R-loop objects impose specific physical constraints on the DNA, as revealed by the presence of stereotypical angles in the surrounding DNA. Biochemical probing and mutagenesis experiments revealed that the formation of R-loop objects at Airn is dictated by the extruded non-template strand, suggesting that R-loops possess intrinsic sequence-driven properties. Consistent with this, we show that R-loops formed at the fission yeast gene sum3 do not form detectable R-loop objects. Our results reveal that R-loops differ by their architectures and that the organization of the non-template strand is a fundamental characteristic of R-loops, which could explain that only a subset of R-loops is associated with replication-dependent DNA breaks.

2019-04-23 | GSE129936 | GEO

Sub1 and RPA associate with RNA Polymerase II at different stages of transcription

Project description:Single stranded DNA binding proteins play many roles in nucleic acid metabolism, but their importance during transcription remains unclear. Quantitative proteomic analysis of RNA polymerase II (RNApII) pre-initiation complexes (PICs) identified Sub1 and the Replication Protein A complex (RPA), both of which bind single-stranded DNA (ssDNA). Sub1, homolog of mammalian coactivator PC4, exhibits strong genetic interactions with factors necessary for promoter melting. Sub1 localizes near the transcription bubble in vitro and binds to promoters in vivo dependent upon PIC assembly. In contrast, RPA localizes to transcribed regions of active genes, strongly correlated with transcribing RNApII but independently of replication. RFA1 interacts genetically with transcription elongation factor genes. Interestingly, RPA levels increase at active promoters in cells carrying a Sub1 deletion or ssDNA binding mutant, suggesting competition for a common binding site. We propose that Sub1 and RPA interact with the non-template strand of RNApII complexes during initiation and elongation, respectively. Chip-chip from wt and sub1D cells with Rfa1 Chromatin immunoprecipitation (ChIP) of Rfa1 in wt and sub1D yeast demonstrated that Rfa1 localization correlates with RNA Polymerase II and is increased at some transcription start sites when Sub1 has been deleted. Comparison of Rfa1 localization in wt vs sub1D yeast

2011-09-26 | E-GEOD-32416 | biostudies-arrayexpress

Sub1 and RPA associate with RNA Polymerase II at different stages of transcription

2011-09-27 | GSE32416 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data