Project Summary/ Abstract Distinct epigenetic mechanisms, including histone modifications, DNA methylation, chromatin remodeling and histone variant exchange, collaboratively and dynamically shape local epigenetic landscape and control gene activities. Alteration of epigenetic pathways and mutations in epigenetic regulators contribute to the pathogenesis of various human diseases. Several studies suggest that these epigenetic pathways are orchestrated to regulate activity-gene expression. Current tools to simultaneously manipulate multiple epigenetic pathways in temporal and locus specific are lacking. Such tools are necessary to precisely dissect the functional interplays and crosstalk between these distinct epigenetic mechanisms for gene regulation. To address this limitation, we propose to establish unique chemical inducible methods that integrate dCas9/gRNA-guided targeting with the orthogonal chemically induced proximity (CIP) technology to simultaneously control different epigenetic regulations including all major epigenetic pathways. We will focus on the following three aims: (i) Develop inducible dual editing platforms for diverse histone modifications; (ii) Develop chemical inducible platforms for chromatin remodeling and nucleosome editing; (iii) Develop chemical inducible editing platforms for DNA methylation and validate the orthogonal manipulation of multiple mechanisms across distinct epigenetic pathways. After finishing this work, we expect to establish a unique toolset that can be readily used by researchers in the field. This platform can be easily adapted and expanded to manipulate new epigenetic factors not tested in this study. This new epigenome editing/remodeling platform will facilitate the advancement of epigenetic research and potentially offer new directions in developing new epigenetic-based therapies.