Identification of epigenetic regulators of fibrotic transformation in cardiac fibroblasts through bulk and single-cell CRISPR screens [scRNA-seq]

Cardiac fibrosis is mediated by the persistent activity of myofibroblasts, which differentiate from resident cardiac fibroblasts in response to tissue damage and stress signals. The signaling pathways and transcription factors regulating fibrotic transformation have been thoroughly studied. By contrast, the roles of chromatin factors in fibroblast differentiation and their contribution to pathogenic cardiac fibrosis remain poorly understood. Here, we have combined bulk and single-cell CRISPR screens to characterize the roles of chromatin factors inex vivofibroblast differentiation. We uncover strong regulators of fibrotic states including Srcap and Tip60 chromatin remodelers, the NSL complex and the co-factors Hcfc1 and Wdr82. We confirm these factors are required for cellular processes underlying fibrosis including collagen synthesis and cell contractility. We use epigenetic profiling to demonstrate these chromatin factors facilitate the activity of pro-fibrotic transcription factors. Finally, using chemical perturbation in primary human cardiac fibroblasts we suggest Tip60 inhibition as a potential therapeutic approach for cardiac fibrosis. Overall design: Primary Cas9 cardiac fibroblasts transduced with Lentiviral-CRISPR library containing sgRNAs targeting specific Chromatin factors were isolated by Fluorescence-activated cell sorting (FACS) according to the presence Cas9 (GFP signal) and sgRNA (BFP signal) and analyzed using Single-Cell 3' (Dual Index) with CRISPR Feature Barcode