Regulation of chromatin remodeling by Notch1 in fibrotic disorders

Notch1 在纤维化疾病中调节染色质重塑

• 批准号: 467040214
• 负责人: Dr. Clara Dees
• 金额: --
• 依托单位: Medizinische Klinik 3 - Rheumatologie und Immunologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/467040214?language=en
• 关键词: Regulation; chromatin; remodeling; Notch1; fibrotic

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).

在本项目中，我们旨在评估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在纤维化疾病中的治疗潜力。在之前的研究中，我们可以证明Notch1信号是SSc成纤维细胞激活和组织纤维化的重要分子途径。基于这些研究，我们在小鼠（Notch1act小鼠）中评估了Notch1的成纤维细胞特异性激活，这些小鼠显示皮肤的进进性纤维化变化。抑制组蛋白去甲基化酶LSD1可改善Notch1激活在体内和体外的促纤维化作用。对有或没有LSD1抑制的Notch1act小鼠和相应的非纤维化对照的皮肤样本进行大量RNA测序，发现了大量差异表达基因（DEGs）。deg的功能分析显示，纤维化相关通路术语、脂质和PPAR信号、丝裂原活化蛋白激酶（MAPK）信号相关术语和SMAD3/4结合位点的富集。为了系统地评估notch1激活和LSD1靶向抑制对组蛋白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耦合测序（ChIPseq）。然后将这些数据与现有的RNAseq数据整合，以鉴定依赖于LSD1的组蛋白甲基化调节的notch1调节基因。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来自不同SSc患者队列的数据集进行比较。此外，我们的目标是利用siRNA、表达载体和小分子抑制剂等标准分子生物学技术，更详细地分析PPAR和MAP激酶信号通路和SMAD3/4信号通路中病理生理相关通路术语的富集。对于分析，我们将采用不同的技术，如共免疫沉淀（CoIP）和报告者测定或ChIP-qPCR。通过在Notch1act小鼠体内抑制上述途径，在体外得到的数据将得到验证。最后，我们的目标是评估靶向LSD1在不同实验性纤维化小鼠模型中的治疗潜力，如博莱霉素诱导的真皮纤维化、TSK1小鼠和慢性移植物抗宿主病（cGvHD）。