title: Measuring Transcription Rate Changes via Time-Course 4-Thiouridine Pulse-Labelling Improves Transcriptional Target Identification.
abstract: Identifying changes in the transcriptional regulation of target genes from high-throughput studies is important for unravelling molecular mechanisms controlled by a given perturbation. When measuring global transcript levels only, the effect of the perturbation [e.g., transcription factor (TF) overexpression or drug treatment] on its target genes is often obscured by delayed feedback and secondary effects until the changes are fully propagated. As a proof of principle, we show that selective measuring of transcripts that are only synthesised after a perturbation [4-thiouridine (4sU) sequencing (4sU-seq)] is a more sensitive method to identify targets and time-dependent transcriptional responses than global transcript profiling. By metabolically labelling RNA in a time-course setup, we could vastly increase the sensitivity of MYCN target gene detection compared to traditional RNA sequencing. The validity of targets identified by 4sU-seq was demonstrated using chromatin immunoprecipitation sequencing and neuroblastoma microarray tumour data. Here, we describe the methodology, both molecular biology and computational aspects, required to successfully apply this 4sU-seq approach.
authors.affiliation: Systems Biology Ireland, Conway Institute of Biomolecular and Biomedical Research and School of Medicine and Medical Science, University College Dublin Conway Institute, Belfield, Dublin 4, Ireland. Electronic address: schwarzl@embl.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Schwarzl
authors.affiliation: Systems Biology Ireland, Conway Institute of Biomolecular and Biomedical Research and School of Medicine and Medical Science, University College Dublin Conway Institute, Belfield, Dublin 4, Ireland.
authors.forename: Desmond G
authors.initials: DG
authors.lastname: Higgins
authors.affiliation: Systems Biology Ireland, Conway Institute of Biomolecular and Biomedical Research and School of Medicine and Medical Science, University College Dublin Conway Institute, Belfield, Dublin 4, Ireland.
authors.forename: Walter
authors.initials: W
authors.lastname: Kolch
authors.affiliation: Systems Biology Ireland, Conway Institute of Biomolecular and Biomedical Research and School of Medicine and Medical Science, University College Dublin Conway Institute, Belfield, Dublin 4, Ireland.
authors.forename: David J
authors.initials: DJ
authors.lastname: Duffy
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling / Q000379:methods*; D005786:Gene Expression Regulation; D006801:Humans; D000071447:N-Myc Proto-Oncogene Protein; D009447:Neuroblastoma / Q000235:genetics* / Q000378:metabolism; D009687:Nuclear Proteins / Q000235:genetics*; D015513:Oncogene Proteins / Q000235:genetics*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D017423:Sequence Analysis, RNA / Q000379:methods; D049490:Systems Biology; D013891:Thiouridine / Q000032:analysis / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C459801:MYCN protein, human; D000071447:N-Myc Proto-Oncogene Protein; D009687:Nuclear Proteins; D015513:Oncogene Proteins; D014157:Transcription Factors; D013891:Thiouridine; D012313:RNA
keywords: 4sU-seq; metabolic labelling; sequencing; systems biology; transcription factor targets