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

2019 ◽  
Author(s):  
Maika Malig ◽  
Stella R. Hartono ◽  
Jenna M. Giafaglione ◽  
Lionel A. Sanz ◽  
Frederic Chedin
Keyword(s):  
Single Molecule ◽  
Distribution Patterns ◽  
Loop Formation ◽  
Topological Constraints ◽  
Single Dna Molecules ◽  
Bisulfite Treatment ◽  
Long Read ◽  
Dna Strand ◽  
Nucleotide Resolution ◽  
Template Dna

ABSTRACTR-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.

scholarly journals Non-destructive enzymatic deamination enables single molecule long read sequencing for the determination of 5-methylcytosine and 5-hydroxymethylcytosine at single base resolution

2019 ◽  
Author(s):  
Zhiyi Sun ◽  
Romualdas Vaisvila ◽  
Bo Yan ◽  
Chloe Baum ◽  
Lana Saleh ◽  
...  
Keyword(s):  
Long Range ◽  
Single Molecule ◽  
Biologically Relevant ◽  
Bisulfite Treatment ◽  
Sequencing Technologies ◽  
Long Read ◽  
Nucleotide Resolution ◽  
Non Destructive ◽  
Methylation Profiling

AbstractThe predominant methodology for DNA methylation analysis relies on the chemical deamination by sodium bisulfite of unmodified cytosine to uracil to permit the differential readout of methylated cytosines. Bisulfite treatment damages the DNA leading to fragmentation and loss of long-range methylation information. To overcome this limitation of bisulfite treated DNA we applied a new enzymatic deamination approach, termed EM-seq (Enzymatic Methyl-seq) to long-range sequencing technologies. Our methodology, named LR-EM-seq (Long Range Enzymatic Methyl-seq) preserves the integrity of DNA allowing long-range methylation profiling of 5-mC and 5-hmC over several kilobases of genomic DNA. When applied to known differentially methylated regions (DMR), LR-EM-seq achieves phasing of over 5 kb resulting in broader and better defined DMRs compared to previously reported. This result demonstrated the importance of phasing methylation for biologically relevant questions and the applicability of LR-EM-seq for long range epigenetic analysis at single molecule and single nucleotide resolution.

scholarly journals Non-denaturing bisulfite treatment and single-molecule real-time sequencing reveals d-loop footprints, their length, position and distribution

2020 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice ◽  
The Relationship

ABSTRACTDisplacement 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.

scholarly journals Epigenetic reprogramming by TET enzymes impacts co-transcriptional R-loops

2021 ◽  
Author(s):  
João C. Sabino ◽  
Madalena R. de Almeida ◽  
Patricia L. Abreu ◽  
Ana M. Ferreira ◽  
Marco M. Domingues ◽  
...  
Keyword(s):  
Stem Cells ◽  
Regulation Of Gene Expression ◽  
Embryonic Stem ◽  
Wide Distribution ◽  
Epigenetic Reprogramming ◽  
Loop Formation ◽  
Genome Wide ◽  
Dna Strand ◽  
Tet Enzymes ◽  
Template Dna

AbstractDNA oxidation by ten-eleven translocation (TET) family enzymes is essential for epigenetic reprogramming. The conversion of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) initiates developmental and cell-type-specific transcriptional programs through mechanisms that include changes in the chromatin structure. Here, we show that the presence of 5hmC in the transcribed DNA promotes the annealing of the nascent RNA to its template DNA strand, leading to the formation of an R-loop. The genome-wide distribution of 5hmC and R-loops show a positive correlation in mouse and human embryonic stem cells and overlap in half of all active genes. Moreover, R-loop resolution leads to differential expression of a subset of genes that are involved in crucial events during stem cell proliferation. Altogether, our data reveal that epigenetic reprogramming via TET activity promotes co-transcriptional R-loop formation, and disclose novel links between R-loops and the regulation of gene expression programs in stem cells.

scholarly journals Competition between the RNA Transcript and the Nontemplate DNA Strand during R-Loop Formation In Vitro: a Nick Can Serve as a Strong R-Loop Initiation Site

2009 ◽  
Vol 30 (1) ◽  
pp. 146-159 ◽  
Author(s):  
Deepankar Roy ◽  
Zheng Zhang ◽  
Zhengfei Lu ◽  
Chih-Lin Hsieh ◽  
Michael R. Lieber
Keyword(s):  
Somatic Hypermutation ◽  
Initiation Site ◽  
Loop Formation ◽  
Class Switch ◽  
Dna Strands ◽  
Dna Strand ◽  
Template Dna ◽  
Rna Transcript

ABSTRACT Upon transcription of some sequences by RNA polymerases in vitro or in vivo, the RNA transcript can thread back onto the template DNA strand, resulting in an R loop. Previously, we showed that initiation of R-loop formation at an R-loop initiation zone (RIZ) is favored by G clusters. Here, using a purified in vitro system with T7 RNA polymerase, we show that increased distance between the promoter and the R-loop-supporting G-rich region reduces R-loop formation. When the G-rich portion of the RNA transcript is downstream from the 5′ end of the transcript, the ability of this portion of the transcript to anneal to the template DNA strand is reduced. When we nucleolytically resect the beginning of the transcript, R-loop formation increases because the G-rich portion of the RNA is now closer to the 5′ end of the transcript. Short G-clustered regions can act as RIZs and reduce the distance-induced suppression of R-loop formation. Supercoiled DNA is known to favor transient separation of the two DNA strands, and we find that this favors R-loop formation even in non-G-rich regions. Most strikingly, a nick can serve as a strong RIZ, even in regions with no G richness. This has important implications for class switch recombination and somatic hypermutation and possibly for other biological processes in transcribed regions.

scholarly journals Analysis of mitochondrial genome methylation using Nanopore single-molecule sequencing

2021 ◽  
Author(s):  
Theresa Lüth ◽  
Christine Klein ◽  
Susen Schaake ◽  
Ronnie Tse ◽  
Sandro Pereira ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Single Molecule ◽  
Biological Significance ◽  
Cpg Methylation ◽  
Nanopore Sequencing ◽  
Sequencing Data ◽  
Low Level ◽  
Bisulfite Treatment ◽  
Synthetic Dna ◽  
Long Read

AbstractThe level and the biological significance of mitochondrial DNA (mtDNA) methylation in human cells is a controversial topic. Using long-read third-generation sequencing technology, mtDNA methylation can be detected directly from the sequencing data, which overcomes previously suggested biases, introduced by bisulfite treatment-dependent methods. We investigated mtDNA from whole blood-derived DNA and established a workflow to detect CpG methylation with Nanopolish. In order to obtain native mtDNA, we adjusted a whole-genome sequencing protocol and performed ligation library preparation and Nanopore sequencing. To validate the workflow, 897bp of methylated and unmethylated synthetic DNA samples at different dilution ratios were sequenced and CpG methylation was detected. Interestingly, we observed that reads with higher methylation in the synthetic DNA did not pass Guppy calling, possibly affecting conclusions about DNA methylation in Nanopore sequencing. We detected in all blood-derived samples overall low-level methylation across the mitochondrial genome, with exceptions at certain CpG sites. Our results suggest that Nanopore sequencing is capable of detecting low-level mtDNA methylation. However, further refinement of the bioinformatical pipelines including Guppy failed reads are recommended.

scholarly journals Bisulfite treatment and single-molecule real-time sequencing reveal D-loop length, position, and distribution

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Shanaya Shital Shah ◽  
Stella R Hartono ◽  
Frédéric Chédin ◽  
Wolf-Dietrich Heyer
Keyword(s):  
Real Time ◽  
Single Molecule ◽  
Detection Methods ◽  
Loop Formation ◽  
Vitro Assay ◽  
Bisulfite Treatment ◽  
Loop Detection ◽  
D Loop ◽  
Donor Choice

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 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-loops with near 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. 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.

Single-molecule regulatory architectures captured by chromatin fiber sequencing

Science ◽  
2020 ◽  
Vol 368 (6498) ◽  
pp. 1449-1454 ◽  
Author(s):  
Andrew B. Stergachis ◽  
Brian M. Debo ◽  
Eric Haugen ◽  
L. Stirling Churchman ◽  
John A. Stamatoyannopoulos
Keyword(s):  
Gene Regulation ◽  
Single Molecule ◽  
Regulatory Elements ◽  
Dna Templates ◽  
Large Numbers ◽  
Long Read ◽  
Chromatin Fibers ◽  
Nucleotide Resolution ◽  
Regulatory Dna ◽  
Transcription Factor Occupancy

Gene regulation is chiefly determined at the level of individual linear chromatin molecules, yet our current understanding of cis-regulatory architectures derives from fragmented sampling of large numbers of disparate molecules. We developed an approach for precisely stenciling the structure of individual chromatin fibers onto their composite DNA templates using nonspecific DNA N6-adenine methyltransferases. Single-molecule long-read sequencing of chromatin stencils enabled nucleotide-resolution readout of the primary architecture of multikilobase chromatin fibers (Fiber-seq). Fiber-seq exposed widespread plasticity in the linear organization of individual chromatin fibers and illuminated principles guiding regulatory DNA actuation, the coordinated actuation of neighboring regulatory elements, single-molecule nucleosome positioning, and single-molecule transcription factor occupancy. Our approach and results open new vistas on the primary architecture of gene regulation.

scholarly journals Defining the location of promoter-associated R-loops at near-nucleotide resolution using bisDRIP-seq

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Jason G Dumelie ◽  
Samie R Jaffrey
Keyword(s):  
Gene Expression ◽  
Loop Formation ◽  
Transcription Start ◽  
Critical Determinant ◽  
Complementary Dna ◽  
Intronless Genes ◽  
High Resolution Map ◽  
Dna Strand ◽  
Nucleotide Resolution ◽  
Mcf 7

R-loops are features of chromatin consisting of a strand of DNA hybridized to RNA, as well as the expelled complementary DNA strand. R-loops are enriched at promoters where they have recently been shown to have important roles in modifying gene expression. However, the location of promoter-associated R-loops and the genomic domains they perturb to modify gene expression remain unclear. To resolve this issue, we developed a bisulfite-based approach, bisDRIP-seq, to map R-loops across the genome at near-nucleotide resolution in MCF-7 cells. We found the location of promoter-associated R-loops is dependent on the presence of introns. In intron-containing genes, R-loops are bounded between the transcription start site and the first exon-intron junction. In intronless genes, the 3' boundary displays gene-specific heterogeneity. Moreover, intronless genes are often associated with promoter-associated R-loop formation. Together, these studies provide a high-resolution map of R-loops and identify gene structure as a critical determinant of R-loop formation.

scholarly journals RAD51 Inhibition Induces R-Loop Formation in Early G1 Phase of the Cell Cycle

2021 ◽  
Vol 22 (7) ◽  
pp. 3740
Author(s):  
Zuzana Nascakova ◽  
Barbora Boleslavska ◽  
Vaclav Urban ◽  
Anna Oravetzova ◽  
Edita Vlachova ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Homologous Recombination ◽  
Chromosome Instability ◽  
Regulation Of Gene Expression ◽  
Protein A ◽  
G1 Phase ◽  
Loop Formation ◽  
Origin Firing ◽  
Dna Strand ◽  
Template Dna

R-loops are three-stranded structures generated by annealing of nascent transcripts to the template DNA strand, leaving the non-template DNA strand exposed as a single-stranded loop. Although R-loops play important roles in physiological processes such as regulation of gene expression, mitochondrial DNA replication, or immunoglobulin class switch recombination, dysregulation of the R-loop metabolism poses a threat to the stability of the genome. A previous study in yeast has shown that the homologous recombination machinery contributes to the formation of R-loops and associated chromosome instability. On the contrary, here, we demonstrate that depletion of the key homologous recombination factor, RAD51, as well as RAD51 inhibition by the B02 inhibitor did not prevent R-loop formation induced by the inhibition of spliceosome assembly in human cells. However, we noticed that treatment of cells with B02 resulted in RAD51-dependent accumulation of R-loops in an early G1 phase of the cell cycle accompanied by a decrease in the levels of chromatin-bound ORC2 protein, a component of the pre-replication complex, and an increase in DNA synthesis. Our results suggest that B02-induced R-loops might cause a premature origin firing.

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