ABSTRACT Skin and lung fibrosis are the hallmarks of systemic sclerosis (SSc, scleroderma), and the reversal of fibrosis is a very challenging clinical problem. Relaxin is a circulating hormone with strong preclinical evidence supporting its role as a potent anti-fibrotic molecule for organ fibrosis. But despite the strong evidence from animal models, relaxin-based therapy in clinical trial failed in reducing the skin fibrosis of SSc patients. One potential molecular explanation for the failure of relaxin in clinical trial is that cells in fibrosis may be insensitive to relaxin. We have found that the mRNA and protein levels of relaxin receptor, RXFP1, are dramatically decreased in the lungs and lung fibroblasts of SSc patients compared to controls. Fibroblasts from fibrotic lungs are impaired in their in vitro sensitivity to a relaxin-like agonist, but enhancement of RXFP1 expression restores relaxin sensitivity in these cells. However, the molecular mechanisms leading to RXFP1 down-regulation is unknown. We have identified a lung fibroblast-specific enhancer in the distal region of the RXFP1 gene that results in >100-fold increase of SV40 promoter-driven luciferase activity in control lung fibroblasts and the enhancement is partially blunted in fibroblasts from SSc (<20-fold). In addition, regions of transcriptionally active histone modifications as well as an upstream CpG island flank this novel enhancer. Therefore, we hypothesize that abnormal function of this distal RXFP1 enhancer, in combination with altered epigenetic changes in SSc lung fibroblasts, is a major driver of RXFP1 down-regulation and that modifications of these regulatory elements and epigenetic events will up-regulation of RXFP1 expression and sensitize SSc fibroblasts to relaxin treatment. We will perform fine mapping of the enhancer region and to identify transcription factors (TFs) responsible for the lung fibroblast-specific enhancement of RXFP1 (Aim 1). The expression levels of Identified TFs will be further correlated with endogenous RXFP1 expression in both control and SSc lungs and fibroblasts. Direct binding of the TFs to the enhancer will be analyzed using EMSA and ChIP assays. We will also determine the role of epigenetic modifications surrounding the enhancer in driving lung fibroblast specific RXFP1 expression (Aim 2). Both histone modification and DNA methylation status will be analyzed in control and SSc lungs and fibroblasts using molecular techniques including our novel method ISH-PLA, a single-cell epigenetic assay allowing detection of histone modifications in tissue sections. The success of this study will provide a novel approach to reverse the ineffectiveness of relaxin-based anti-fibrotic treatment in SSc by identifying potential therapeutic targets for restoring RXFP1 expression in fibrotic lungs of these patients.