Transcriptomic and Epigenetic Profiling of Fibroblasts in Idiopathic Pulmonary Fibrosis

Abstract

Idiopathic pulmonary fibrosis (IPF), a devastating, fibroproliferative, chronic lung disorder, is associated with expansion of fibroblasts/myofibroblasts, which leads to excessive production and deposition of extracellular matrix. IPF is typically clinically identified as end-stage lung disease, after fibrotic processes are well-established and advanced. Fibroblasts have been shown to be critically important in the development and progression of IPF. We hypothesize that differential chromatin access can drive genetic differences in IPF fibroblasts relative to healthy fibroblasts. To this end, we performed assay of transposase-accessible chromatin sequencing to identify differentially accessible regions within the genomes of fibroblasts from healthy and IPF lungs. Multiple motifs were identified to be enriched in IPF fibroblasts compared with healthy fibroblasts, including binding motifs for TWIST1 and FOXA1. RNA sequencing identified 93 genes that could be annotated to differentially accessible regions. Pathway analysis of the annotated genes identified cellular adhesion, cytoskeletal anchoring, and cell differentiation as important biological processes. In addition, single nucleotide polymorphism analysis showed that linkage disequilibrium blocks of IPF risk single nucleotide polymorphisms with IPF-accessible regions that have been identified to be located in genes that are important in IPF, including MUC5B, TERT, and TOLLIP. Validation studies in isolated lung tissue confirmed increased expression for TWIST1 and FOXA1 in addition to revealing SHANK2 and CSPR2 as novel targets. Thus, modulation of differential chromatin access may be an important mechanism in the pathogenesis of lung fibrosis.

Keywords: ATAC sequencing; RNA sequencing; epigenetics; fibroblasts; idiopathic pulmonary fibrosis.

Figure 1.

RNA sequencing analysis of healthy and idiopathic pulmonary fibrosis (IPF) fibroblasts (FBs). (A) Volcano plot and (B) heatmap of differentially expressed genes (DEGs) in healthy and IPF FBs by RNA sequencing. Cutoff FDR was <0.05 and fold change >1.5. (C) Annotation of differentially expressed genes in healthy and IPF FBs using DAVID. BP = biological process; DAVID = database for annotation, visualization and integrated discovery; ECM = extracellular matrix; FDR = false discovery rate; KEGG = Kyoto Encyclopedia of Genes and Genomes.

Figure 2.

Assay for transposase-accessible chromatin sequencing analysis of healthy and IPF FBs. (A) Heatmap of differentially accessible regions (DARs) in healthy and IPF FBs by assay for transposase-accessible chromatin sequencing. Cutoff FDR was <0.05 and fold change >1.5. (B) Enriched motifs and related transcription factors (TFs) in differentially accessible regions in IPF fibroblasts using Homer and MEME-ChIP.

Figure 3.

Networks of enriched TFs and their potential target genes. (A) TFs enriched in IPF FB more DARs; (B) TFs enriched in IPF FB less DARs.

Figure 4.

(A–I) Expression levels for the TFs: TWIST1 (A), FOXA1 (B), FOXP1 (C), ZBTB18 (D), CEBPA (E), GATA6 (F), MECOM (G), TGIF (H) and MEIS1 (I) from control or IPF lung tissue. Significance level *P < 0.05 refers to comparisons between Control and IPF.

Figure 5.

(A–I) Expression levels for predicted target genes for FOXA1: CSRP2 (A), SHANK2 (B), INSC (C), LOXL2 (D); the TWIST1 targets: SVIL1 (E), KAZALD1 (F), and the fibrotic markers: COL1A2 (G), FN1 (H), and POSTN (I) from control or IPF lung tissue. Significance level *P < 0.05 refers to comparisons between Control and IPF.