Differential Dna Methylation Drives Allelic Architecture For Grb10-Ddc Locus In The Developing Heart

DNA is organized into spatially distinct units by architectural protein CTCF to enrich for intradomain enhancer-promoter contacts, but developmental and tissue specific regulation of architecture in vivo is poorly defined. By profiling neonatal mouse tissues at the imprinted Grb10-Ddc locus, putative tissue- and allele-specific chromatin was revealed, which we interrogated by deletions in vivo. We uncovered a new tissue-specific intronic insulator at Grb10 that is distinct from the classic differentially methylated imprinting control region, acquiring its own allele-specific DNA methylation during development, ultimately regulating gene expression of the entire cluster in heart and muscle. Termed CTCF-Binding Region 2.3 (CBR2.3), this insulator assembles a paternal-specific contact domain with Ddc located 150 kb away, restricting a newly validated cardiac enhancer to the paternal Ddc promoter. Paternal deletion of CBR2.3 in mice reconfigures the chromatin topology to mimic the maternal chromosome, resulting in Grb10-Ddc misexpression and cardiac phenotypes. This in vivo validation of a robust allelic and tissue-specific insulator offers mechanistic insight into how genes utilize different layers of spatial organization to achieve variation in gene expression in development.