Fig 5. Zbtb7a transduces regulation of accessibility upstream and independently of transcriptional activation.

(A) Left: mRNA expression levels of 153 direct p65 target genes (73 “TA3-responsive” plus 80 “non-TA3-responsive”), in TNF-α-treated p65-knockout fibroblasts and in fibroblasts reconstituted with p65 or p65 TA3. Middle: p65 ChIP signals, and right: Zbtb7a ChIP signals, at promoters of p65 target genes in TNF-α-treated normal fibroblasts. mRNA levels are log2 microarray signal differences to non-reconstituted p65-knockout fibroblasts, for 3 biological replicates. (B) mRNA expression differences between TNF-α-treated control and Zbtb7a-knockdown fibroblasts expressing p65 TA3, at distinct gene sets. Dots in violins indicate mean values. Significance of expression difference between TA3-responsive and control genes: P = 3.0 × 10⁻¹⁴. (C) GFP reporter expression in TNF-α-treated control (left) or Zbtb7a-knockout (right) fibroblasts, expressing p65 C-terminal regions fused to the DBD of Gal4 and cotransfected with plasmids carrying 1 kb promoter sequences from the Cxcl2 gene, in which NFκB binding motifs are replaced by the Gal4-UAS. Zbtb7a-knockout and congenic-control fibroblasts are both derived on a p53-knockout background, to prevent premature senescence [20]. Error bars indicate SEM. (D) Mean TA3-induced DNase hypersensitivity levels across TA3-responsive (left) or non-TA3-responsive (right) p65 target promoters, in TNF-α-treated control and Zbtb7a-knockdown fibroblasts. (E) DNase hypersensitivity levels induced by p65 TA3 at TA3-responsive, non-TA3-responsive, or control (non-NFκB target) promoters, in TNF-α-treated control and Zbtb7a-knockdown fibroblasts. Induced levels represent differences in mean cut frequencies within ±600 bp surrounding the TSS, compared to p65-knockout fibroblasts. Lines in boxplots indicate median values; whiskers extend to the most extreme data within 1.5× the IQR from the box; outliers are not shown. Significance of difference to Zbtb7a knockdown at non-TA3-responsive P = 4.7 × 10⁻². (F) Mean induced DNase hypersensitivity levels across p65 target promoters, in TNF-α-treated fibroblasts expressing p65 TA3 or a loss-of-function TA3 mutant that does not interact with Zbtb7a (“p65 TA3 mutant”). Additional details of statistical analysis are provided as Supporting information, and numerical values underlying figures are reported in S1 Data. ChIP, chromatin immunoprecipitation; DBD, DNA-binding domain; GFP, green fluorescent protein; IQR, interquartile range; NFκB, nuclear factor kappa B; SEM, standard error of the mean; TNF-α, tumour necrosis factor alpha; TSS, transcription start site.