scholarly journals Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq)

2021 ◽  
Author(s):  
Jane Mellor ◽  
Phillip Lorenz ◽  
Anna Lamstaes ◽  
Harry J Fischl ◽  
Shidong Xi ◽  
...  
Keyword(s):  
Low Cost ◽  
Cost Effective ◽  
Pulse Labelling ◽  
Single Nucleotide ◽  
Functional Regions ◽  
Nascent Rna ◽  
Nucleotide Resolution ◽  
Nascent Transcripts ◽  
The Impact ◽  
Single Nucleotide Resolution

Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3' most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3' RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ~100nt transcripts that map to the 5' end of genes. We show that isolation of these short nascent RNA and sequencing the 5' and 3' ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution.

scholarly journals Plants regenerated from tissue culture contain stable epigenome changes in rice

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Hume Stroud ◽  
Bo Ding ◽  
Stacey A Simon ◽  
Suhua Feng ◽  
Maria Bellizzi ◽  
...  
Keyword(s):  
Tissue Culture ◽  
Phenotypic Variability ◽  
Whole Genome ◽  
Single Nucleotide ◽  
Protein Coding ◽  
Protein Coding Genes ◽  
Regenerated Plants ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

Most transgenic crops are produced through tissue culture. The impact of utilizing such methods on the plant epigenome is poorly understood. Here we generated whole-genome, single-nucleotide resolution maps of DNA methylation in several regenerated rice lines. We found that all tested regenerated plants had significant losses of methylation compared to non-regenerated plants. Loss of methylation was largely stable across generations, and certain sites in the genome were particularly susceptible to loss of methylation. Loss of methylation at promoters was associated with deregulated expression of protein-coding genes. Analyses of callus and untransformed plants regenerated from callus indicated that loss of methylation is stochastically induced at the tissue culture step. These changes in methylation may explain a component of somaclonal variation, a phenomenon in which plants derived from tissue culture manifest phenotypic variability.

scholarly journals Cisplatin-DNA adduct repair of transcribed genes is controlled by two circadian programs in mouse tissues

2018 ◽  
Vol 115 (21) ◽  
pp. E4777-E4785 ◽  
Author(s):  
Yanyan Yang ◽  
Ogun Adebali ◽  
Gang Wu ◽  
Christopher P. Selby ◽  
Yi-Ying Chiou ◽  
...  
Keyword(s):  
Excision Repair ◽  
Therapeutic Index ◽  
Future Research ◽  
Repair Capacity ◽  
Single Nucleotide ◽  
Circadian Time ◽  
Mouse Tissues ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

Cisplatin is a major cancer chemotherapeutic drug. It kills cancer cells by damaging their DNA, mainly in the form of Pt-d(GpG) diadducts. However, it also has serious side effects, including nephrotoxicity and hepatotoxicity that limit its usefulness. Chronotherapy is taking circadian time into account during therapy to improve the therapeutic index, by improving efficacy and/or limiting toxicity. To this end, we tested the impact of clock time on excision repair of cisplatin-induced DNA damage at single-nucleotide resolution across the genome in mouse kidney and liver. We found that genome repair is controlled by two circadian programs. Repair of the transcribed strand (TS) of active, circadian-controlled genes is dictated by each gene’s phase of transcription, which falls across the circadian cycle with prominent peaks at dawn and dusk. In contrast, repair of the nontranscribed strand (NTS) of all genes, repair of intergenic DNA, and global repair overall peaks at Zeitgeber time ZT08, as basal repair capacity, which is controlled by the circadian clock, peaks at this circadian time. Consequently, the TS and NTS of many genes are repaired out of phase. As most cancers are thought to have defective circadian rhythms, these results suggest that future research on timed dosage of cisplatin could potentially reduce damage to healthy tissue and improve its therapeutic index.

scholarly journals Reproducible inference of transcription factor footprints in ATAC-seq and DNase-seq datasets via protocol-specific bias modeling

2018 ◽  
Author(s):  
Aslihan Karabacak Calviello ◽  
Antje Hirsekorn ◽  
Ricardo Wurmus ◽  
Dilmurat Yusuf ◽  
Uwe Ohler
Keyword(s):  
Transcription Factor ◽  
Open Chromatin ◽  
Specific Sequence ◽  
Single Nucleotide ◽  
Genome Wide ◽  
Distinct Sequence ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Sequence Bias ◽  
Single Nucleotide Resolution

ABSTRACTDNase-seq and ATAC-seq are broadly used methods to assay open chromatin regions genome-wide. The single nucleotide resolution of DNase-seq has been further exploited to infer transcription factor binding sites (TFBS) in regulatory regions via footprinting. Recent studies have demonstrated the sequence bias of DNase I and its adverse effects on footprinting efficiency. However, footprinting and the impact of sequence bias have not been extensively studied for ATAC-seq. Here, we undertake a systematic comparison of the two methods and show that a modification to the ATAC-seq protocol increases its yield and its agreement with DNase-seq data from the same cell line. We demonstrate that the two methods have distinct sequence biases and correct for these protocol-specific biases when performing footprinting. Despite differences in footprint shapes, the locations of the inferred footprints in ATAC-seq and DNase-seq are largely concordant. However, the protocol-specific sequence biases in conjunction with the sequence content of TFBSs impacts the discrimination of footprint from background, which leads to one method outperforming the other for some TFs. Finally, we address the depth required for reproducible identification of open chromatin regions and TF footprints.

scholarly journals NickSeq for genome-wide strand-specific identification of DNA single-strand break sites with single nucleotide resolution

2019 ◽  
Author(s):  
Joshua J. Elacqua ◽  
Navpreet Ranu ◽  
Sarah E. Dilorio ◽  
Paul C. Blainey
Keyword(s):  
Genome Editing ◽  
Nuclease Activity ◽  
Single Strand ◽  
Dna Breaks ◽  
Single Nucleotide ◽  
Single Strand Break ◽  
Genome Wide ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

ABSTRACTDNA single-strand breaks (SSBs), or ‘nicks’, are the most common form of DNA damage. Nicks occur at rates of tens of thousands per cell per day, and result from many sources including oxidative stress and endogenous enzyme activities. Accumulation of nicks, due to high rates of occurrence or defects in repair enzymes, has been implicated in multiple diseases. However, improved methods for nick analysis are needed to learn how their locations and number affect cells, disease progression, and health outcomes. In addition to natural processes including DNA repair, leading genome-editing technologies rely on nuclease activity, including nick generation, at target sites. There is currently a pressing need for methods to study unintended nicking activity genome-wide to evaluate the impact of emerging genome editing tools on cells and organisms. Here we developed a new method, NickSeq, for efficient strand-specific profiling of nicks in complex DNA samples with single nucleotide resolution and low false-positive rates. NickSeq produces deep sequence datasets enriched for reads near nick sites and establishes a readily detectable mutational signal that allows for determination of the nick site and strand. In this work, we apply NickSeq to profile off-target activity of the Nb.BsmI nicking endonuclease and an engineered spCas9 nickase. NickSeq will be useful in exploring the relevance of spontaneously occurring or repair-induced DNA breaks in human disease, DNA breaks caused by DNA damaging agents including therapeutics, and the activity of engineered nucleases in genome editing and other biotechnological applications.

scholarly journals Biophysical Principles of Lineage Factor PU.1 Binding Revealed by NextPBMs

2018 ◽  
Author(s):  
Nima Mohaghegh ◽  
David Bray ◽  
Jessica Keenan ◽  
Ashley Penvose ◽  
Kellen K. Andrilenas ◽  
...  
Keyword(s):  
Dna Binding ◽  
Binding Modes ◽  
Single Nucleotide ◽  
Post Translational Modifications ◽  
Genome Wide ◽  
A Cell ◽  
Nucleotide Resolution ◽  
The Impact ◽  
Single Nucleotide Resolution

ABSTRACTDetermining the biophysical principles that shape transcription factor (TF) binding in a cell-specific manner is key to quantitative models of gene expression. High-throughput (HT) in vitro methods measuring protein-DNA binding are invaluable for relating TF binding affinity to genome-wide binding; however, the impact of cell-specific post-translational modifications (PTMs) and cofactors are not routinely assessed. To address these limitations, we describe a new HT approach, called nextPBMs (nuclear extract protein-binding microarrays), to characterize TF binding that accounts for PTMs and endogenous cofactors. We use nextPBMs to examine the DNA binding of the lineage factor PU.1/Spi1 and IRF8 in human monocytes. We identify two binding modes for PU.1 in monocytes – autonomous binding unaffected by PTMs and cooperative binding with IRF8, and identify a single cooperative mode for IRF8. We characterize the DNA binding of PU.1:IRF8 complexes, and show how nextPBMs can be used to discover cell-specific cofactors and characterize TF cooperativity at single-nucleotide resolution. We show that chromatin state and cofactors both influence the affinity requirements for PU.1 binding sites. Furthermore, we find that the influences of cooperative (IRF8) and collaborative (C/EBPα) cofactors on PU.1-binding-site affinity are independent and additive.

Motivating Compliance: Firm Response to Mandatory Disclosure Policies

2018 ◽  
Vol 32 (2) ◽  
pp. 103-119
Author(s):  
Colleen M. Boland ◽  
Chris E. Hogan ◽  
Marilyn F. Johnson
Keyword(s):  
Conflict Of Interest ◽  
Low Cost ◽  
Cost Effective ◽  
Data Availability ◽  
Policy Adoption ◽  
Future Research ◽  
Regulated Firms ◽  
Firm Response ◽  
Disclosure Rules ◽  
The Impact

SYNOPSIS Mandatory existence disclosure rules require an organization to disclose a policy's existence, but not its content. We examine policy adoption frequencies in the year immediately after the IRS required mandatory existence disclosure by nonprofits of various governance policies. We also examine adoption frequencies in the year of the subsequent change from mandatory existence disclosure to a disclose-and-explain regime that required supplemental disclosures about the content and implementation of conflict of interest policies. Our results suggest that in areas where there is unclear regulatory authority, mandatory existence disclosure is an effective and low cost regulatory device for encouraging the adoption of policies desired by regulators, provided those policies are cost-effective for regulated firms to implement. In addition, we find that disclose-and-explain regulatory regimes provide stronger incentives for policy adoption than do mandatory existence disclosure regimes and also discourage “check the box” behavior. Future research should examine the impact of mandatory existence disclosure rules in the year that the regulation is implemented. Data Availability: Data are available from sources cited in the text.

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