DNA methylation is a fundamental epigenetic modification to regulate 10mammalian gene expression, where each type of cell creates a specific methylation profile during its differentiation. Hepatocyte differentiation is well characterized at the transcriptome level, although epigenetic controls during the differentiation process have yet to be investigated. We analyzed omics data, including DNA methylation profiles, during hepatocyte differentiation. We found GATA6 as a key transcription factor (TF) for its binding motif-depen-dent chromatin activation and DNA demethylation in definitive endoderm dif-ferentiation, an initial step in hepatocyte lineage commitment.We developed a bioinformatics pipeline to identify TFs involved in DNA de-methylation. We found that specific TF families are involved in DNA demeth-ylation in a cell lineage-specific manner. We validated more than 30 TFs pos-sessing DNA demethylation induction activity, suggesting that many TFs are involved in regulation of cell type-specific DNA methylation profiles. Further-more, we succeeded in obtaining insight into TF-mediated binding site-specific DNA demethylation, where more than two TF families, sharing similar but distinct binding motifs, are expressed in different cell lineages and/or different differentiation stages, creating distinct DNA methylation profiles of terminally differentiated cells.We have begun to explore the role and outcomes of disordered DNA de-methylation. We generated two iPS cell lines with either a RUNX1 knock-out or a RUNX1 mutation without DNA demethylation ability to understand the role of RUNX1-mediated DNA demethylation in hematopoiesis. Further, we found that deficiency of Ten-Eleven Translocation-2 (TET2), known as a DNA demethylation factor, results in aberrant megakaryocyte-erythroid progeni-tor (MEP) cells. We are exploring the link between the MEP abnormality and pathogenesis of myeloproliferative neoplasms.In studies of the epithelial-to-mesenchymal transition (EMT) process, we identified previously unrecognized cell states within a subpopulation of mam-mary epithelial cells using single-cell ATAC-seq and RNA-seq. Further, we suc-cessfully identified candidate epigenome regulators during EMT using a large-scale CRISPR-Cas9 screen. In drug-induced cell reprogramming studies, we found that transplantation of reprogrammed neuron-like cells improved brain functions, such as locomotor function and memory function, in a chronic isch-emic stroke mouse model.Figure: A schematic illustration of a model for the interrelation between GATA6-mediated DNA demethylation and chromatin statusGATA6 first binds to GATA6 binding motifs in permissive heterochromatin sites, then opens and activates the chromatin at the binding sites and finally completes the DNA demethylation.Recent Major PublicationsSuzuki T, Furuhata E, Maeda S, Kishima M, Miyajima Y, Tanaka Y, Lim J, Nishimura H, Nakanishi Y, Shojima A, Suzuki H. Master Transcription Factors Regulate the DNA Methylation Landscape During Hepatocyte Differentia-tion. bioRxiv https://www.biorxiv.org/content/10.1101/2020.12.16.423165v1 (2020)Nakano R, Kitanaka T, Namba S, Kitanaka N, Sato M, Shibukawa Y, Masuhiro Y, Kano K, Matsumoto T, Sugiya H. All-trans retinoic acid induces reprogramming of canine dedifferentiated cells into neuron-like cells. Plos One 15, e0229892 (2020)Pervolarakis N, Nguyen QH, Williams J, Gong Y, Gutier-rez G, Sun P, Jhutty D, Zheng GXY, Nemec CM, Dai X, Watanabe K, Kessenbrock K. Integrated Single-Cell Tran-scriptomics and Chromatin Accessibility Analysis Reveals Regulators of Mammary Epithelial Cell Identity. Cell Rep 33, 108273 (2020)Laboratory for Cellular Function Conversion TechnologyTeam Leader: Harukazu Suzuki
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