Enhancer-based modulation of cardiac fibroblasts

不同类型的心肌细胞对心脏功能是必不可少的。心肌细胞提供收缩力量，而成纤维细胞提供心脏结构支架所必需的细胞外基质。在心脏病中，成纤维细胞的激活是病理性心肌纤维化发生发展的关键事件。到目前为止，成纤维细胞激活的潜在转录机制在心脏中仍未解决。对转录调控至关重要的是增强子元件。这些元件结合转录因子，增强远端基因启动子的活性。增强子元件具有高度的细胞类型和激活状态特异性，因此是未来干预的一个有前景的靶点。本项目的目的是利用表观遗传学方法和CRISPR干扰(CRISPRi)来识别和调节对成纤维细胞激活至关重要的增强子元件。为了达到这一目标，我们计划了三个项目部分。目标1是利用全基因组染色质状态分析，确定肥厚性心脏(TAC)中与成纤维细胞激活相关的增强子元件，并在药物刺激后模拟神经激素激活。我们将进行染色质构象分析(Hi-ChIP)以连接增强子和靶基因。本项目部分将确定参与成纤维细胞激活的增强子元件，并将解决受影响的增强子-启动子接触。目的2是测试成纤维细胞增强剂的功能相关性。我们将使用sgRNA引导的核酸酶缺陷Cas9融合到抑制器域(CRISPRi)来沉默目标1中确定的大量增强子。因此，我们将采用两阶段CRISPRi筛查策略。第一阶段将确定干扰成纤维细胞激活的候选sgRNAs。第二阶段将结合CRISPRi实验和单细胞RNA测序，破译单个和组合sgRNAs对成纤维细胞激活基因程序的影响。本项目将提供基于CRISPRi的方法来干扰成纤维细胞的激活。目的3是验证和优化sgRNA序列的特异性，并证明基于CRISPRi的增强子在体外和体内沉默的有效性。本工作的假设是通过增强子的功能遗传扰动来控制成纤维细胞的活性。该项目将是迈向心脏纤维化功能性基因治疗的重要一步。

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.