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Patients with chronic obstructive pulmonary disease (COPD) are still waiting for curative treatments. Considering the environmental cause of COPD (e.g., cigarette smoke) and disease phenotypes, including stem-cell senescence and impaired differentiation, we hypothesized that COPD will be associated with altered epigenetic signaling in lung cells. We generated genome-wide DNA methylation maps at single CpG resolution of primary human lung fibroblasts (HLFs) isolated from distal parenchyma of ex-smoker controls and COPD patients, with both mild and severe disease. The epigenetic landscape is markedly changed in lung fibroblasts across COPD stages, with DNA methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA sequencing of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair, and extracellular matrix organization. Notably, we identified epigenetic and transcriptional dysregulation already in mild COPD patients, providing unique insights into early disease. Integration of profiling data identified 110 candidate regulators of disease phenotypes, including epigenetic factors. Using phenotypic screens, we verified the regulator capacity of multiple candidates and linked them to repair processes in the human lung.Our study provides first integrative high-resolution epigenetic and transcriptomic maps of human lung fibroblasts across stages of COPD. We reveal novel transcriptomic and epigenetic signatures associated with COPD onset and progression and identify new candidate regulators involved in the pathogenesis of chronic respiratory diseases. The presence of various epigenetic factors among the candidates demonstrates that epigenetic regulation in COPD is an exciting research field that holds promise for novel therapeutic avenues for patients.

Despite decades of intensive research, patients with COPD are still desperately waiting for curative treatments. For these to be developed, better understanding of molecular changes driving disease development is required. Based on key features and the environmental cause of COPD (cigarette smoke), we hypothesised that the disease will be triggered by altered epigenetic signalling in lung cells. Using cutting-edge, low-input whole genome bisulfite sequencing technology, we generated first unbiased DNA methylation maps of parenchymal fibroblasts isolated from lungs of smoker controls and COPD patients, both with early and established disease. We demonstrate that epigenetic landscape is severely changed in fibroblasts across COPD stages, with methylation changes occurring predominantly in regulatory regions, including promoters and enhancers. RNA-seq analysis of matched fibroblasts demonstrated dysregulation of genes involved in proliferation, DNA repair and extracellular matrix organisation. Excitingly, we identify differentially methylated regions and gene subsets dysregulated already in mild COPD patients, providing unique insight into early disease. Integration of profiling data, together with upstream regulator analysis identified 112 candidate regulators of COPD phenotypes. Using high-content phenotypic screens in primary lung fibroblasts from normal and COPD donors, we linked the function of multiple of these genes to key processes in COPD, establishing them as potential novel targets for therapy. Interestingly, many of these candidates are epigenetic factors, emphasising the critical role of epigenetic regulation in COPD.

The Shank proteins are crucial scaffolding elements of the post-synaptic density (PSD). One of the best-characterized domains in Shank is the PDZ domain, which binds to C-terminal segments of several other PSD proteins. We carried out a detailed structural analysis of Shank3 PDZ domain-peptide complexes, to understand determinants of binding affinity towards different ligand proteins. Ligand peptides from four different proteins were cocrystallized with the Shank3 PDZ domain, and binding affinities were determined calorimetrically. In addition to conserved class I interactions between the first and third C-terminal peptide residue and Shank3, side chain interactions of other residues in the peptide with the PDZ domain are important factors in defining affinity. Structural conservation suggests that the binding specificities of the PDZ domains from different Shanks are similar. Two conserved buried water molecules in PDZ domains may affect correct local folding of ligand recognition determinants. The solution structure of a tandem Shank3 construct containing the SH3 and PDZ domains showed that the two domains are close to each other, which could be of relevance, when recognizing and binding full target proteins. The SH3 domain did not affect the affinity of the PDZ domain towards short target peptides, and the schizophrenia-linked Shank3 mutation R536W in the linker between the domains had no effect on the structure or peptide interactions of the Shank3 SH3-PDZ unit. Our data show the spatial arrangement of two adjacent Shank domains and pinpoint affinity determinants for short PDZ domain ligands with limited sequence homology.