SRA

Summary: We report that overexpression of the TET1 catalytic domain induces massive global DNA demethylation in HEK293T cells but this is not seen with full length TET1, despite production of 5-hydroxymethylcytosine (hmC). Genome-wide mapping reveals that hmC production is relatively inhibited as DNA methylation level increases, which can be explained by the preferential binding of TET1 to unmethylated CpG islands (CGIs) through its CXXC domain. Consistent with it, overexpression of full length TET1 specifically decreases DNA methylation in sparsely methylated (1-10%) CGIs, while TET1 knockdown results in increased DNA methylation spreading from methylated edges into hypomethylated CGIs. Thus, in adult cells TET1 is a maintenance DNA demethylase that prevents DNA methylation spreading into CpGs. Overall Design: Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the DNA methylation profiles of HEK293T cells overexpressing mTET1-CD, TET1-CD, mTET1-FL, or TET1-FL. In this approach, genomic DNA is sequentially cut at CCCGGG sites with the methylation sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5’CCGG overhangs), creating different end sequences that represent methylation status of the CCCGGG sites. These end sequences are analyzed by next generation sequencing, and thereafter the methylation status at individual CCCGGG sites across the genome can be determined.