A roadmap of constitutive NF-κB activity in Hodgkin lymphoma: Dominant roles of p50 and p52 revealed by genome-wide analyses

Abstract

Background: NF-κB is widely involved in lymphoid malignancies; however, the functional roles and specific transcriptomes of NF-κB dimers with distinct subunit compositions have been unclear.

Methods: Using combined ChIP-sequencing and microarray analyses, we determined the cistromes and target gene signatures of canonical and non-canonical NF-κB species in Hodgkin lymphoma (HL) cells.

Results: We found that the various NF-κB subunits are recruited to regions with redundant κB motifs in a large number of genes. Yet canonical and non-canonical NF-κB dimers up- and downregulate gene sets that are both distinct and overlapping, and are associated with diverse biological functions. p50 and p52 are formed through NIK-dependent p105 and p100 precursor processing in HL cells and are the predominant DNA binding subunits. Logistic regression analyses of combinations of the p50, p52, RelA, and RelB subunits in binding regions that have been assigned to genes they regulate reveal a cross-contribution of p52 and p50 to canonical and non-canonical transcriptomes. These analyses also indicate that the subunit occupancy pattern of NF-κB binding regions and their distance from the genes they regulate are determinants of gene activation versus repression. The pathway-specific signatures of activated and repressed genes distinguish HL from other NF-κB-associated lymphoid malignancies and inversely correlate with gene expression patterns in normal germinal center B cells, which are presumed to be the precursors of HL cells.

Conclusions: We provide insights that are relevant for lymphomas with constitutive NF-κB activation and generally for the decoding of the mechanisms of differential gene regulation through canonical and non-canonical NF-κB signaling.

Keywords: B lymphocytes; ChIP sequencing; Transcription factor; cell death; consensus sequence; enhancer; gene expression; inflammation; lymphoma; promoter.

Figures

Fig. 1

Dominant contribution of p50 and p52 in the constitutive NF-κB activity in HL cells. a Left: RNAi-mediated knockdown (KD) of NIK (MAP3K14). L1236 cells were incubated with siRNA against MAP3K14 and harvested 1 day after the end of the siRNA treatment. Protein levels of the precursor proteins p105 and p100, their products p50 and p52, as well as phospho-Ser-866/870-p100 and phospho-Ser-933-p105 were analyzed in whole extracts by western blot (WB). CDK4 was used as loading control. Right: EMSA analysis of L1236 whole cell extracts after KDs of single NF-κB subunits and double KD of p50 and p52. Control of KD efficiencies is shown in Additional file 2: Figure S1B. b WB analysis of nuclear (N) and cytoplasmic (C) distribution of NF-κB subunits in HL cell lines, as indicated (top labels). p105 and PARP1 serve as purity controls for nuclear and cytoplasmic extracts, respectively. c Immunohistochemical detection of p105/p50 and p100/p52 in representative biopsies from HL patients. Arrows indicate nuclear abundance of p50 (left panel) and p52 (right panel) in malignant Reed-Sternberg cells compared to surrounding benign cells. A total of 20 biopsies were used, all were stained for p105/p50 and 18 biopsies were stained for p100/p52

Fig. 2

ChIP-sequencing analysis of genome wide distribution of RelA, RelB, p50, and p52 binding regions. a Venn diagram showing the total number and intersections of ChIP-seq regions for p50, p52, RelA, and RelB in L1236 cells. See Additional file 4: Table S2 and Additional file 2: Figure S2A for ChIP-seq data and gene assignments. b Genome-wide NF-κB subunit occupancy profiles. Left: Regions (peak summit + -500 bp) were grouped based on their profiles of ChIP enrichment over input for all four subunits using k-means clustering (k = 8). Gray bars on top indicate the ChIPed subunit. Each row represents a region that was identified as a peak for at least one of the four subunits. The distinct clusters are indicated by numbers on the left. Right: Regions were also classified by the combination of subunits based on the peak calling results. Gray bars on top indicate which combination of subunits was called. The color-coded heatmap shows the percentage of regions in each cluster with a specific combination of subunits. c Visualization of representative ChIP-seq regions (UCSC genome browser). Top, NFKBIA gene with overlapping peaks for p50, p52, RelA, and RelB in the promoter and first intron. Bottom, intron 2 of the CDH1 gene showing selective binding of p50 and p52 to two ChIP-seq regions

Fig. 3

MEME de novo motif analysis revealed redundant consensus sites for each NF-κB subunit. a Occurrence of de novo detected NF-κB-like motifs in the indicated numbers of ChIP-seq regions analyzed by MEME. b NF-κB motifs detected by MEME de novo search in each of the four NF-κB subunit datasets of L1236 cells. See Additional file 5: Table S3 for CLOVER motif analysis data

Fig. 4

Enriched biological processes and genes with significance for HL biology controlled by p50/RelA and p52/RelB. a, b Genes activated (a) or repressed (b) by p50/RelA or p52/RelB in L1236 cells, respectively, and functional enrichment analysis of using Gene Ontology for biological processes (GO) analysis. Top panels: Venn diagrams displaying numbers of total, unique, and overlapping target genes for the two dimer combinations used for GO analysis (solid and stippled arrows, respectively). Direct target genes of p50/RelA and p52/RelB were defined as bound by at least p50 and p52, respectively, by ChIP-sequencing and differentially regulated upon KD of the specific dimers (FDR <0.05), and at least 10 % expression difference between the knockdown and control experiment). See Additional file 10: Table S8 for GO analysis data. c Schematic representation of canonical and non-canonical target genes that are known to be relevant for HL biology. Among both up- and downregulated genes, two functional groups were highly represented in HL literature: (1) cytokines and receptors, and (2) components of NF-κB-subordinated pathways [16, 19, 29, 50]

Fig. 5

Prediction of gene expression patterns from combinatorial binding of NF-κB subunits using logistic regression analyses. a Left: Regulation by non-canonical NF-κB versus no regulation. Right: Regulation by canonical NF-κB versus no regulation. Note that both, up- and downregulated genes are included in the categories ‘canonical’ regulated or ‘non-canonical’ regulated genes. Different parameter choices were systematically analyzed to evaluate the performance of the prediction framework for the logistic regression models (see Additional file 2: Figure S5). The y-axis shows the combinations of NF-κB subunits, which are listed as binary vectors (1 = presence, 0 = absence). The x-axis shows parameter estimates of the logistic regression models, which can be interpreted as log odds ratios and are indicated by circles. Vertical lines show confidence intervals (95 %). Filled circles indicate that parameters are significantly different from zero (P <0.01). Missing estimates and confidence intervals occur when the specific subunit combination was not observed in the dataset. Bar plots on the right side indicate the percentage of bootstrap samples in which each parameter was significantly different from zero (P <0.01). The dashed line marks 90 %. See Additional file 1: Supplemental Experimental Procedures for details. b Schematic presentation of the dependency of gene activation or repression on the distance and subunit occupancies of NF-κB binding regions

Fig. 6

Non-canonical as well as canonical subunits inhibit apoptosis of HL cells. a HL cell lines (L1236, L540 and KM-H2) were treated with two distinct siRNA sequences against p50/RelA and p52/RelB, as indicated and harvested 3 days after the end of the siRNA treatments. Overall redox activity of the cells was measured using Alamar Blue assay. Error bars represent SEM (n = 3). See Additional file 2: Figures S6A and B for time-course experiments. For time-course experiments see Additional file 2: Figures S6A and B. b WB analysis of p50/RelA or p52/RelB KD efficiencies and expression of the initiator caspase 8 (p18), the initiator caspase 9 (p10), and the effector caspase 3 (p17) in the samples of L1236 cells treated with siRNAs as described above. Protein levels of the two apoptosis inhibitors (c-FLIPS/L and Bcl-XL) identified as NF-κB target genes are also shown. α-tubulin was used as loading control

Fig. 7

Canonical and non-canonical NF-κB target genes differentiate primary HL from other lymphoid malignancies. The canonical and non-canonical NF-κB signatures identified in L1236 cells were evaluated using public gene expression data from patient samples (normal and malignant B cells [28]) available in GEO (GSE12453), by comparing their expression in HL samples to the rest. The heat map expression levels are encoded by the base 2 logarithmic scale color bar (right). A total of 123 NF-κB regulated genes were differentially expressed in HL samples and presented concordant expression changes (activation by NF-κB dimers and upregulation in HL samples or repression by NF-κB dimers and downregulation in HL samples). The gene names (left) and lymphoid cell types (top) are indicated. BL, Burkitt’s lymphoma; DLBCL, diffuse large B-cell lymphoma; FL, follicular lymphoma; NLPHL, nodular lymphocyte-predominant HL; TCRBL, T cell-rich B cell lymphoma. Germinal center (GC) B cells are indicated (bottom). See also Additional file 11: Table S9