scholarly journals A novel enrichment strategy reveals unprecedented number of novel transcription start sites at single base resolution in a model prokaryote and the gut microbiome

2015 ◽  
Author(s):  
Laurence Ettwiller ◽  
John Buswell ◽  
Erbay Yigit ◽  
Ira Schildkraut
Keyword(s):  
Transcription Start ◽  
Single Base ◽  
Transcription Start Sites ◽  
E Coli ◽  
Rna Transcripts ◽  
Genome Wide ◽  
Single Base Resolution ◽  
Mouse Cecum ◽  
First Time ◽  
Enrichment Strategy

We have developed Cappable-seq that specifically captures primary RNA transcripts by enzymatically modifying the 5' triphosphorylated end of RNA with a selectable tag. We first applied Cappable-seq to E. coli , achieving up to 50 fold enrichment of primary transcripts and identifying an unprecedented 16539 transcription start sites (TSS) genome-wide at single base resolution. We also applied Cappable-seq to a mouse cecum sample and for the first time identified TSS in a microbiome. Furthermore, Cappable-seq universally depletes ribosomal RNA and reduces the complexity of the transcriptome to a single quantifiable tag per TSS enabling digital profiling of gene expression in any microbiome.

scholarly journals Genome-Wide Identification of Transcription Start Sites, Promoters and Transcription Factor Binding Sites in E. coli

PLoS ONE ◽  
2009 ◽  
Vol 4 (10) ◽  
pp. e7526 ◽  
Author(s):  
Alfredo Mendoza-Vargas ◽  
Leticia Olvera ◽  
Maricela Olvera ◽  
Ricardo Grande ◽  
Leticia Vega-Alvarado ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Binding Sites ◽  
Transcription Factor Binding Sites ◽  
Transcription Factor Binding ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
E Coli ◽  
Factor Binding ◽  
Genome Wide

scholarly journals Bacterial Epigenomics: Coming of Age

mSystems ◽  
2021 ◽  
Author(s):  
Pedro H. Oliveira
Keyword(s):  
Dna Methylation ◽  
Coming Of Age ◽  
Innate Immune ◽  
Physiological Significance ◽  
Single Base ◽  
Genome Wide Analysis ◽  
Genome Wide ◽  
Single Base Resolution

Epigenetic DNA methylation in bacteria has been traditionally studied in the context of antiparasitic defense and as part of the innate immune discrimination between self and nonself DNA. However, sequencing advances that allow genome-wide analysis of DNA methylation at the single-base resolution are nowadays expanding and have propelled a modern epigenomic revolution in our understanding of the extent, evolution, and physiological significance of methylation.

scholarly journals Timing Polymerase Pausing with TV-PRO-seq

2018 ◽  
Author(s):  
Jie Zhang ◽  
Massimo Cavallaro ◽  
Daniel Hebenstreit
Keyword(s):  
High Resolution ◽  
Human Cells ◽  
Rrna Genes ◽  
Dna Motifs ◽  
Single Base ◽  
Chromatin States ◽  
Genome Wide ◽  
A Genome ◽  
Single Base Resolution ◽  
Polymerase Pausing

Transcription of many genes in metazoans is subject to polymerase pausing, which corresponds to the transient arrest of transcriptionally engaged polymerase. It occurs mainly at promoter proximal regions and is not well understood. In particular, a genome-wide measurement of pausing times at high resolution has been lacking.We present here an extension of PRO-seq, time variant PRO-seq (TV-PRO-seq), that allowed us to estimate genome-wide pausing times at single base resolution. Its application to human cells reveals that promoter proximal pausing is surprisingly short compared to other regions and displays an intricate pattern. We also find precisely conserved pausing profiles at tRNA and rRNA genes and identified DNA motifs associated with pausing time. Finally, we show how chromatin states reflect differences in pausing times.

scholarly journals Genome-Wide Identification of Estrogen Receptor α-Binding Sites in Mouse Liver

2008 ◽  
Vol 22 (1) ◽  
pp. 10-22 ◽  
Author(s):  
Hui Gao ◽  
Susann Fält ◽  
Albin Sandelin ◽  
Jan-Åke Gustafsson ◽  
Karin Dahlman-Wright
Keyword(s):  
Estrogen Receptor ◽  
Binding Sites ◽  
Mouse Liver ◽  
Estrogen Receptor Α ◽  
Estrogen Signaling ◽  
Transcription Start ◽  
Expression Levels ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Binding Regions

Abstract We report the genome-wide identification of estrogen receptor α (ERα)-binding regions in mouse liver using a combination of chromatin immunoprecipitation and tiled microarrays that cover all nonrepetitive sequences in the mouse genome. This analysis identified 5568 ERα-binding regions. In agreement with what has previously been reported for human cell lines, many ERα-binding regions are located far away from transcription start sites; approximately 40% of ERα-binding regions are located within 10 kb of annotated transcription start sites. Almost 50% of ERα-binding regions overlap genes. The majority of ERα-binding regions lie in regions that are evolutionarily conserved between human and mouse. Motif-finding algorithms identified the estrogen response element, and variants thereof, together with binding sites for activator protein 1, basic-helix-loop-helix proteins, ETS proteins, and Forkhead proteins as the most common motifs present in identified ERα-binding regions. To correlate ERα binding to the promoter of specific genes, with changes in expression levels of the corresponding mRNAs, expression levels of selected mRNAs were assayed in livers 2, 4, and 6 h after treatment with ERα-selective agonist propyl pyrazole triol. Five of these eight selected genes, Shp, Stat3, Pdgds, Pck1, and Pdk4, all responded to propyl pyrazole triol after 4 h treatment. These results extend our previous studies using gene expression profiling to characterize estrogen signaling in mouse liver, by characterizing the first step in this signaling cascade, the binding of ERα to DNA in intact chromatin.

scholarly journals Nascent RNA sequencing identifies a widespread sigma70-dependent pausing regulated by Gre factors in bacteria

2020 ◽  
Author(s):  
Zhe Sun ◽  
Alexander Yakhnin ◽  
Peter C. FitzGerald ◽  
Carl E. Mclntosh ◽  
Mikhail Kashlev
Keyword(s):  
Rna Polymerase ◽  
Transcription Start Site ◽  
Environmental Cues ◽  
Start Site ◽  
Transcription Start ◽  
E Coli ◽  
Eukaryotic Genes ◽  
Genome Wide ◽  
Transcript Cleavage

ABSTRACTPromoter-proximal pausing regulates expression of many eukaryotic genes and serves as checkpoints for assembly of elongation/splicing machinery. Little is known how broadly the pausing is employed in transcriptional regulation in bacteria. We applied NET-seq combined with RNase I footprinting for genome-wide analysis of σ70-dependent transcription pauses in Escherichia coli. Many E. coli genes appear to contain clusters of strong backtracked pauses at 10-20-bp distance from the transcription start site caused by retention of σ70 subunit in RNA polymerase. The pauses in 10-15-bp register of the promoter are dictated by binding of σ70 to canonical −10 element, 6-7 nt spacer and “YR+1Y” motif centered at transcription start site all characteristic for strong E. coli promoters. The promoters for the pauses in 16-20-bp register contain an additional −10-like sequence positioned on the same face of the DNA duplex as the original −10 element suggesting that σ70 hopping was responsible for these pauses. Our in vitro analysis reveals that RNA polymerase backtracking and DNA scrunching are involved in these pauses that are relieved by Gre transcript cleavage factors. The genes coding for transcription factors are enriched in these pauses suggesting that σ70 and Gre proteins regulate transcription in response to changing environmental cues.

scholarly journals Dynamics of transcription-dependent H3K36me3 marking by the SETD2:IWS1:SPT6 ternary complex

2019 ◽  
Author(s):  
Katerina Cermakova ◽  
Eric A. Smith ◽  
Vaclav Veverka ◽  
H. Courtney Hodges
Keyword(s):  
Ternary Complex ◽  
Spatial Distributions ◽  
Epigenetic Memory ◽  
Dynamic Features ◽  
Transcription Start ◽  
Accurate Representation ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Genome Wide Data ◽  
Tight Correlation

AbstractSETD2 contributes to gene expression by marking gene bodies with H3K36me3, which is thought to assist in the concentration of transcription machinery at the small portion of the coding genome. Despite extensive genome-wide data revealing the precise localization of H3K36me3 over gene bodies, the physical basis for the accumulation, maintenance, and sharp borders of H3K36me3 over these sites remains rudimentary. Here we propose a model of H3K36me3 marking based on stochastic transcription-dependent placement and transcription-independent spreading. Our analysis of the spatial distributions and dynamic features of these marks indicates that transcription-dependent placement dominates the establishment of H3K36me3 domains compared to transcription-independent spreading processes, and that turnover of H3K36me3 limits its capacity for epigenetic memory. By adding additional terms for asymmetric histone turnover occurring at transcription start sites, our model provides a remarkably accurate representation of H3K36me3 levels and dynamics over gene bodies. Furthermore, we validate our findings by revealing that loss of SPT6 impairs the transcription-coupled activity of the SETD2:IWS1:SPT6 ternary complex, thereby reducing the tight correlation between transcription and H3K36me3 levels at gene bodies.

scholarly journals Mirror bisulfite sequencing — a method for single-base resolution of hydroxymethylcytosine

2015 ◽  
Author(s):  
Xueguang Sun ◽  
Darany Tan ◽  
Tzu Hung Chung ◽  
Xi-Yu Jia
Keyword(s):  
Bisulfite Sequencing ◽  
Biological Role ◽  
Human Tissues ◽  
Bisulfite Conversion ◽  
Single Base ◽  
Cpg Site ◽  
Genome Wide ◽  
Single Base Resolution ◽  
Dna Strand

While the role of 5-methylcytosine has been well studied, the biological role of 5-hydroxymethylcytosine still remains unclear due to the limited methods available for single-base detection of 5-hydroxymethylcytosine (5hmC). Here, we present Mirror bisulfite sequencing detects 5-hydroxymethylcytosines at a single CpG site by synthesizing a DNA strand to mirror the parental strand. This semi-conservative duplex is sequentially treated with β-glucosyltransferase and M.SssI methylase. A glucosyl-5hmCpG in the parental strand inhibits methylation of the mirroring CpG site, and after bisulfite conversion, a thymine in the mirroring strand indicates a 5hmCpG site in the parental strand whereas a cytosine indicates a non-5hmC site. Using this method, the 5hmC levels of various human tissues and paired liver tissues were mapped genome-wide.

scholarly journals DChIPRep, an R/Bioconductor package for differential enrichment analysis in chromatin studies

2016 ◽  
Author(s):  
Christophe D Chabbert ◽  
Lars M Steinmetz ◽  
Bernd Klaus
Keyword(s):  
Input Data ◽  
Enrichment Analysis ◽  
Bioconductor Package ◽  
Analytic Framework ◽  
Transcription Start ◽  
Transcription Start Sites ◽  
Genome Wide ◽  
Bioconductor Project ◽  
User Friendly ◽  
Genome Wide Study

The genome–wide study of epigenetic states requires the integrative analysis of histone modification ChIP–seq data. Here, we introduce an easy–to–use analytic framework to compare profiles of enrichment in histone modifications around classes of genomic elements, e.g. transcription start sites (TSS). Our framework is available via the user–friendly R/Bioconductor package DChIPRep. DChIPRep uses biological replicate information as well as chromatin Input data to allow for a rigorous assessment of differential enrichment. DChIPRep is available for download through the Bioconductor project at http://bioconductor.org/packages/DChIPRep. Contact [email protected]

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