E-Box Accessibility in Myofibroblasts in Pulmonary Fibrosis

ABSTRACT Idiopathic pulmonary fibrosis (IPF) is a progressive and devastating disorder characterized by scarring in the lung, which leads to impaired gas exchange, respiratory failure, and death. The accumulation and differentiation of fibroblasts to the contractile and highly synthetic myofibroblast is a central event in the pathogenesis of pulmonary fibrosis. Single cell-RNA sequencing technologies have defined the transcriptomic profiles of multiple populations of fibroblasts in IPF lungs with unprecedented granularity. Single cell RNA-Seq cannot, however, define collectively how these genes in the “myofibroblast program” are regulated. For this level of information, complementary “multiomic” techniques are needed. To begin to explain how myofibroblasts in IPF are regulated, we have performed single nucleus assay for transposase-accessible chromatin sequencing (snATAC-seq) of IPF lungs and have discovered that the E-Box motif (CAGATG) is the most enriched in DARs in myofibroblasts compared to non-myogenic fibroblasts. And of the E-box transcription factors, we have discovered that TWIST1 is the most favorably predicted to bind to these E-boxes. We have previously published experimental evidence that TWIST1 is important in the pathogenesis of pulmonary fibrosis. These observations lead us hypothesize that the myofibroblast program in pulmonary fibrosis is regulated by two parallel and essential events: the accessibility of E-Box motifs and the expression of the E- Box-binding transcription factors including TWIST1. To test this hypothesis, we propose three specific aims. The first is to determine the “regulatory architecture” of open chromatin in myofibroblasts in IPF. We will determine transcriptional activity of the differentially-accessible regions of open chromatin identified by our ATAC-Seq data. We will employ massively-parallel reporter assays, a novel technique in fibroblast biology. Second, we will determine how expression levels of the E-Box transcription factors regulate myofibroblast differentiation. Third, we will develop E-box decoys as a mechanistic way to disrupt myofibroblast effector functions. Herein, we present a new model where the significant enrichment of E-Box motifs in regions of open chromatin and the up-regulation of E-box transcription factors represent the essential elements of a conserved and critical regulatory hub in the myofibroblast program in IPF.