Regulation of chromatin remodeling by Notch1 in fibrotic disorders

在本项目中，我们的目标是评估Notch信号与纤维化中组蛋白甲基化的表观遗传转录调控的相互作用，并分析组蛋白去甲基化酶抑制剂在纤维化疾病中的治疗潜力。在先前的研究中，我们可以证明Notch 1信号是SSc中成纤维细胞活化和组织纤维化的重要分子途径。基于这些研究，我们评估了小鼠（Notch 1act小鼠）中Notch 1的成纤维细胞特异性激活，该小鼠显示皮肤的进行性纤维化变化。抑制组蛋白去甲基化酶LSD 1可改善Notch 1激活在体外和体内的促纤维化作用。对来自有或没有LSD 1抑制的Notch 1act小鼠和相应的非纤维化对照的皮肤样品进行批量RNA测序，鉴定了大量差异表达基因（DEG）。DEG的功能分析显示了纤维化相关途径术语以及脂质和PPAR信号传导的富集，以及关于丝裂原活化蛋白激酶（MAPK）信号传导的术语的富集，以及SMAD 3/4结合位点的富集。我们计划在有和没有LSD 1抑制的Notch 1act小鼠中进行染色质免疫沉淀结合测序（ChIPseq）。然后将这些数据与现有的RNAseq数据整合，以识别依赖于LSD 1的组蛋白甲基化调控的Notch 1调控基因。这些数据将在人样本中通过培养的成纤维细胞中的ChIP-qPCR并通过将获得的鼠数据集与从不同SSc患者队列获得的数据集进行比较来验证。此外，我们的目标是分析富集的病理生理相关的途径条款有关的PPAR和MAP激酶信号通路和SMAD 3/4信号更详细地使用标准的分子生物学技术，如分子靶向的信号中间体的siRNA，表达载体和小分子抑制剂。对于分析，我们将采用不同的技术，如免疫共沉淀（CoIP）和报告基因测定或ChIP-qPCR。体外获得的数据将通过抑制Notch 1act小鼠中的上述途径在体内得到证实。最后，我们的目标是评估靶向LSD 1在不同的实验性纤维化小鼠模型中的治疗潜力，例如博来霉素诱导的皮肤纤维化、TSK 1小鼠和慢性移植物抗宿主病（cGvHD）。

In the project proposed herein, we aim to evaluate the interaction of Notch signaling with epigenetic transcriptional regulation by histone methylation in fibrosis, and to analyze the therapeutic potential of histone demethylase inhibition in fibrotic diseases.In previous studies, we could demonstrate that Notch1 signaling is an important molecular pathway for fibroblast activation and tissue fibrosis in SSc. Based on these studies, we evaluated fibroblast-specific activation of Notch1 in mice (Notch1act mice), which showed progressive fibrotic changes of the skin. Inhibition of the histone demethylase LSD1 ameliorated the pro-fibrotic effects of Notch1 activation in vitro and in vivo. Bulk RNA sequencing on skin samples from Notch1act mice with or without LSD1 inhibition and respective non-fibrotic controls identified a plethora of differentially expressed genes (DEGs). Functional analyses of the DEGs showed an enrichment of fibrosis-relevant pathway terms as well as of lipid and PPAR signaling, and of terms concerning mitogen-activated protein kinase (MAPK) signaling, and an enrichment of SMAD3/4 binding sites.To assess the effect of Notch1-activation and of targeted inhibition of LSD1 on histone 3 lysine 4 (H3K4)- and H3K9-methylation systematically, we plan to perform chromatin immunoprecipitation coupled with sequencing (ChIPseq) in Notch1act mice with and without inhibition of LSD1. These data will then be integrated with the existing RNAseq data to identify Notch1-regulated genes regulated by histone methylation dependent on LSD1. These data will be verified in human samples by ChIP-qPCR in cultured fibroblasts and by comparing the obtained murine datasets with datasets obtained from different SSc patient cohorts. Further, we aim to analyze the enrichment of pathophysiological relevant pathway terms concerning PPAR and MAP kinase signaling pathways and SMAD3/4 signaling in more detail using standard molecular biology techniques such as molecular targeting of signaling intermediates by siRNA, expression vectors and small molecule inhibitors. For analyses, we will employ different techniques such as co-immunoprecipitation (CoIP) and reporter assays or ChIP-qPCR. The data obtained in vitro will be confirmed in vivo by inhibiting the above mentioned pathways in Notch1act mice. Finally, we aim to evaluate the therapeutic potential of targeting LSD1 in different murine models of experimental fibrosis, such as bleomycin-induced dermal fibrosis, TSK1 mice and in chronic graft-versus-host-disease (cGvHD).