Measuring Transcription Rate Changes via Time-Course 4-Thiouridine Pulse-Labelling Improves Transcriptional Target Identification. Issue 21 (23rd October 2015)

• Record Type: Journal Article
• Authors: Schwarzl, Thomas; Higgins, Desmond G.; Kolch, Walter; Duffy, David J.
• Is Part Of: Journal of molecular biology. Volume 427:Issue 21(2015:Nov. 01)
• Page Start: 3368
• Page End: 3374
• Publication Date: 2015-10-23
• Subjects: 4sU 4-thiouridine -- DE differentially expressed -- NB neuroblastoma -- TF transcription factor -- RNA-seq RNA sequencing -- 4sU-seq 4sU sequencing -- ChIP chromatin immunoprecipitation -- ChIP-seq chromatin immunoprecipitation sequencing; metabolic labelling -- 4sU-seq -- sequencing -- transcription factor targets -- systems biology; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2015.09.006
• Languages: English
• ISSNs: 0022-2836

Abstract

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. Graphical abstract: Highlights: Discovering TF targets using a time-course experimental design. Metabolic pulse-labelling combined with high-throughput time-course sequencing. Improved TF target discovery compared to traditional RNA sequencing.