Fig. 4: Intragenic RELA-binding sites associate with alternative splicing events.

a Alternative splicing modifications of the CD44 exon v10 upon in HEK cells expressing or not Tax and knocked down or not for RELA or DDX5/17 expressions. TaxM22 and siRNA-mediated RELA depletion were used to assess the dependency of splicing events after NF-κB activation. Histograms represent the results of exon-specific quantitative RT-PCR measurements computed as a relative exon inclusion (alternatively spliced exon vs constitutive exon reflecting the total gene expression level). b Schematic representation of the human CD44 gene. Black and white boxes represent constitutive and alternative exons, respectively, as previously annotated (50). The orange box represents the kB site localized at –218 bp from the TSS and the 40 bp fragment deleted by CRISPR/Cas9 in CD44ΔkB HEK cells. c qChIP analysis of RELA occupancy across the promoter, the exon v10, and the constitutive exon E16 of CD44. RELA enrichment is expressed as the fold-increase in signal relative to the background signal obtained using a control IgG. d Relative exon inclusion of CD44 exon v10 was quantified by qRT-PCR in parental cells and its CD44∆kB counterparts. e Distribution of alternative exons that are regulated or not by Tax and DDX5/17 in RELA-enriched intragenic regions. The analysis was restricted to alternative exons expressed in HEK cells and regulated or not by Tax. Boxes extend from the 25th to 75th percentiles, the mid line represents the median and the whiskers indicate the maximum and the minimum values. f Bootstrapped distribution of median distance between intragenic RELA peaks and either Tax-regulated exons (red line, 1079 bp) or randomly chosen exons (10⁵ repetitions) (blue). p-values were determined by sample t-test. g Consensus de novo motif for RELA-binding sites <1 kb of Tax-regulated exons. Data are presented as the mean ± SEM values from biological replicates. Each black square represents a biological replicate. Statistical significance was determined with two-way ANOVA followed by Fisher’s LSD test (*p < 0.05, **p < 0.01, ***p < 0.001) (a, c, and d) and two-tailed Wilcoxon test (e, ****p < 0.0001). Exact p-values for Tax vs CTL: a <0.0001, c parental, promoter: 0.0248 and V10: 0.0005; CD44∆kB, V10: 0.021; d parental: 0.0028; CD44∆kB: 0.0054. Source data are provided as a Source Data file.