Regulation of chromatin remodeling by Notch1 in fibrotic disorders

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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).