Control mechanisms of 5-hydroxymethylcytosine metabolism in human cells

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Project Summary The 5-hydroxymethylcytosine (5hmC) in mammalian DNA is drawing significant attention in epigenetics because of its indispensable roles in gene expression regulation. The loss of 5hmCs in various cancers including glioblastomas and lymphomas has been linked to the poor survival rate as well as the resistance to anti-cancer therapeutics. Although there is amassing evidence of 5hmCs as a novel epigenetic marker, there is very little understanding on how 5hmCs are enriched at specific loci in the genome. In this project, we aim to identify the factors controlling the DNA demethylation pathway, which result in the formation of locus-specific 5hmCs in human neurons. For this purpose, proteins controlling the activity of ten-eleven translocase (TET) family enzymes in the DNA demethylation pathway will be identified. To examine the function of candidate proteins, we will take advantage of human induced-neurons (hiNs) derived from human induced-pluripotent stem cells (hiPSCs). In addition, we will elucidate the role and fate of 5hmCs formed during DNA damage repair process using blue light-inducible CRISPR/Cas9 technique. This robust experimental platform will enable the rapid production of isogenic and homogenous neurons and other cell types. With this cell-based assay system, high-throughput assays can be developed that will advance biomedical researches and drug discoveries. Finally, we expect the successful completion of this project will expand our understanding on DNA demethylation and their roles in epigenetics, development, and various human diseases.