Biology subjects

Chedin, F.

Publications and source records attributed to Chedin, F..

16 recordsLinked to original sources

bioRxiv2026

Sequence and Structural Alignments Reveal Insights into ANKLE2 Evolution and Function

ANKLE2 is an enigmatic protein with emerging roles in cell division, development, and virus replication. While ANKLE2 orthologs are present in all animals, its domain composition has evolved over time. ANKLE2s two namesake domains, the ankyrin repeat and LEM domains, have clear and defined roles; however nearly all ANKLE2 orthologs have at least three other structured domains with poorly understood purposes. In this study, we performed sequence and structural alignments of ANKLE2 orthologs to improve our understanding of the proteins evolution and function. We identified that ANKLE2s transmembrane domain likely evolved more recently and coincided with loss of VAPA interaction as a membrane anchoring mechanism. We show that despite stark differences in amino acid sequence, the structure of the LEM and ankyrin repeat domains are highly conserved across ANKLE2 orthologs. To investigate ANKLE2s uncharacterized domains, we performed structural alignments to identify similar proteins. This revealed surprising similarities between portions of ANKLE2 and nuclease or nucleic acid-binding proteins. However, ANKLE2 lacks key motifs imparting function in these domains, which was confirmed by experimental interrogation. We further identified that loss of ANKLE2 is correlated with changes in DNA damage response and micronuclei formation. We believe this methodology demonstrates the power of combining structural predictions with classical molecular techniques in exploring poorly understood proteins. ImportanceANKLE2 is a scaffolding protein present in all animals; however much of its function is poorly understood. By evaluating ANKLE2 sequence and structure from many different organisms and comparing its various domains with other proteins, we gain insight into how ANKLE2 evolved and what cellular roles it might be fulfilling. Further, this approach can be used to investigate other understudied or uncharacterized proteins.

molecular biology↗

bioRxiv2025

RNase H1 levels dramatically affect mitochondrial genome maintenance with little impact on nuclear R-loops in murine B cells

Overexpression of RNase H1, a ribonuclease that degrades RNA:DNA hybrids and R-loops, can suppress genome instability phenotypes in a range of maladaptive conditions. This has been interpreted to suggest that genotoxic co-transcriptional R-loops arise under these conditions and are resolved by RNase H1. Here, we manipulated RNase H1 levels using conditional knockout and overexpression models in primary murine B cells and mapped the resulting genomic R-loop landscapes. Rnaseh1 deletion resulted in a dramatic loss of mitochondrial replication and compromised B cell responses, consistent with a critical mitochondrial function for RNase H1. Genome-wide R-loops were, however, not significantly affected. More surprisingly, overexpressing active nuclear RNase H1 did not lead to significant reduction of R-loop levels or change their distribution. These results were confirmed using a human cell line in which active, nuclear RNase H1 can be induced. Our findings indicate that co-transcriptional R-loops are not efficiently resolved by RNase H1 and suggest that the identity of the RNA/DNA hybrids at the root of the genome instability phenotypes suppressed by RNase H1 may need to be re-interpreted.

genomics↗

bioRxiv2025

Protein-mediated stabilization and nicking of the non-template DNA strand dramatically affect R-loop formation in vitro

R-loops are an important class of non-B DNA structures that form co-transcriptionally. Using in vitro transcription and unbiased quantitative sequencing readouts, we show that the addition of single-strand DNA binding proteins co-transcriptionally can drive a 3- to 5-fold increase of R-loop frequency without significant changes to R-loop distribution. We propose that this is caused by stabilizing and preventing the collapse of short nascent R-loops. This suggests that R-loop formation is highly dynamic and highlights single strand binding proteins as players in cellular R-loop regulation. We further show that non-template strand DNA nicks are powerful initiators of R-loop formation, increasing R-loop frequencies by up to two orders of magnitude. Atomic force microscopy (AFM) revealed that the non-template strand in nick-initiated structures is often flayed away from the RNA:DNA hybrid and engaged in self-pairing, creating unique forked R-loop features. DNA nicks, one of the most frequent DNA lesions in cells, are therefore potential hotspots for opportunistic R-loop initiation and may cause the formation of a novel class of R-loops. Overall, this work highlights the importance of the displaced single-strand on R-loop initiation and dynamics. SIGNIFICANCE STATEMENTR-loops are three-stranded DNA:RNA hybrid structures that form during transcription and play critical roles in both gene regulation and genome stability. This study reveals that single-stranded DNA-binding protein (SSB) increases R-loop frequency by stabilizing nascent R-loops, revealing a role for this protein in R-loop dynamics. Additionally, single strand DNA breaks, common cellular lesions, are identified as potent initiators of R-loops, increasing their rate of formation by up to 100-fold. Using atomic force microscopy, the study uncovers unique forked structures in nick-initiated R-loops, representing a novel class of secondary R-loop structures. These findings provide new insights into how cellular factors and DNA damage influence R-loop formation, with implications for understanding genome instability in health and disease.

molecular biology↗

bioRxiv2024

The R-loop Grammar predicts R-loop formation under different topological constraints

R-loops are transient three-stranded nucleic acids that form during transcription when the nascent RNA hybridizes with the template DNA, freeing the DNA non-template strand. There is growing evidence that R-loops play important roles in physiological processes such as control of gene expression, and that they contribute to chromosomal instability and disease. It is known that R-loop formation is influenced by both the sequence and the topology of the DNA substrate, but many questions remain about how R-loops form and the 3-dimensional structures that they adopt. Here we represent an R-loop as a word in a formal grammar called the R-loop grammar and predict R-loop formation. We train the R-loop grammar on experimental data obtained by single-molecule R-loop footprinting and sequencing (SMRF-seq). Despite not containing explicit topological information, the R-loop grammar accurately predicts R-loop formation on plasmids with varying starting topologies and outperforms previous methods in R-loop prediction. Author summaryR-loops are prevalent triple helices that play regulatory roles in gene expression and are involved in various diseases. Our work improves the understanding of the relationship between the nucleotide sequence and DNA topology in R-loop formation. We use a mathematical approach from formal language theory to define an R-loop language and a set of rules to model R-loops as words in that language. We train the resulting R-loop grammar on experimental data of co-transcriptional R-loops formed on different DNA plasmids of varying topology. The model accurately predicts R-loop formation and outperforms prior methods. The R-loop grammar distills the effect of topology versus sequence, thus advancing our understanding of R-loop structure and formation.

genomics↗

bioRxiv2021

Quality-controlled R-loop meta-analysis reveals the characteristics of R-Loop consensus regions

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA during transcription. While the pathological consequences of R-loops have been well-studied to date, the locations, classes, and dynamics of physiological R-loops remain poorly understood. R-loop mapping studies provide insight into R-loop dynamics, but their findings are challenging to generalize. This is due to the narrow biological scope of individual studies, the limitations of each mapping modality, and, in some cases, poor data quality. In this study, we reprocessed 693 R-loop mapping datasets from a wide array of biological conditions and mapping modalities. From this data resource, we developed an accurate method for R-loop data quality control, and we reveal the extent of poor-quality data within previously published studies. We then identified a set of high-confidence R-loop mapping samples and used them to define consensus R-loop sites called "R-loop regions" (RL regions). In the process, we revealed the stark divergence between S9.6 and dRNH-based R-loop mapping methods and identified biologically meaningful subtypes of both constitutive and variable R-loops. Taken together, this work provides a much-needed method to assess R-loop data quality and reveals intriguing aspects of R-loop biology.

bioinformatics↗

bioRxiv2021

Exploration and analysis of R-loop mapping data with RLBase

R-loops are three-stranded nucleic acid structures formed from the hybridization of RNA and DNA during nascent transcription. In 2012, Ginno et al. introduced the first R-loop mapping method, DNA:RNA immunoprecipitation (DRIP) sequencing. Since that time, dozens of studies have implemented R-loop mapping and new high-resolution techniques have been developed. The resulting datasets have tremendous potential to reveal the causes and consequences of R-loops genome-wide. However, poor quality and variability between mapping approaches pose serious barriers to the meta-analysis of these data. In our recent work, we reprocessed 693 R-loop mapping samples, devising new quality methods, defining a set of high-confidence mapping samples, and then deriving R-loop regions, consensus sites of R-loop formation. This analysis yielded the largest R-loop data resource to date along with novel computational approaches for R-loop mapping analysis. Now, we introduce RLBase, an innovative web server which builds upon those data and software by providing users with the capability to (1) explore hundreds of public R-loop mapping datasets, (2) explore consensus R-loop regions, (3) analyze user-supplied datasets to generate an HTML quality report, and (4) download all the processed data for the 693 samples we previously reprocessed and standardized. In addition to RLBase, we also describe the other software which, along with RLBase, provides a computational framework for R-loop bioinformatics. RLBase, and the rest of these software (termed "RLSuite"), are provided freely under an MIT license and made publicly available: https://gccri.bishop-lab.uthscsa.edu/rlsuite/. RLBase is directly accessible via the following URL: https://gccri.bishop-lab.uthscsa.edu/rlbase/.

bioinformatics↗

bioRxiv2021

Genomic patterns of transcription-replication interactions in mouse primary B cells

Conflicts between transcription and replication machinery are a potent source of replication stress and genome stability; however, no technique currently exists to identify endogenous genomic locations prone to transcription-replication interactions. Here, we report a novel method to identify genomic loci prone to transcription-replication interactions termed transcription-replication immunoprecipitation on nascent DNA sequencing, TRIPn-Seq. TRIPn-Seq employs the sequential immunoprecipitation of RNA polymerase 2 phosphorylated at serine 5 (RNAP2s5) followed by enrichment of nascent DNA previously labeled with bromodeoxyuridine. Using TRIPn-Seq, we mapped 1,009 unique transcription-replication interactions (TRIs) in mouse primary B cells characterized by a bimodal pattern of RNAP2s5, bidirectional transcription, an enrichment of RNA:DNA hybrids, and a high probability of forming G-quadruplexes. While TRIs themselves map to early replicating regions, they exhibit enhanced Replication Protein A association and replication fork termination, marks of replication stress. TRIs colocalize with double-strand DNA breaks, are enriched for deletions, and accumulate mutations in tumors. We propose that replication stress at TRIs induces mutations potentially contributing to age-related disease, as well as tumor formation and development.

genomics↗

bioRxiv2020

SF3B1-targeted Splicing Inhibition Triggers Global Alterations in Transcriptional Dynamics and R-Loop Metabolism

Efficient co-transcriptional splicing is thought to suppress genome-destabilizing R-loops. Inhibition of SF3B1, a core U2 spliceosome component, by Pladienolide B (PladB) in human K562 cells caused widespread intron retention and modest R-loops gains. Minimal overlap existed between these events, suggesting that unspliced introns by themselves do not cause excessive R-loops. R-loop gains were instead driven by extensive readthrough transcription at a subset of stress-response genes, defining a new class of aberrant "downstream of genes" (DoG) R-loops. Such DoG R-loops were temporally and spatially uncoupled from loci experiencing DNA damage. Unexpectedly, the predominant response to splicing inhibition was a global R-loop loss. This resulted from increased promoter-proximal pausing and defective transcription elongation associated with premature termination. Similar results were observed upon depletion of Aquarius, a U2 spliceosome-associated factor previously thought to suppress R-loops. Thus, U2 spliceosome-targeted splicing inhibition triggered profound alterations in transcriptional dynamics, leading to unexpected disruptions in R-loop landscapes. HIGHLIGHTSO_LIIntron retention caused by SF3B1 inhibition does not trigger excessive R-loops C_LIO_LIStress-response genes shows readthrough transcription and R-loop gains C_LIO_LIR-loop gains and DNA damage are temporally and spatially uncoupled C_LIO_LIU2 snRNP inhibition causes broad reduction in transcription and dominant R-loop loss C_LI

molecular biology↗

bioRxiv2020

NON-DENATURING BISULFITE TREATMENT AND SINGLE-MOLECULE REAL-TIME SEQUENCING REVEALS D-LOOP FOOTPRINTS, THEIR LENGTH, POSITION AND DISTRIBUTION

Displacement loops (D-loops) are signature intermediates formed during homologous recombination. Numerous factors regulate D-loop formation and disruption, thereby influencing crucial aspects of DNA repair, including donor choice and the possibility of a crossover outcome. While D-loop detection methods exist, it is currently unfeasible to assess the relationship between D-loop editors and D-loop characteristics such as length and position. Here, we developed a novel in vitro assay to characterize the length and position of individual D-loop with base-pair resolution and deep coverage, while also revealing their distribution in a population. Non-denaturing bisulfite treatment modifies the cytosines on the displaced strand of the D-loop to uracil, leaving a permanent signature for the displaced strand. Subsequent single-molecule real-time sequencing uncovers the cytosine conversion patch as a D-loop footprint, revealing D-loop characteristics at unprecedented resolution. The D-loop Mapping Assay is widely applicable with different substrates and donor types and can be used to study factors that influence D-loop properties.

biochemistry↗

bioRxiv2020

Rdh54/Tid1 Inhibits Rad51-Rad54-Mediated D-loop Formation and Limits D-loop Length

Displacement loops (D-loops) are intermediates formed during homologous recombination that play a pivotal role in the fidelity of repair. Rdh54 (a.k.a. Tid1), a Rad54 paralog in Saccharomyces cerevisiae, is well-known for its role with Dmc1 recombinase during meiotic recombination. Yet contrary to Dmc1, Rdh54 is also present in somatic cells where its function is less understood. While Rdh54 enhances the Rad51 DNA strand invasion activity in vitro, it is unclear how it interplays with Rad54-mediated invasions. Here, we show that Rdh54 inhibits D-loop formation by Rad51 and Rad54 in an ATPase-independent manner. Using a novel D-loop Mapping Assay, we further demonstrate that Rdh54 uniquely restricts the lengths of Rad54-mediated D-loops. The alterations in D-loop properties appear to be important for cell survival and mating-type switch in haploid yeast, whereas Rdh54 expression is suppressed in diploids. We propose that Rdh54 and Rad54 compete for potential binding sites within the Rad51 filament, where Rdh54 acts as a physical roadblock to Rad54s translocation activity, limiting D-loop formation and D-loop length.

biochemistry↗

bioRxiv2020

Recognition of cellular RNAs by the S9.6 antibody creates pervasive artefacts when imaging RNA:DNA hybrids

The contribution of RNA:DNA hybrid metabolism to cellular processes and disease states has become a prominent topic of study. The S9.6 antibody recognizes RNA:DNA hybrids with a subnanomolar affinity, making it a broadly used tool to detect and study RNA:DNA hybrids. However, S9.6 also binds double-stranded RNA in vitro with significant affinity. Though frequently used in immunofluorescence microscopy, the possible reactivity of S9.6 with non-RNA:DNA hybrid substrates in situ, particularly RNA, has not been comprehensively addressed. Furthermore, S9.6 immunofluorescence microscopy has been methodologically variable and generated discordant imaging datasets. In this study, we find that the majority of the S9.6 immunofluorescence signal observed in fixed human cells arises from RNA, not RNA:DNA hybrids. S9.6 staining was quantitatively unchanged by pre-treatment with the human RNA:DNA hybrid-specific nuclease, RNase H1, despite experimental verification in situ that S9.6 could recognize RNA:DNA hybrids and that RNase H1 was active. S9.6 staining was, however, significantly sensitive to pre-treatments with RNase T1, and in some cases RNase III, two ribonucleases that specifically degrade single-stranded and double-stranded RNA, respectively. In contrast, genome-wide maps obtained by high-throughput DNA sequencing after S9.6-mediated DNA:RNA Immunoprecipitation (DRIP) are RNase H1-sensitive and RNase T1- and RNase III-insensitive. Altogether, these data demonstrate that the S9.6 antibody, though capable of recognizing RNA:DNA hybrids in situ and in vitro, suffers from a lack of specificity that precludes reliable imaging of RNA:DNA hybrids and renders associated imaging data inconclusive in the absence of controls for its promiscuous recognition of cellular RNAs.

molecular biology↗

bioRxiv2019

High-Throughput Single-Molecule R-loop Footprinting Reveals Principles of R-loop Formation

R-loops are a prevalent class of non-B DNA structures that form during transcription upon reannealing of the nascent RNA to the template DNA strand. R-loops have been profiled using the S9.6 antibody to immunoprecipitate DNA:RNA hybrids. S9.6-based DNA:RNA immunoprecipitation (DRIP) techniques revealed that R-loops form dynamically over conserved genic hotspots. We developed an orthogonal profiling methodology that queries R-loops via the presence of long stretches of single-stranded DNA on the looped-out strand. Non-denaturing sodium bisulfite treatment catalyzes the conversion of unpaired cytosines to uracils, creating permanent genetic tags for the position of an R-loop. Long read, single-molecule PacBio sequencing allows the identification of R-loop footprints at near nucleotide resolution in a strand-specific manner on single DNA molecules and at ultra-deep coverage. Single-molecule R-loop footprinting (SMRF-seq) revealed a strong agreement between S9.6-and bisulfite-based R-loop mapping and confirmed that R-loops form from unspliced transcripts over genic hotspots. Using the largest single-molecule R-loop dataset to date, we show that individual R-loops generate overlapping sets of molecular clusters that pile-up through larger R-loop-prone zones. SMRF-seq further established that R-loop distribution patterns are driven by both intrinsic DNA sequence features and DNA topological constraints, revealing the principles of R-loop formation.

genetics↗

bioRxiv2019

RNA strand invasion activity of the Polycomb complex PRC2

Epigenetic regulation is conveyed through information encoded by specific chromatin features. Non-canonical nucleic acid structures could in principle also convey biological information but their role(s) in epigenetic regulation is not known. Polycomb Group (PcG) proteins form memory of transient transcriptional repression events that is necessary for development. In Drosophila, PcG proteins are recruited to specific DNA sequences, Polycomb Response Elements (PREs). PREs are switchable memory elements that can exist in repressed, active, or unengaged states 1,2. How PcG activities are targeted to PREs to maintain repressed states only in appropriate developmental contexts has been difficult to elucidate. Biochemically, PcG protein complexes modify chromatin to maintain gene repression 1,3,4. However, PcG proteins also interact with both RNA and DNA, and RNA is implicated in the targeting of PcG function. We find that R-loops, three-stranded nucleic acid structures formed when an RNA hybridizes to its complementary DNA and displaces the other DNA strand 5, form at many PREs in Drosophila embryos, and correlate with the repressive state. R-loops are recognized by the PcG complex PRC1 in vitro. Unexpectedly, we find that the PcG complex PRC2 has RNA strand invasion activity, which can drive formation of RNA-DNA hybrids, the key component of R-loops. Our results suggest a new mechanism for targeting PcG function through R-loop formation by PRC2 and recognition by PRC1. More generally, our findings suggest formation and recognition 6 of non-canonical nucleic acid structures as an epigenetic mechanism.

biochemistry↗

bioRxiv2019

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

Three-stranded R-loop structures have been associated with genomic instability phenotypes. What underlies their wide-ranging effects on genome stability remains poorly understood. Here we combined biochemical and atomic force microscopy approaches with single molecule R-loop footprinting to demonstrate that R-loops formed at the model Airn locus in vitro adopt a defined set of three-dimensional conformations characterized by distinct shapes and volumes, which we call R-loop objects. Interestingly, 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 sequence of 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.

biophysics↗

bioRxiv2018

DNA Topoisomerase I differentially modulates R-loops across the human genome

BackgroundCo-transcriptional R-loops are abundant non-B DNA structures in mammalian genomes. DNA Topoisomerase I (Top1) is often thought to regulate R-loop formation owing to its ability to resolve both positive and negative supercoils. How Top1 regulates R-loop structures at a global level is unknown.\n\nResultsHere, we performed high-resolution strand-specific R-loop mapping in human cells depleted for Top 1 and found that Top1 depletion resulted in both R-loop gains and losses at thousands of transcribed loci, delineating two distinct gene classes. R-loop gains were characteristic for long, highly transcribed, genes located in gene-poor regions anchored to Lamin B1 domains and in proximity to H3K9me3-marked heterochromatic patches. R-loop losses, by contrast, occurred in gene-rich regions overlapping H3K27me3-marked active replication initiation regions. Interestingly, Top1 depletion coincided with a block of the cell cycle in G0/G1 phase and a trend towards replication delay.\n\nConclusionsOur findings reveal new properties of Top1 in regulating R-loop homeostasis and suggest a potential role for Top1 in controlling replication initiation via R-loop formation.

genomics↗

bioRxiv2017

The affinity of the S9.6 antibody for double-stranded RNAs impacts the mapping of R-loops in fission yeast.

R-loops, which result from the formation of stable DNA:RNA hybrids, can both threaten genome integrity and act as physiological regulators of gene expression and chromatin patterning. To characterize R-loops in fission yeast, we used the S9.6 antibody-based DRIPc-seq method to sequence the RNA strand of R-loops and obtain strand-specific R-loop maps at near nucleotide resolution. Surprisingly, preliminary DRIPc-seq experiments identified mostly RNase H-resistant but exosome-sensitive RNAs that mapped to both DNA strands and resembled RNA:RNA hybrids (dsRNAs), suggesting that dsRNAs form widely in fission yeast. We confirmed in vitro that S9.6 can immuno-precipitate dsRNAs and provide evidence that dsRNAs can interfere with its binding to R-loops. dsRNA elimination by RNase III treatment prior to DRIPc-seq allowed the genome-wide and strand-specific identification of genuine R-loops that responded in vivo to RNase H levels and displayed classical features associated with R-loop formation. We also found that most transcripts whose levels were altered by in vivo manipulation of RNase H levels did not form detectable R-loops, suggesting that prolonged manipulation of R-loop levels could indirectly alter the transcriptome. We discuss the implications of our work in the design of experimental strategies to probe R-loop functions.

molecular biology↗