🤑 S1-DRIP-seq identifies high expression and polyA tracts as major contributors to R-loop formation

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Chromatin Immunoprecipitation. Immunofuorescence with s antibody. Genomic extraction for R loops detection. Dot blot for​.


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r-loop dot blot

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Dot blot: R-Loop. General notes. The pCALM3_2 vector used to generate R-​loops was kindly provided by Prof. Frederic Chedin at UC Davis.


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r-loop dot blot

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antisense lncRNA can be generated by R-loops that form when nascent transcript invades B S validation by dot blot western. B 50 RACE.


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Dot blot analysis was performed according to the protocol reported elsewhere (​41). Genomic DNA was isolated by standard extraction with.


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Quantifying R-Loop Formation Using Dot Blots. When properly controlled using RNase H treatment and accurate DNA quantification, dot blot.


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Traditionally, R-loops have been analyzed by electron microscopy, dot-blot hybridization and immunostaining. Although useful for visualizing.


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Dot blot analysis was performed according to the protocol reported elsewhere (​41). Genomic DNA was isolated by standard extraction with.


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To address this, quiescent cells were exposed to CPT and R-loop levels were assessed by a DNA slot blot assay employing S antibody.


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Dot blot analysis was performed according to the protocol reported elsewhere (​41). Genomic DNA was isolated by standard extraction with.


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antisense lncRNA can be generated by R-loops that form when nascent transcript invades B S validation by dot blot western. B 50 RACE.


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r-loop dot blot

Hybrids were stabilized by treatment with S1 nuclease, which preferentially degraded single-stranded nucleic acids prior to sonication. The ability to map R loops was made feasible by the discovery of the S9. Other hybrid regions showed asymmetry that extended beyond the most terminal bins or was internal to the ORF and sufficiently varied that it was not possible to define any additional specific categories. We found similar hybrid levels accumulated at the regions coding for mature transcripts and the transcribed spacers, an indication that these hybrids were not likely to have formed artifactually after lysis with processed rRNA transcripts Supplemental Fig. This striking correlation was strengthened when we relaxed the stringency for calling hybrid-prone regions to include those identified in two out of four biological replicates and also accounted for hybrids present in duplicated genes that had previously fallen into the repetitive DNA category. These results are consistent with high levels of gene transcription driving hybrid formation independently of genomic context. Induction of expression with galactose Gal; hatched bars promotes hybrid formation at the endogenous GAL7 gene and at the ectopic SMC3 locus when under the control of the Gal promoter. Given the centrality of R loops in many genomic functions, the precise mapping of where they form and understanding why they form are important biological questions. The proportion of major genome features identified as hybrid-prone. The dotted line indicates the proportion of all features identified as hybrid-prone 9. Yeast genomic DNA was either not treated N. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. The regions identified represent the strongest occurrences of hybrids in the genome, and it is possible that weaker or more transient hybrids occurred elsewhere but were missed by our analyses. B Hybrid-prone features. B Dot blot showing the effect of S1 treatment on genomic R loops. As a second assessment, we determined the percentage of ORFs in each expression category that formed hybrids Fig. The other two studies mapped hybrids in the genome of the budding yeast Saccharomyces cerevisiae , a particularly powerful model used to identify and characterize factors that modulate R-loop formation Luna et al. While it is known that most fungal species have considerable positive AT skew across the lengths of genes McLean and Tirosh , particularly in lower expression categories Supplemental Fig.{/INSERTKEYS}{/PARAGRAPH} With the exception of hybrids in telomeric regions, which occurred largely over the terminal TG 1—3 repeats, no evidence of a significant GC skew pattern was found at hybrid-prone regions Supplemental Fig. Their work provided an excellent paradigm for use of the S9. Using a rapid dot blot R-loop assay Supplemental Fig. The use of cross-linking agents or an alternative fragmentation protocol with nonspecific nucleases did not abrogate this loss data not shown. Under repressed conditions in which Gal-inducible genes are poorly expressed Cloutier et al. While informative, this correlation did not address whether high gene expression was critical to initiate hybrid formation. To quantitatively assess the potential asymmetric distribution of hybrids, we looked for biases in the density of reads along the lengths of hybrid-prone ORFs. A The percentage of ORFs prone to hybrid formation in each expression category. The discrepancies between the two genomic studies as well as previous data likely resulted from inherent limitations in the methodologies that caused low hybrid signal to background noise. Here we present a new S1-DRIP-seq S1 nuclease DRIP with deep sequencing method for mapping hybrids in budding yeast that dramatically improves the signal to noise ratio at hybrid regions. In contrast, few additional hybrid-prone ORFs were identified in the other expression categories. No significant differences were observed except in the highest expression category, where the median GC content for hybrid-prone ORFs was slightly higher than that of nonhybrid ORFs Supplemental Fig. The dotted line is positioned at the expected value 7. Thus, GC content along with expression level might have contributed to hybrid formation in a subset of highly expressed genes but was unlikely to be an important determinant for most of the hybrid-prone regions. We next sought to investigate the pattern of hybrid formation at various representative hybrid-prone regions in the genome. Heat maps of the hybrid-prone ORFs categorized by expression level corroborated this, as ORFs in the two highest expression categories appeared to have more uniformly distributed hybrid regions Supplemental Fig. The frequent presence of hybrids in these classes of structural RNAs represented a significant enrichment and confirmed a propensity for hybrid formation in these genomic features Fig. To test whether the structure of S1-treated R loops would affect sequencing results, we devised a synthetic spike-in mimicking the structure Supplemental Fig. Hybrid regions were also strongly enriched for genes encoding structural RNAs. Thus, hybrid-forming regions were much more likely to occur in ORFs in the two highest expression categories. The signal from hybrid-forming regions was sensitive to RNase H treatment Fig. To eliminate the possibility of methodology-based artifacts, several hybrid regions were also verified throughout this work using a second DRIP methodology relying on restriction enzyme fragmentation RE-DRIP-qPCR in place of S1 treatment and sonication. At the 35S rRNA locus, a long primary transcript is rapidly processed into three mature rRNA transcripts through the elimination of transcribed spacer regions Henras et al. These limitations also precluded these studies from identifying factors dictating hybrid formation. To directly assess potential causality, we tested whether low-expressed genes with no detectable hybrids would form hybrids when expression levels were increased using an inducible gene expression system. These studies revealed that R loops are highly correlated with unmethylated CpG island promoters exhibiting a strong strand asymmetry in the distribution of guanines and cytosines GC skew. D The proportion of each asymmetric category and the remaining genes that is in either the highest two expression categories high, FPKM of — or all the remaining lower expressed categories medium—low, FPKM of —0. Overall, the sequence read densities of peak regions identified by S1-DRIP-seq were significantly enriched over immunoprecipitation background, which enabled detection of distinct sites of R-loop formation at high base-pair resolution in the nuclear genome Fig. Two different hybrid-forming regions—a Ty1 retrotransposon and HSP gene—cloned onto a yeast artificial chromosome YAC still form hybrids. This antibody has been used in three different genome-wide studies to map R loops. The heat map displays the hybrid signal along individual ORFs, sorted according to total signal strength. The sixth row shows features annotated in the Saccharomyces Genome Database, with features overlapping with hybrid regions indicated below. Hybrids were then immunoprecipitated with S9. While the different structural and mitochondrial RNAs might have different characteristics predisposing them to hybrid formation, they all share a common attribute, which is high levels of transcription. However, when levels of expression were pushed above the hybrid-associated threshold by growth in Gal, hybrids were detected in both GAL7 and SMC3 but not at a control locus Fig. Forty-two percent of the ORF-associated hybrid regions were in ORFs in the two highest expression categories, an approximately four times enrichment over expected if hybrid formation was independent of transcription levels Fig. Genomic distribution of hybrid-prone regions. B Hybrid-prone ORFs with asymmetric hybrid signals. We used the dot blot assay to determine the amount of S1 nuclease that would sufficiently preserve DNA:RNA hybrids during sonication without impinging on initial hybrid levels. R loops have emerged as prominent genomic feature in many organisms, from bacteria to humans Santos-Pereira and Aguilera R loops can act as precursors to genomic instability, modulators of gene expression, and regulators of chromatin epigenetic marks Huertas and Aguilera ; Li and Manley ; Nakama et al. In human cells, genomic regions with GC skew are prone to hybrid formation Ginno et al. The high signal to noise ratio in the S1-DRIP-seq data was a significant improvement on the data obtained by ChIP-seq and DRIP-chip experiments that had likely led to the underestimation and overestimation of the number of hybrid-prone sites, respectively Supplemental Fig. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. Furthermore, both studies failed to detect major differences between wild-type and RNase H-defective cells. This method allows for quantitative recovery of R loops and precise mapping of hybrid locations, allowing us to elucidate the parameters and sequence features that predispose parts of the genome to R-loop formation in vivo. We speculated that the energy introduced by sonication promoted branch migration, causing the displacement of the RNA and reannealing of the ssDNA molecules. Next, we looked for the presence of AT skew in hybrid-prone regions. However, marked differences between the two genome-wide studies and previous work on R loops in yeast were observed. Understanding the parameters dictating R-loop formation in vivo has been hampered by the limited quantitative and spatial resolution of current genomic strategies for mapping R loops. C Asymmetric hybrid formation. To further investigate the link between hybrid formation and gene expression, we rank-ordered the expression level of all yeast ORFs based on transcript abundance, divided them into 20 expression categories, and cross-compared them with hybrid-forming regions van Dijk et al. We conclude that when gene expression levels exceed a certain threshold, transcription itself becomes a determinant of hybrid formation. Reads were aligned to the reference genome using the Bowtie2 algorithm; hybrid-prone regions were identified in uniquely mapped reads using the model-based analysis of ChIP-seq MACS2 peak-calling algorithm. Using these correlative studies, we discovered and, through genetic manipulations, validated two features—high expression levels and polyA tracts—as causal determinants for hybrid formation that account for a large fraction of hybrids in the yeast genome. The robust correlation between high transcription levels and hybrid formation implied a causal relationship between them. We conclude that DNA fragments resulting from S1-DRIP are efficient substrates for sequencing and show little sequencing bias stemming from their nonstandard structure. Yeast ORFs were divided into 20 categories based on their expression FPKM [fragments per kilobase per million mapped fragments] , and each bar indicates the percentage of all hybrid-prone ORFs found in each expression category. {PARAGRAPH}{INSERTKEYS}R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. We began by comparing the GC content of hybrid-prone ORFs in the different expression categories with their nonhybrid counterparts. Taken together, our results demonstrated that hybrids form at Ty elements in the genome of wild-type cells, but their levels are kept low by RNase H. Note that the normalized read coverage value over the rDNA is amplified because of the tandem repeat nature of the locus and that the hybrid signal at each rDNA repeat in the absence of RNase H is actually similar to that at other hybrid-prone features in the genome Fig. For example, the DRIP-chip study inferred that a large portion of the nuclear genome around one-third is prone to hybrid formation, while the ChIP-seq study suggested that a small fraction of loci could form hybrids; notably, transfer RNAs tRNAs , rDNA, and a few highly expressed genes. Thus, even with this relaxed stringency, most transcripts failed to lead to detectable hybrid formation. Finally, extensive R-loop formation was also detected in the mitochondrial genome Supplemental Fig. To test whether high expression alone is sufficient to drive hybrid formation, we introduced two highly expressed hybrid regions—a Ty element and HSP —onto a yeast artificial chromosome YAC. Thus, the hybrid map generated by S1-DRIP-seq led to the identification of the first global genomic features causal for R-loop formation in yeast. Immunoprecipitation of the spike-in using the S9. B The percentage of ORFs in each expression category that overlaps a hybrid. Successful capture of both strands indicated that the plus strand was extended during end repair to form a more canonical dsDNA substrate for sequencing Supplemental Fig. The distribution of hybrids at ORFs. Sequenced reads were normalized to the genome-wide mean across all nonpeak regions, and regions reported here as hybrid-prone and used in our analyses were identified in at least three of the four biological replicates. Taken together, these analyses revealed that the two highest expression categories are highly predictive of whether ORFs form hybrids. ORFs in the 18 remaining expression categories span over three orders of magnitude of expression level, yet there is no correlation with hybrid formation. These ORFs spanned a wide range of gene expression levels. For genes transcribed below that threshold, additional factors other than transcription level must be involved in hybrid formation. The distribution of sequence reads observed at genomic hybrid regions was as predicted based on the spike-in sequencing results Supplemental Fig. We found that regions associated with medium—low expression categories had a positive AT skew, leading to an enrichment of A bases in the coding strand Fig. P -values were generated by a one-tailed Fisher's test. Improvement of both the spatial and quantitative mapping of hybrids provided by our S1-DRIP-seq methodology demonstrated its utility for identifying features linked to hybrid formation genome-wide. However, the limited spatial resolution associated with the methodology may have precluded the discovery of additional hybrid-forming features.