title: Native elongating transcript sequencing reveals human transcriptional activity at nucleotide resolution.
abstract: Major features of transcription by human RNA polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates. Together, human NET-seq exposes the topography and regulatory complexity of human gene expression.
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Mayer
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Julia
authors.initials: J
authors.lastname: di Iulio
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Seth
authors.initials: S
authors.lastname: Maleri
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Umut
authors.initials: U
authors.lastname: Eser
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98104, USA.
authors.forename: Jeff
authors.initials: J
authors.lastname: Vierstra
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98104, USA.
authors.forename: Alex
authors.initials: A
authors.lastname: Reynolds
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98104, USA.
authors.forename: Richard
authors.initials: R
authors.lastname: Sandstrom
authors.affiliation: Department of Genome Sciences, University of Washington, Seattle, WA 98104, USA; Department of Medicine, Division of Oncology, University of Washington, Seattle, WA 98104, USA.
authors.forename: John A
authors.initials: JA
authors.lastname: Stamatoyannopoulos
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Electronic address: churchman@genetics.med.harvard.edu.
authors.forename: L Stirling
authors.initials: LS
authors.lastname: Churchman
mesh_terms: D017398:Alternative Splicing; D004742:Enhancer Elements, Genetic; D005091:Exons; D006367:HeLa Cells; D006801:Humans; D011401:Promoter Regions, Genetic; D012319:RNA Polymerase II / Q000378:metabolism*; D016372:RNA, Antisense / Q000235:genetics; D017423:Sequence Analysis, RNA / Q000379:methods; D061805:Transcription Elongation, Genetic; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: D016372:RNA, Antisense; D014157:Transcription Factors; D012319:RNA Polymerase II