Altered AP-1, RUNX, and EGR chromatin dynamics drive human fibrotic lung disease

Objectives: Pulmonary fibrosis can result from autoimmune, inflammatory, or idiopathic conditions, including systemic sclerosis-associated interstitial lung disease (SSc-ILD). Previous studies have implicated myofibroblasts and SPP1hi macrophages as drivers of fibrosis in lungs, as well as other tissues. Single-cell RNA-sequencing has delineated fibroblast and macrophage transcriptomes but provides limited insight into the transcriptional control of profibrotic gene programmes. Methods: To determine the chromatin accessibility dynamics and transcription factors driving profibrotic gene programmes in human pulmonary fibrosis, we performed multiomic single-nucleus assay for transposase-accessible (ATAC)/RNA-sequencing on explanted SSc-ILD and control lungs. Using the neural network tool ChromBPNet, we analysed pseudobulk fibroblast and macrophage populations to infer transcription factor binding dynamics at single base-pair resolution. Hierarchical causal modelling for single-cell multiomics data (HALO), a novel algorithm developed to derive low-dimensional representations of sparse multiomics data, revealed transcription factor-regulatory element-gene networks. Results: ChromBPNet inferred increased transcription factor binding to profibrotic genes, including CTHRC1 and ADAM12 in fibroblasts, and SPP1 and CCL18 in macrophages. SSc-ILD fibroblasts showed increased binding and enhancer activity by AP-1, RUNX, and EGR transcription factors to profibrotic genes, whereas macrophages displayed enhanced binding and activity of AP-1 and basic helix-loop-helix (bHLH)-ZIP transcription factors in genes regulating the SPP1hi phenotype. HALO confirmed AP-1, RUNX, and EGR activity controlling profibrotic gene programmes. Conclusions: These data provide comprehensive insights into the complexity of transcriptional control of SSc-ILD gene expression programmes and define specific AP-1, RUNX, and EGR enhancers in key myofibroblast marker genes CTHRC1 and ADAM12, and a bHLH-ZIP enhancer in SPP1hi macrophages.