Transcriptional outcomes and kinetic patterning of gene expression in response to NF-κB activation
Fig 3
Modes of transcriptional activation by RELA.
(A) Selected RELA target genes were assayed for effects of ERK inhibition on inducible transcription. qRT-PCR was carried out using RNA extracted from BJAB cells activated with P+I for indicated times in the absence (blue lines) or presence (red lines) of PD0325901. The top line shows representative genes whose maximal activation occurs between 1 and 4 h, during the period when nuclear RELA levels diminish. The bottom line shows representative genes that are transiently induced by P+I; data shown are the average of 2 independent experiments with qRT-PCR carried out in duplicate; error bars represent the standard error of the mean between experiments. (B) Representative examples of transcription factors identified as direct RELA targets in activated BJAB cells. Two lines at the top of each panel show RNA-Seq tracks obtained from cells that were pretreated with Tet (+Tet) or not (-Tet) for 24 h, followed by activation with P+I. Only time points at which RNA expression changed maximally are shown; complete time courses for each gene are provided in S3H Fig. Y axis numbers denote normalized reads per million. The next 4 lines show representative RELA ChIP-Seq tracks over the entire time course. Peak calling was carried out with MACS2, using input DNA as the control. Transcription orientation (arrows) and gene organization in hg19 are noted below each set. The y axis denotes normalized reads per 10 million. ChIP followed by quantitative PCR validation and NF-κB dependence of these genes is shown in S3E Fig. IRF and KLF family members have been previously proposed as NF-κB target genes. HES1 and ZNF267 were identified in this study. (C) Kinetic patterns of gene induction of direct (304) and indirect (502) RELA target genes in activated BJAB cells. Cells were activated with P+I for the indicated times, followed by RNA-Seq. Mean normalized reads for direct and indirect target genes from 2 independent experiments are plotted for each time point. Error bars represent the standard error of the mean. Genome scale datasets are available on the GEO website (http://www.ncbi.nlm.nih.gov/geo/) (Accession number GSE117259). Underlying data for Fig 3A and C are provided in S1A and S1B Data. ChIP-Seq, chromatin immunoprecipitation and sequencing; ERK, extracellular signal–regulated kinase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; GEO, Gene Expression Omnibus; NF-κB, nuclear factor kappa B; P+I, phorbol 12-myristate 13-acetate and ionomycin; qRT-PCR, quantitative real-time PCR; RNA-Seq, RNA sequencing; Tet, tetracycline.