biostudies-literatureJuan AMNICHD NIH HHSNHLBI NIH HHSNational Heart, Lung, and Blood InstituteNICHDNational Institutes of HealthNational Institute of General Medical SciencesNIGMS NIH HHSNational Institute of Child Health and Human Development3613-3631.e7https://www.ebi.ac.uk/biostudies/studies/S-EPMC9547965biostudies-literatureUnknown82(19)Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architectuMolecular cellTissue-specific Grb10/Ddc insulator drives allelic architecture for cardiac development.PMC95479655T32HD083185-02R37 GM051279GM051279R01 GM051279F31 HD095583R35 HL140018T32 HD083185F31 HD095583-01A1Epstein JALeu NARosier CLBartolomei MSThorvaldsen JLRurik JGJuan AMKrapp CFoong YHLi LLan YfalseTissue-specific Grb10/Ddc insulator drives allelic architecture for cardiac development.Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architectu2022-01-01T00:00:00Z2022 Oct2026-05-28T01:36:57.296Z2025-04-04T12:49:16.384ZS-EPMC95479653610863210.1016/j.molcel.2022.08.021