Inducible transcription and RELA binding in activated B lymphoma cells.

BJAB human B lymphoma cells were treated with P+I for 1 or 4 h, followed by analyses of inducible transcription and RELA binding by RNA-Seq and ChIP-Seq, respectively. (A) Gene expression changes at either 1 or 4 h post activation compared to unactivated cells. Two biological replicate experiments were carried out, and differential gene expression was analyzed using EBSeq. (B) Inducibly up- or down-regulated transcripts were partitioned into 6 patterns (middle panel, top, or bottom, respectively) by k-means clustering (see also S1B–S1E Fig). Each graph represents the gene expression pattern at 0, 1, and 4 h. Numbers of genes in each pattern are indicated below in black. The letter “A” after a pattern number indicates genes that are activated by P+I, and the letter “R” indicates genes that are repressed by P+I. Red numbers identify putative RELA target genes according to criteria discussed in the text and summarized in parts D and E below. (C) Representative examples of RNA-Seq profile of genes corresponding to patterns 1A and 5A of up-regulated genes (right panel, top, and middle) and from pattern 1R of down-regulated genes (right panel, bottom). The y axis represents normalized reads per million. Chromosomal locations of genes in hg19 are shown above RNA-Seq tracks. (D) Two replicate RELA ChIP-Seq experiments were carried out as described in the Methods section. Peaks were called using MACS2 with input DNA as control at FDR ≤ 0.05. Peaks with peak score ≥ 100 that were common to both biological replicates were used for further analysis. ChIP-Seq experiments were annotated with respect to genomic location using HOMER. Total numbers of RELA peaks are noted above the bars, and activation times are indicated below. (E) Relationship of inducible transcription and RELA binding. Genes were categorized based on the level of inducible activation (top 2 rows) or repression (bottom 2 rows). Genes in each category (second column) that bound RELA (third column) were assigned based on criteria noted in the text. The majority of RELA binding was induced upon cell activation (fourth column). Newly identified RELA-bound up- and down-regulated genes are listed in S1 Table. (F) Representative browser tracks of RELA binding to genes whose transcriptional responses are shown in part C. The y axis represents normalized reads per 10 million. Chromosomal locations of each gene in hg19 are shown above ChIP-Seq tracks. RNA-Seq and ChIP-Seq data are available on the GEO website (http://www.ncbi.nlm.nih.gov/geo/) (Accession number GSE117259). ChIP-Seq, chromatin immunoprecipitation and sequencing; FDR, false discovery rate; GEO, Gene Expression Omnibus; P+I, phorbol 12-myristate 13-acetate and ionomycin; RNA-Seq, RNA sequencing; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region.