Respiratory virus induced IL‐6 expression is regulated by an extragenic suppressor

Yang, Jun; Brasier, Allan; Zhang, Yueqing

Interleukin‐6 (IL‐6) is a pleiotropic inflammatory cytokine that plays a pivotal role in the activation and regulation of proper immune response during respiratory viral infections. Aberrant IL‐6 expression, however, may promote virus survival and/or exacerbation of clinical symptoms, such as the inflammatory cytokine storms observed in severe COVID‐19 patients infected with SARS‐CoV‐2. IL‐6 is now regarded as a prominent target for clinical intervention but less is known about how IL‐6 itself is regulated in various disorders. Using precision nuclear run‐on next generation sequencing (PRO‐Seq) to measure the transcriptionally engaged RNA polymerases (RNA Pol II) in human primary small airway epithelial cells (hSAECs), we have discovered an upstream enhancer‐like region in the IL‐6 gene locus. This is a region that resides within an open chromatin domain. It has DNase I hypersensitivity and enriches in activating H3K27Ac mark, but lack of active transcription mark H3K36me3. Interestingly, it displays increased ratio of H3K4me1 over H3K4me3, a phenomenon usually associated with active enhancers. We were therefore surprised to observe that rhinovirus (RV) infection disrupts active RNA Pol II binding in this region, despite the fact that RNA Pol II is increased in the body of the IL‐6 gene. These findings suggest that the upstream sequence may be functioning as a suppressor of IL‐6 expression. To test this possibility, we targeted the KRAB transcriptional silencer to the upstream sequence using CRISPRi (KRAB/dCas9 directed by sgRNA). hSAECs expressing control sgRNA or the sequence‐specific sgRNA were infected with RV and IL‐6 mRNA measured by Q‐RT‐PCR. Intriguingly, the sequence‐specific sgRNA enhanced RV‐ induced IL‐6 expression. These results suggest that the upstream element is not an enhancer but a functionally active suppressor of IL‐6 expression and that the mechanism of IL‐6 expression involves a previously unknown de‐repression step. It will advance us to understand how dynamic interactions between cis‐regulatory elements regulate the threshold, magnitude and duration of IL‐6 gene transcription in response to distinct factors.