C71-23 Long-read Sequencing Reveals Differential Patterns of Methylation at Targets of Chromatin-modifying Enzymes in the Idiopathic Pulmonary Fibrosis Lung Epithelium

Abstract Rationale Idiopathic pulmonary fibrosis (IPF) is characterized by fibrotic scarring and reduced respiratory function associated with loss of normal alveolar epithelial cell populations. Genomic and transcriptomic approaches have been used to describe pathways and mediators associated with disease progression. However, the role of epithelial cell epigenetic regulation in IPF remains largely underexplored and could provide therapeutic strategies to guide adaptive alveolar differentiation and repair. DNA methylation regulates promoter activity, with the loss of 5-methyl-cytosine (5mC) generally associated with increased transcription. We hypothesize that alterations in the 5mC profiles of the lung epithelium of IPF subjects drive changes in gene expression that contribute to fibrotic disease. Methods CD326+ epithelial cells were isolated from male IPF and age-matched donor lungs rejected for transplant (N = 5 control and N = 4 IPF). Genomic DNA was isolated, libraries were prepared with the Oxford Nanopore Ligation sequencing protocol and sequenced on a PromethION 2. Basecalling was performed with Dorado, and 5mC calls were filtered with Modkit to exclude reads with confidence scores below the 10th percentile of the overall distribution. Methylation analysis was conducted with methylKit, requiring ≥10× coverage per site. 5mC values were evaluated as the percentage of methylated bases of the total bases sequenced within 100-bp regions. Differentially methylated regions with a ≥ 10% methylation difference and FDR-adjusted p 0.01 were considered significant. Smoking history was included as a covariate for statistical analysis. Promoter regions were defined as − 2000 to + 300 bp relative to transcription start sites. CpG shores were defined as +/-2000 bp relative to CpG islands. Transcription factor binding site enrichment analysis was performed using Enrichr gene list analysis tools. Results Differential methylation analysis of promoter regions and CpG islands and shores (CpG regions) revealed 3,789 genes with hypomethylation and 2,070 genes with hypermethylation in the IPF epithelium. Binding site enrichment analysis linked hypomethylated regions to targets of TP63 and SMAD2/3, consistent with their established increase in signaling activity in the IPF epithelium. Targets of KDM2B, a histone demethylase and polycomb repressive complex (PRC) regulator, were also hypomethylated. Hypermethylated regions were enriched for targets of PRC-associated enzymes SUZ12, JARID2, RNF2, and EZH2, suggesting increased PRC activity. Conclusion Here, we demonstrate that regulators of methylation may serve as novel targetable modulators of transcriptional programs associated with IPF progression. Our data suggest increased KDM2B and PRC activity, implicating chromatin modification pathways in the IPF epithelium. This abstract is funded by: NIH/NHLBI U01HL175444