Regulation of chromatin remodeling by Notch1 in fibrotic disorders

在本文提出的项目中，我们旨在评估Notch信号与组蛋白甲基化的表观遗传转录调控在纤维化中的相互作用，并分析组蛋白去甲基酶抑制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Notch1信号是SSC中成纤维细胞激活和组织纤维化的重要分子途径。在这些研究的基础上，我们评估了Notch1在显示皮肤进行性纤维化变化的小鼠(Notch1act小鼠)中的成纤维细胞特异性激活。抑制组蛋白去甲基酶LSD1可改善Notch1激活在体外和体内的促纤维化作用。对有或没有LSD1抑制的Notch1act小鼠和各自的非纤维化对照组的皮肤样本进行批量RNA测序，发现了过多的差异表达基因(DEG)。对DEGS的功能分析显示，与纤维化相关的途径术语以及脂质和PPAR信号丰富，丝裂原激活蛋白激酶(MAPK)信号丰富，SMAD3/4结合位点丰富。为了系统地评估Notch1激活和靶向抑制LSD1对组蛋白3赖氨酸4(H3K4)和H3K9甲基化的影响，我们计划在有和没有LSD1抑制的Notch1act小鼠中进行染色质免疫沉淀结合测序(ChIPseq)。然后，这些数据将与现有的RNAseq数据相结合，以识别依赖于LSD1的组蛋白甲基化调控的Notch1调控基因。这些数据将在人类样本中通过培养成纤维细胞的CHIP-qPCR进行验证，并将获得的小鼠数据集与从不同SSC患者队列获得的数据集进行比较。此外，我们的目标是利用标准的分子生物学技术，如siRNA、表达载体和小分子抑制剂对信号中间产物进行分子靶向，更详细地分析PPAR和MAP激酶信号通路以及SMAD3/4信号通路中与病理生理相关的术语的丰富情况。对于分析，我们将使用不同的技术，如共免疫沉淀(CoIP)和报告分析或芯片定量聚合酶链式反应。体外获得的数据将通过抑制Notch1act小鼠的上述途径在体内得到证实。最后，我们的目标是评估靶向LSD1在不同的实验性纤维化模型中的治疗潜力，例如博莱霉素诱导的皮肤纤维化，TSK1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).