ATAC-seq revealing chromatin accessibility and novel motifs linked to corneal fibrosis.

Abstract

Corneal fibrosis results in loss of corneal transparency and vision impairment. Dynamic chromatin accessibility is a key regulatory mechanism driving fibrotic process, allowing transcription factors to access specific DNA sequences to activate or shut down transcription of pro- and anti-fibrotic genes. Genome-wide chromatin accessibility profiling was employed to explore the epigenomic landscape in alkali-induced fibrosis in rabbit cornea to identify key fibrotic regulators. Chromatin accessible regions (peaks) were identified using MACS2 and annotated relative to genomic features using ChIPseeker. de novo motif enrichment was evaluated by HOMER. Differentially accessible regions (DARs) were quantified with csaw and edgeR followed by pathway enrichment analysis using clusterProfiler. Of the total 121,305 peaks identified across all samples, 5890 DARs (FDR<0.05) were identified exhibiting reduced accessibility. Distinct chromatin condensation was observed at promoter region in fibrotic (5&#xa0;%) vs. na&#xef;ve (15&#xa0;%) corneas. Functional enrichment analysis revealed that cell cycle, metabolism and stress response regulation genes were the most affected. de novo motifs analysis identified motifs with high similarities to KLF17 (p&#xa0;=&#xa0;1e), NRF2 (p&#xa0;=&#xa0;1e), and ETV6 (p&#xa0;=&#xa0;1e) as potential regulators of corneal fibrosis. The qRT-PCR evaluation of motifs in human corneal myofibroblasts showed low expression of KLF17 (p&#xa0;<&#xa0;0.05) and NRF2 (p&#xa0;=&#xa0;0.23), and elevated levels of ETV6 (p&#xa0;=&#xa0;0.64) compared to fibroblast cells. ATAC-sequences are made available at the NCBI Gene Expression Omnibus (GEO accession no. GSE305524). Fibrosis triggers extensive epigenomic reprogramming, characterized by promoter-to-enhancer regulatory shifts and selective activation of fibrosis-associated transcriptional networks providing promising avenue for therapeutic interventions managing corneal fibrosis.