Nucleotide resolution 4-thiouridine sequencing by SNU-Seq and sf4sU-Seq reveals the transcriptional responsiveness of an epigenetically primed human genome.

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: Single-Nucleotide resolution 4sU sequencing (SNU-Seq) and size-fractionated 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.