Bromodomain-containing Protein 4 Regulates Innate Inflammation in Airway Epithelial Cells via Modulation of Alternative Splicing

Abstract

Bromodomain-containing Protein 4 (BRD4) is a transcriptional regulator which coordinates gene expression programs controlling cancer biology, inflammation, and fibrosis. In airway viral infection, non-toxic BRD4-specific inhibitors (BRD4i) block the release of pro-inflammatory cytokines and prevent downstream remodeling. Although the chromatin modifying functions of BRD4 in inducible gene expression have been extensively investigated, its roles in post-transcriptional regulation are not as well understood. Based on its interaction with the transcriptional elongation complex and spliceosome, we hypothesize that BRD4 is a functional regulator of mRNA processing. To address this question, we combine data-independent analysis - parallel accumulation-serial fragmentation (diaPASEF) with RNA-sequencing to achieve deep, integrated coverage of the proteomic and transcriptomic landscape of human small airway epithelial cells exposed to viral challenge and treated with BRD4i. The transcript-level data was further interrogated for alternative splicing analysis, and the resulting data sets were correlated to identify pathways subject to post-transcriptional regulation. We discover that BRD4 regulates alternative splicing of key genes, including Interferon-related Developmental Regulator 1 (IFRD1) and X-Box Binding Protein 1 (XBP1), related to the innate immune response and the unfolded protein response, respectively. These findings extend the transcriptional elongation-facilitating actions of BRD4 in control of post-transcriptional RNA processing in innate signaling.

Targeted Degradation of CDK9 Potently Disrupts the MYC Transcriptional Network

“…These observations are reflective of important mechanistic differences driven in part by complete abrogation of CKD9 kinase and scaffolding functions by the degrader. The molecular consequence of which is a breakdown of the BRD4-P-TEFb complex, an insight that aligns with the rapid and sustained degradation of critical early response genes regulated by BRD4 (e.g., MYC and IFRD1) by the degrader 2,34,35 . Moreover, the kinetics of action of degradation versus inhibition of CDK9 is likely to be an important determinant.…”

“…We observed subtle temporal differences between CDK9 degradation and inhibition. Degradation had a potent effect on early growth signaling and innate inflammation as evidenced by the negative enrichment of TNFA Signaling Via NFKB, and the Unfolded Protein Response pathways 34 (Figure 3C). It appears that CDK9 degradation rapidly downregulated BRD4-mediated processes, which was expected given the structural and functional relationship between the P-TEFb and BRD4 [34][35][36] .…”