Deletion of -DMR Enhances Migration and Invasion of MLTC-1 Depending on the CTCF Binding Sites.

The imprinted domain on mouse chromosome 12 contains three well-characterized paternally methylated differentially methylated regions (DMRs): IG-DMR, -DMR, and -DMR. These DMRs control the expression of many genes involved in embryonic development, inherited diseases, and human cancer in this domain. The first maternal methylation DMR discovered in this domain was the -DMR, the targets and biological function of which are still unknown. Here, using an enhancer-blocking assay, we first dissected the functional parts of the -DMR and showed that its insulator activity is dependent on the CCCTC-binding factor (CTCF) in MLTC-1. Results from RNA-seq showed that the deletion of the -DMR and its compartment CTCF binding sites, but not GGCG repeats, lead to the downregulation of numerous genes on chromosome 12, in particular the drastically reduced expression of and in the domain, while differentially expressed genes are enriched in the MAPK pathway. In vitro assays revealed that the deletion of the -DMR and CTCF binding sites enhances cell migration and invasion by decreasing Dlk1 and activating the Notch1-Rhoc-MAPK/ERK pathway. These findings enhance research into gene regulation in the domain by indicating that the -DMR functions as a long-range regulatory element which is dependent on CTCF binding sites and affects multiple genes in this domain.