Nucleotide resolution 4-thiouridine sequencing by SNU-Seq and sf4sU-Seq reveals the transcriptional responsiveness of an epigenetically primed human genome
Source: PubMed Central Open Access, NCBI / U.S. National Library of Medicine
Abstract Genomes are pervasively transcribed, leading to stable and unstable transcripts that influence 3-dimensional genome organization and gene regulation. High sensitivity and nucleotide resolution are required to resolve mammalian transcriptomes. Here, we exploit the sensitivity of 4-thiouridine (4sU) in two nucleotide-resolution methods:ingle-ucleotide resolution 4ssequencing (SNU-Seq) andize-ractionated 4sU-Seq (sf4sU-Seq). sf4sU-Seq involves gel isolation of abundant 4sU-labelled promoter-proximal transcripts, enabling nucleotide resolution mapping of transcription start sites and promoter-proximal pauses (PPPs) around +63 nucleotides on Pol II-transcribed loci. SNU-Seq maps the precise position of polymerases on transcription units, including paused Pol II at the PPP, validated using sf4sU-Seq, and enables the discovery of thousands of divergently transcribed intragenic and intergenic regions of open chromatin, many uncharacterized. Conversely and consistent with extensive epigenetic priming, hundreds of the >10 000 regions of acetylated open chromatin lacking detectable transcription using SNU-Seq, show IFNγ-dependent induction of divergent transcription, linked at selected loci to the formation of promoter-enhancer loops. At other primed regions, formation of promoter–enhancer loops is coincident with divergent transcription at the enhancer but precedes transcription of pre-mRNA from the promoter, supporting distinct priming mechanisms. Thus, 4sU-based methods, cou
Abstract
Abstract Genomes are pervasively transcribed, leading to stable and unstable transcripts that influence 3-dimensional genome organization and gene regulation. High sensitivity and nucleotide resolution are required to resolve mammalian transcriptomes. Here, we exploit the sensitivity of 4-thiouridine (4sU) in two nucleotide-resolution methods:ingle-ucleotide resolution 4ssequencing (SNU-Seq) andize-ractionated 4sU-Seq (sf4sU-Seq). sf4sU-Seq involves gel isolation of abundant 4sU-labelled promoter-proximal transcripts, enabling nucleotide resolution mapping of transcription start sites and promoter-proximal pauses (PPPs) around +63 nucleotides on Pol II-transcribed loci. SNU-Seq maps the precise position of polymerases on transcription units, including paused Pol II at the PPP, validated using sf4sU-Seq, and enables the discovery of thousands of divergently transcribed intragenic and intergenic regions of open chromatin, many uncharacterized. Conversely and consistent with extensive epigenetic priming, hundreds of the >10 000 regions of acetylated open chromatin lacking detectable transcription using SNU-Seq, show IFNγ-dependent induction of divergent transcription, linked at selected loci to the formation of promoter-enhancer loops. At other primed regions, formation of promoter–enhancer loops is coincident with divergent transcription at the enhancer but precedes transcription of pre-mRNA from the promoter, supporting distinct priming mechanisms. Thus, 4sU-based methods, coupled to chromatin analysis, enable detailed characterization of genome structure, transcription, and responsiveness. Graphical Abstract Graphical Abstract For image description, please refer to the figure legend and surrounding text. http://www.w3.org/1999/xlink float portrait gkag652gra.jpg float ga1 portrait graphical