Enhancer-based modulation of cardiac fibroblasts

• Applicant: Professor Dr. Ralf Gilsbach
• Subject Area: Pharmacology ; Cardiology, Angiology
• Term: from 2019 to 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 428316638

The different cardiac cell types are essential for heart function. Cardiac myocytes provide contraction force, while fibroblasts provide extracellular matrix essential for the structural scaffold of the heart. In heart disease, activation of fibroblasts is the key event for the development of pathological cardiac fibrosis. So far, the underlying transcriptional mechanisms of fibroblast activation remain unresolved in the heart. Of central importance for transcriptional control are enhancer elements. These elements bind transcription factors and enhance the activity of distal gene promoters. Enhancer elements are highly cell type- and activation state-specific and thus represent a promising target for future interventions.The aim of this project is to identify and modulate enhancer elements crucial for fibroblast activation using epigenetic methods and CRISPR interference (CRISPRi). To reach this aim we plan three project parts.Aim 1 is to identify enhancer elements associated with fibroblast activation in hypertrophic hearts (TAC) and after pharmacological stimulation mimicking neurohormonal activation using genome wide chromatin state analysis. We will perform chromatin conformation analysis (Hi-ChIP) to link enhancers and target genes. This project part will identify enhancer elements involved in fibroblast activation and will resolve affected enhancer-promoter contacts. Aim 2 is to test the functional relevance of fibroblast enhancers. We will silence a large set of enhancers identified in aim 1 using a sgRNA-guided nuclease deficient Cas9 fused to a repressor domain (CRISPRi). Therefore, we will apply a two-stage CRISPRi screening strategy. The first stage will identify candidate sgRNAs interfering with fibroblast activation. The second stage will combine CRISPRi experiments with single cell RNA sequencing to decipher the effect of individual as well as combinatorial sgRNAs on gene programs of fibroblast activation. This project part will provide CRISPRi-based methods to interfere with fibroblast activation.Aim 3 is to validate and optimize the specificity of sgRNA sequences and prove the efficacy of CRISPRi-based enhancer silencing in vitro and in vivo.The hypothesis of this work is the control of fibroblast activity using functional genetic perturbation of enhancers. This project will be an important step towards functional genetic therapy of cardiac fibrosis.

• DFG Programme: Research Grants