Abstract Introduction Misexpression of airway epithelial genes such as MUC5B, ERN2, and PIGR is implicated in IPF pathogenesis. However, transcriptional control of airway epithelial gene expression is poorly understood, which limits our understanding of the underlying mechanisms that lead to gene misexpression in disease. We applied integrated multi-omics methods to identify airway epithelial transcriptional regulatory mechanisms relevant to IPF. Methods We performed ATAC-seq on primary airway epithelial cell cultures at several time points during differentiation evoked by air liquid interface (ALI) culture. We used transcription factor enrichment analysis (TFEA) to identify specific transcription factor families as candidate drivers of changes in chromatin accessibility during the ALI time course. RT-qPCR, RNA-seq and western blotting was applied to characterize expression patterns of selected transcription factors, and ChIP-seq was used to annotate the airway epithelia ELF3 cistrome. We integrated our data with nascent sequencing data to identify direct transcription targets of ELF3, which we validated using reporter assays. Immunohistochemistry and mass spectrometry were used to study cellular control of ELF3 activity, and spatial transcriptomics data was used to correlate ELF3 expression with specific genes in lung tissue from controls and subjects with IPF. Results Through bioinformatic analysis (TFEA) of ATAC-seq data generated from ALI cultures over a time course, we identified the ETS family as a major driver of changes in chromatin accessibility during airway epithelia differentiation. RNA and western analysis showed that the specific ETS factors, ELF3, EHF and ETS2, are expressed at high, relatively constant levels in undifferentiated and differentiated airway epithelia, and immunohistochemistry indicated that ELF3 nuclear localization increases under ALI culture conditions. Targets of ELF3 that are specific to differentiated airway cells were identified by ChIP-seq. These include ERN2 and PIGR, and PIGR expression was correlated with ELF3 and EHF levels in lung tissue from subjects with IPF. Conclusions ETS family members are critical drivers of chromatin remodeling during airway epithelial differentiation, and the activity of a key epithelial ETS factor, ELF3, is regulated by cellular localization during differentiation. ELF3 directly occupies genomic binding sites located near a number of airway epithelial specific genes, including PIGR and ERN2, which is implicated in UPR responses to MUC5B misexpression in IPF. Further relevance of ELF3 to IPF is supported by spatial transcriptomics data which associates ELF3 and PIGR expression in IPF. In aggregate, we identify post-translational control mechanisms as regulating ELF3 activity, and direct targets of ELF3 that are relevant to IPF. This abstract is funded by: NIH
Gerber et al. (Fri,) studied this question.