Project Summary Continuous differentiation of intestinal stem cells (ISCs) maintains the integrity of intestinal epithelium while producing diverse functional cells at rapid pace. This epithelium also displays remarkable cell plasticity, including the ability of mature cells to dedifferentiate into stem cells upon loss of ISCs. As many genetic and signaling drivers of this cell fate reversion are being discovered, cell intrinsic epigenetic controls that must be rewired for cell plasticity, remain largely unknown. Using state-of-the-art single-cell sc-Multiome assays that reveal chromatin accessibility (scATAC-seq) and gene expression (scRNA-seq) in parallel, we have recently produced high-quality data comparing native epithelial cells and cells undergoing regeneration upon ISC ablation. Using these data and innovative computational methods, we will chart the chromatin and gene expression dynamic underlying cell fate changes in intestinal regeneration, and identify key epigenetic modulators and transcription factor controllers of this process. In conjunction with these analyses, we propose to delineate promoter and enhancer relevant epigenetic controls (histone modifications) through the time course of regeneration. Aberrant chromatin structure can cause gene dysregulation and contribute to tumorigenesis, including colorectal cancer. DNA methylation is the most frequently modulated epigenetic modality in colorectal as well as many other cancers. However, determinants of the reciprocal interactions between DNA methylation and mutational changes or other epigenetic layers have not been studied critically. Also missing is the knowledge of how early in tumorigenesis does the DNA methylation change occur and how uniform are the changes in DNA methylation across cells. Technological limitations have been the most significant barrier for such analyses, as current methods to characterize DNA methylation at single-cell level suffer from low CpG coverage limited to gene promoters and GpG islands, which precludes understanding of the true breadth of DNA methylation change, particularly at gene regulatory enhancers. We propose to override these limitations through development of a novel assay for characterizing DNA methylation as well as RNA expression at single-cell level. We combine chemical methods geared to preserve DNA integrity and gentler nuclei handling based on magnetic bead capture in our method called scTaMT-seq, which will provide a robust protocol for analyzing DNA methylation in unprecedented detail at highest possible resolution. This technology will propel the understanding of epigenomic impact on various biological processes, particularly cancer.