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Global repositioning of transcription start sites in a plant-fermenting bacterium.

ABSTRACT: Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5' mRNA fragments with template-switching reverse transcription. We find the locations and expression levels of TSS for hundreds of genes change during metabolism of different plant substrates. We show that TSS reveals riboswitches, non-coding RNA and novel transcription units. We identify sequence motifs associated with carbon source-specific TSS and use them for regulon discovery, implicating a LacI/GalR protein in control of pectin metabolism. We discuss how the high resolution and specificity of Capp-Switch enables study of condition-specific changes in transcription initiation in bacteria.

SUBMITTER: Boutard M

PROVIDER: S-EPMC5171806 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Global repositioning of transcription start sites in a plant-fermenting bacterium.

Boutard Magali M Ettwiller Laurence L Cerisy Tristan T Alberti Adriana A Labadie Karine K Salanoubat Marcel M Schildkraut Ira I Tolonen Andrew C AC

Nature communications 20161216

Bacteria respond to their environment by regulating mRNA synthesis, often by altering the genomic sites at which RNA polymerase initiates transcription. Here, we investigate genome-wide changes in transcription start site (TSS) usage by Clostridium phytofermentans, a model bacterium for fermentation of lignocellulosic biomass. We quantify expression of nearly 10,000 TSS at single base resolution by Capp-Switch sequencing, which combines capture of synthetically capped 5' mRNA fragments with temp ...[more]

PMID: 27982035

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Project description:The overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616, we have performed a global analysis of TSS and TSS-Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5' cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering, and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with <10 read counts, 39.3% of TSS overlapped with 5' ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50 bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions (±50 bp) revealed 46% of the predicted TSS were "novel" and previously un-annotated. Using whole-genome bisulfite sequencing data from the same tissues, we were able to determine that a proportion of these "novel" TSS were hypo-methylated (32.2%) indicating that they are likely to be reproducible rather than "noise". This global analysis of TSS in sheep will significantly enhance the annotation of gene models in the new ovine reference assembly. Our analyses provide one of the highest resolution annotations of transcript regulation and diversity in a livestock species to date.

Project description:Cap Analysis of Gene Expression (CAGE) in combination with single-molecule sequencing technology allows precision mapping of transcription start sites (TSSs) and genome-wide capture of promoter activities in differentiated and steady state cell populations. Much less is known about whether TSS profiling can characterize diverse and non-steady state cell populations, such as the approximately 400 transitory and heterogeneous cell types that arise during ontogeny of vertebrate animals. To gain such insight, we used the chick model and performed CAGE-based TSS analysis on embryonic samples covering the full 3-week developmental period. In total, 31,863 robust TSS peaks (>1 tag per million [TPM]) were mapped to the latest chicken genome assembly, of which 34% to 46% were active in any given developmental stage. ZENBU, a web-based, open-source platform, was used for interactive data exploration. TSSs of genes critical for lineage differentiation could be precisely mapped and their activities tracked throughout development, suggesting that non-steady state and heterogeneous cell populations are amenable to CAGE-based transcriptional analysis. Our study also uncovered a large set of extremely stable housekeeping TSSs and many novel stage-specific ones. We furthermore demonstrated that TSS mapping could expedite motif-based promoter analysis for regulatory modules associated with stage-specific and housekeeping genes. Finally, using Brachyury as an example, we provide evidence that precise TSS mapping in combination with Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-on technology enables us, for the first time, to efficiently target endogenous avian genes for transcriptional activation. Taken together, our results represent the first report of genome-wide TSS mapping in birds and the first systematic developmental TSS analysis in any amniote species (birds and mammals). By facilitating promoter-based molecular analysis and genetic manipulation, our work also underscores the value of avian models in unravelling the complex regulatory mechanism of cell lineage specification during amniote development.

Dataset Information

Global repositioning of transcription start sites in a plant-fermenting bacterium.

Publications

Global repositioning of transcription start sites in a plant-fermenting bacterium.

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