RNA sequencing can be used to monitor nascent transcripts to provide insight into gene transcription independently of mRNA stability. In Cell, Smale and colleagues use RNA sequencing of nascent transcripts, chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) and binding-motif data sets to delineate unique features of the transcription regulation of individual genes. Beyond statistical enrichment, careful analysis of motif strength, nascent versus existing mRNA, ChIP-seq peak strength and chromatin availability at promoters of primary and secondary response genes induced substantially (over tenfold) in lipid A–stimulated bone marrow–derived macrophages identifies distinct and unique regulatory mechanisms at promoters regulated by the transcription factors NF-κB and IRF3. As such, a motif-strength threshold is required for the binding of NF-κB to promoters in vivo and delayed induction of particular genes is due to a requirement for nucleosome remodeling. Such quantitative, gene-centric analysis also reveals that although IRF3 and NF-κB cooperatively bind hundreds of genes, they co-regulate a very restricted number of genes, indicative of a highly targeted and subtle regulatory input.

Cell 165, 165–179 (2016)