Transcriptional outcomes and kinetic patterning of gene expression in response to NF-κB activation
Fig 2
Attenuation of classical NF-κB-dependent transcription by dnIκBα expression.
Two clones of BJAB cells were generated to perturb canonical NF-κB signaling by Tet-inducible dnIκBα expression. (A) Western blot analysis of parent BJAB cells and 1 clone treated with Tet for the indicated times (h); whole-cell extracts were fractionated by SDS-PAGE and transferred to NC membranes, which were probed with anti-IκBα or anti-β-actin (loading control) antibodies. (B) RELA induction in dnIκBα-expressing BJAB cells. Clone 1 cells were activated with P+I for 1 or 4 h either in the absence of Tet (-Tet) or after Tet pretreatment for 24 h (+Tet). Nuclear extracts were fractionated with SDS-PAGE, transferred to membranes, and probed with anti-RELA or anti-hnRNP antibodies (loading control). Results with 1 representative clone are shown. (C) Relationship of dnIκBα-responsive transcripts to RELA binding. Total RNA isolated from each BJAB clone activated in the presence or absence of Tet was subject to RNA-Seq (2 biological replicates from each clone). Genes that were differentially expressed in each clone because of dnIκBα expression were identified based on FDR ≤ 0.05 in EBSeq. Inducible activation of 806 genes was reduced by dnIκBα expression in both clones; of these, 304 bound RELA (middle panel, green), and 502 (middle panel, gray) did not bind RELA. Time-dependent expression patterns of RELA-binding genes in the absence (blue lines) or presence (green lines) of Tet-induced dnIκBα were determined by k-means clustering (right). The letters “Ad” after the pattern number indicate direct target genes that were activated by RELA, and the letters “Ai” indicate indirect target genes that were RELA dependent. Numbers of genes in each category are shown in black below the graphs. Numbers in red identify genes that were changed more than 2-fold in the absence of Tet. Time-dependent expression patterns of genes that were down-regulated by IκBα but did not bind RELA are shown to the left. Numbers and color coding are as described above. (D) Representative RNA-Seq and ChIP-Seq tracks for RELA target genes identified in C (green). Two previously known NF-κB target genes (TNFAIP3 and STAT5) with different expression kinetics are shown on the top line, and 2 newly identified RELA target genes (HERPUD1 and NR1D1) are shown on the bottom line. RNA-Seq tracks show the effects of dnIκBα expression (+Tet) at the time of maximum expression (1 h for pattern 3Ad and 4 h for pattern 2Ad). RNA-Seq tracks for all time points +/− Tet are provided in S2G Fig. All time points are shown for RELA ChIP-Seq tracks to visualize the dynamics of RELA recruitment and loss from the genome over the experimental time course. Numbers above the tracks refer to gene location in hg19. A complete list of all 304 RELA target genes identified in this study is provided in S2 Table. Genome scale datasets are available on the GEO website (http://www.ncbi.nlm.nih.gov/geo/) (Accession number GSE117259). ChIP-Seq, chromatin immunoprecipitation and sequencing; dnIκBα, dominant negative mutated IκBα; FDR, false discovery rate; GEO, Gene Expression Omnibus; hnRNP, heterogeneous nuclear ribonucleoprotein; IκBα, NFKB inhibitor alpha; NC, nitrocellulose; NF-κB, nuclear factor kappa B; P+I, phorbol 12-myristate 13-acetate and ionomycin; RNA-Seq, RNA sequencing; Tet, tetracycline.