Differential DNA Methylation Drives Allelic Architecture for Grb10-Ddc Locus in the Developing Heart

While it is understood that the genome is organized into large domains, little is known about cell-type specific interactions at individual loci in mammalian development. Imprinted loci represent a compelling feature of chromatin conformation, illustrating how genes are controlled in local domains to drive parental-specific transcriptional programs important for growth: a clear example being the imprinted Grb10-Ddc locus. Using hybrid mouse crosses and genetic engineering, we uncovered an insulator at Grb10 that is tissue-specific and dynamically controlled by DNA methylation. Insulator CBR2.3 acquires paternal-specific hypomethylation post-implantation, assembling a higher-order structure on the paternal chromosome. Deletion of paternal CBR2.3 converts the chromatin domain to the maternal configuration, redirecting Grb10-Ddc enhancer function, resulting in major cardiac phenotypes. We conclude that CBR2.3 functions as a shared, paternal and tissue-specific insulator for Grb10-Ddc expression in heart, contributing to fundamentally different chromosomal conformations that impact enhancer activity in vivo. Capture-C on F1 hybrid hearts derived from reciprocal crosses between C57BL/6 (B6) mice and animals homozygous for the Japanese Fancy (JF1/Ms) strain.