ABSTRACT: This study profiles RNA:DNA hybrid formation in human and mouse cell lines. DRIPc-seq (strand-specific R-loop mapping) was performed on human NT2 cells and mouse 3T3 cells. DRIP-seq (R-loop mapping) was performed on human NT2 and K562 and mouse E14 and 3T3 cell lines. MethylC-seq and RNA-seq were performed on NT2.
Project description:R loops form when transcripts hybridize to homologous DNA on chromosomes, yielding a DNA:RNA hybrid and a displaced DNA single strand. R loops impact the genome of many organisms, regulating chromosome stability, gene expression, and DNA repair. 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. We report a novel whole-genome method, S1-DRIP-seq (S1 nuclease DNA:RNA immunoprecipitation with deep sequencing), for mapping hybrid-prone regions in budding yeast Saccharomyces cerevisiae Using this methodology, we identified ?800 hybrid-prone regions covering 8% of the genome. Given the pervasive transcription of the yeast genome, this result suggests that R-loop formation is dictated by characteristics of the DNA, RNA, and/or chromatin. We successfully identified two features highly predictive of hybrid formation: high transcription and long homopolymeric dA:dT tracts. These accounted for >60% of the hybrid regions found in the genome. We demonstrated that these two factors play a causal role in hybrid formation by genetic manipulation. 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.
Project description:R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.
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.
Project description:Restricted contact with the external environment has allowed the development of microbial communities adapted to the oligotrophy of caves. However, nutrients can be transported to caves by drip water and affect the microbial communities inside the cave. To evaluate the influence of aromatic compounds carried by drip water on the microbial community, two limestone caves were selected in Brazil. Drip-water-saturated and unsaturated sediment, and dripping water itself, were collected from each cave and bacterial 16S rDNA amplicon sequencing and denaturing gradient gel electrophoresis (DGGE) of naphthalene dioxygenase (ndo) genes were performed. Energy-dispersive X-ray spectroscopy (EDX) and atomic absorption spectroscopy (AAS) were performed to evaluate inorganic nutrients, and GC was performed to estimate aromatic compounds in the samples. The high frequency of Sphingomonadaceae in drip water samples indicates the presence of aromatic hydrocarbon-degrading bacteria. This finding was consistent with the detection of naphthalene and acenaphthene and the presence of ndo genes in drip-water-related samples. The aromatic compounds, aromatic hydrocarbon-degrading bacteria and 16S rDNA sequencing indicate that aromatic compounds may be one of the sources of energy and carbon to the system and the drip-water-associated bacterial community contains several potentially aromatic hydrocarbon-degrading bacteria. To the best of our knowledge, this is the first work to present compelling evidence for the presence of aromatic hydrocarbon-degrading bacteria in cave drip water.
Project description:This study describes the first use of concurrent high-precision temperature and drip rate monitoring to explore what controls the temperature of speleothem forming drip water. Two contrasting sites, one with fast transient and one with slow constant dripping, in a temperate semi-arid location (Wellington, NSW, Australia), exhibit drip water temperatures which deviate significantly from the cave air temperature. We confirm the hypothesis that evaporative cooling is the dominant, but so far unattributed, control causing significant disequilibrium between drip water and host rock/air temperatures. The amount of cooling is dependent on the drip rate, relative humidity and ventilation. Our results have implications for the interpretation of temperature-sensitive, speleothem climate proxies such as δ(18)O, cave microecology and the use of heat as a tracer in karst. Understanding the processes controlling the temperature of speleothem-forming cave drip waters is vital for assessing the reliability of such deposits as archives of climate change.
Project description:BACKGROUND:Chulalongkorn Stroke Center is a comprehensive stroke center (CSC) located in Bangkok, Thailand. Our stroke network consists of different levels of spoke hospitals, ranging from community hospitals where thrombolytic treatment is not available, to those capable of onsite thrombolytic therapy. This study aimed to assess the time to treatment and outcomes among acute ischemic stroke patients who received thrombolytic treatment in the Chulalongkorn Stroke Network by 1.) Direct arrival at the CSC (mothership) 2.) Telestroke-assisted thrombolytic treatment with secondary transfer to the CSC (drip-and-ship) 3.) Referral from community hospital to the CSC for thrombolytic treatment (ship-and-drip). METHODS:Acute ischemic stroke patients who received thrombolytic treatment during January 2016-December 2017 in the Chulalongkorn Stroke Network were studied. Time to treatment and clinical outcomes were compared among treatment groups. RESULTS:There were 273 patients in the study including 147, 87, and 39 patients in mothership, drip-and-ship, and ship-and-drip paradigms, respectively. The door-to-needle-time (DTN) and onset-to-needle-time (OTN) times were significantly longest in ship-and-drip group (146.5?±?62/205.03?±?44.88 mins) compared to mothership (38?±?23/155.2?±?60.54 mins) and drip-and-ship (63.0?±?44/166.09?±?87 mins), P?<?0.05. There was no significant difference regarding functional independence defined by modified Rankin Scale (mRS)???2 at 3?months (P?=?0.12), in-hospital mortality (P?=?0.37), mortality at 3?months (P?=?0.73), and symptomatic intracerebral hemorrhage (P?=?0.24) among groups. CONCLUSION:Thrombolytic treatment with drip and ship method under teleconsultation is feasible in Thailand. There was no difference of clinical outcome among the 3 treatment paradigms. However, DTN time and OTN time were longest in the ship-and-drip paradigm.
Project description:DNA Double Strands Breaks (DSBs) are highly detrimental since they can lead to mutations and chromosomes rearrangements (amplification, deletion, translocation and chromosome loss). Here, we set to assess the role of senataxin, a RNA:DNA helicase involved in the regulation of transcription and the maintenance of genome integrity, at sites of DNA Double Strand Breaks. We performed ChIP-Seq mapping of senataxin before and after damage, genome-wide DNA:RNA hybrids (DRIP) mapping before and after damage. We also performed RNA-Seq and RNA pol II mapping (total and phosphorylated on serine 2 of the CTD) by ChIP-Seq in undamaged cells to discriminate between damage in active versus inactive regions.
Project description:Cough affects all individuals at different times, and its economic burden is substantial. Despite these widespread adverse effects, cough research relies on animal models, which hampers our understanding of the fundamental cause of cough. Postnasal drip is speculated to be one of the most frequent causes of chronic cough; however, this is a matter of debate. Here we show that mechanical stimuli by postnasal drip cause chronic cough. We distinguished human cough from sneezes and expiration reflexes by airflow patterns. Cough and sneeze exhibited one-peak and two-peak patterns, respectively, in expiratory airflow, which were also confirmed by animal models of cough and sneeze. Transgenic mice with ciliary dyskinesia coughed substantially and showed postnasal drip in the pharynx; furthermore, their cough was completely inhibited by nasal airway blockade of postnasal drip. We successfully reproduced cough observed in these mice by injecting artificial postnasal drip in wild-type mice. These results demonstrated that mechanical stimulation by postnasal drip evoked cough. The findings of our study can therefore be used to develop new antitussive drugs that prevent the root cause of cough.
Project description:BACKGROUND:Drip loss is a key aspect of meat quality. Transcriptome profiles of muscle with divergent drip loss would offer important insight into the genetic factors responsible for the trait. In this study, drip loss and other meat quality traits of 28 purebred Duroc pigs were measured, muscles of these individuals were RNA sequenced, and eight individuals with extremely low and high drip loss were selected for analyzing their transcriptome differences and identifying potential candidate genes affecting drip loss. RESULTS:As a result, 363 differentially expressed (DE) genes were detected in the comparative gene expression analysis, of which 239 were up-regulated and 124 were down-regulated in the low drip loss group. The DE genes were further filtered by correlation analysis between their expression and drip loss values in the 28 Duroc pigs measured and comparison of them with QTLs affecting drip loss. Consequently, of the 363 DE genes, 100 were identified as critical DE genes for drip loss. Functional analysis of these critical DE genes revealed some GO terms (extracellular matrix, cell adhesion mediated by integrin, heterotypic cell-cell adhesion), pathway (ECM-receptor interaction), and new potential candidate genes (TNC, ITGA5, ITGA11, THBS3 and CD44) which played an important role in regulating the variation of drip loss, and deserved to carry further studies to unravel their specific mechanism on drip loss. CONCLUSIONS:Our study revealed some GO terms, pathways and potential candidate genes affecting drip loss. It provides crucial information to understand the molecular mechanism of drip loss, and would be of help for improving meat quality of pigs.