Distinct IL-1α-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1α induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-κB at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1α-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1α/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-κB recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 “master” enhancer in the regulation of sustained IL-1α signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNFα-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-κB

Single-Cell Analysis of Multiple Steps of Dynamic NF-κB Regulation in Interleukin-1α-Triggered Tumor Cells Using Proximity Ligation Assays

The frequently occurring heterogeneity of cancer cells and their functional interaction with immune cells in the tumor microenvironment raises the need to study signaling pathways at the single cell level with high precision, sensitivity, and spatial resolution. As aberrant NF-κB activity has been implicated in almost all steps of cancer development, we analyzed the dynamic regulation and activation status of the canonical NF-κB pathway in control and IL-1α-stimulated individual cells using proximity ligation assays (PLAs). These systematic experiments allowed the visualization of the dynamic dissociation and re-formation of endogenous p65/IκBα complexes and the nuclear translocation of NF-κB p50/p65 dimers. PLA combined with immunostaining for p65 or with NFKBIA single molecule mRNA-FISH facilitated the analysis of (i) further levels of the NF-κB pathway, (i) its functionality for downstream gene expression, and (iii) the heterogeneity of the NF-κB response in individual cells. PLA also revealed the interaction between NF-κB p65 and the P-body component DCP1a, a new p65 interactor that contributes to efficient p65 NF-κB nuclear translocation. In summary, these data show that PLA technology faithfully mirrored all aspects of dynamic NF-κB regulation, thus allowing molecular diagnostics of this key pathway at the single cell level which will be required for future precision medicine

Distinct IL-1 alpha-responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

How cytokine-driven changes in chromatin topology are converted into gene regulatory circuits during inflammation still remains unclear. Here, we show that interleukin (IL)-1 alpha induces acute and widespread changes in chromatin accessibility via the TAK1 kinase and NF-kappa B at regions that are highly enriched for inflammatory disease-relevant SNPs. Two enhancers in the extended chemokine locus on human chromosome 4 regulate the IL-1 alpha-inducible IL8 and CXCL1-3 genes. Both enhancers engage in dynamic spatial interactions with gene promoters in an IL-1 alpha/TAK1-inducible manner. Microdeletions of p65-binding sites in either of the two enhancers impair NF-kappa B recruitment, suppress activation and biallelic transcription of the IL8/CXCL2 genes, and reshuffle higher-order chromatin interactions as judged by i4C interactome profiles. Notably, these findings support a dominant role of the IL8 master enhancer in the regulation of sustained IL-1 alpha signaling, as well as for IL-8 and IL-6 secretion. CRISPR-guided transactivation of the IL8 locus or cross-TAD regulation by TNF alpha-responsive enhancers in a different model locus supports the existence of complex enhancer hierarchies in response to cytokine stimulation that prime and orchestrate proinflammatory chromatin responses downstream of NF-kappa B