During inflammation, the liver responds to pro-inflammatory signals by producing and secreting acute phase reactants (APR) involved in the inflammatory process. The notable increase in the level of these proteins is due to transcriptional induction that is jointly mediated by the pro-inflammatory cytokines IL-1β, TNFα and IL-6. Although critical to restoring homeostasis during infection or trauma, the hepatic response to these cytokines can be deleterious in pathologies involving chronic inflammation such as cancer.

We set out to decipher how pro-inflammatory cytokines cooperate to elicit a transcriptional response in hepatocytes. We examined the transcriptome of primary mouse hepatocytes treated with different combinations of cytokines and found several gene expression patterns showing a complex cross-talk between cytokine signaling pathways. Specifically, we found that a dual treatment of IL-1β and IL-6 results in a synergistic expression of 164 genes, many of which are APR genes. TNFα had an effect similar to IL-1β, albeit weaker. A sequential rather than a combined treatment attenuated the synergistic effect, suggesting direct cooperation between the two cytokines in inducing transcription (as opposed to a cascade of transcription). Assaying enhancer activity by H3K27ac ChIP-seq revealed the dynamic nature of cytokine-responsive enhancers, with hundreds of enhancers changing their activity level following either treatment. Importantly, enhancers becoming more active upon dual IL-1/IL-6 treatment were enriched with binding sequences for STAT3, CEBP, AP1 and NFkB. Because IL-6 is the major pro-inflammatory cytokine and its transcriptional effects are mediated by the STAT3 transcription factor (TF), we assayed STAT3 binding patterns by ChIP-seq. As expected, STAT3 binding was elevated following IL-6 treatment genome-wide. Surprisingly, in 351 enhancers, STAT3 binding was markedly increased in the dual treatment compared to IL-6 treatment alone, suggesting a supporting role for IL-1β in assisting STAT3 loading at a subset of enhancers. This sub-population of enhancers had lower motif scores compared to unassisted STAT3 binding sites, indicating optimal STAT3 binding at these enhancers might require some facilitation by other factors. Accordingly, these enhancers were less active at the untreated state, but increased in activity following IL-1β treatment, an effect we termed ‘IL-1β priming’.

Our results are consistent with a scenario whereby IL-1β-activated TFs (C/EBPβ, AP1 and/or NFκB) assist STAT3 loading by binding a subset of enhancers, activating them thereby augmenting STAT3 binding. This unexplored mechanism of transcriptional regulation by cytokines may be targeted to alleviate the deleterious effects of pro-tumorigenic inflammatory programs.

Citation Format: Ido Goldstein, Songjoon Baek, Myong-Hee Sung, Gordon L. Hager. Pro inflammatory cytokines induce synergistic gene expression by assisted loading of STAT3 at a subset of primed enhancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5510. doi:10.1158/1538-7445.AM2017-5510