A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus [4C-seq]

Dysregulation of imprinted gene loci also referred to as loss of imprinting (LOI) can result in severe developmental defects and other diseases, but the molecular mechanisms that ensure imprint stability remain incompletely understood. Here, we dissect the functional components of the imprinting control region of the essential Dlk1-Dio3 locus (called IG-DMR) and the mechanism by which they ensure imprinting maintenance. Using pluripotent stem cells carrying an allele-specific reporter system, we demonstrate that the IG-DMR consists of two antagonistic regulatory elements: a paternally methylated CpG-island that prevents the activity of Tet dioxygenases and a maternally unmethylated regulatory element, which serves as a non-canonical enhancer and maintains expression of the maternal Gtl2 lncRNA by precluding de novo DNA methyltransferase function. Targeted genetic or epigenetic editing of these elements leads to LOI with either bi-paternal or bi-maternal expression patterns and respective allelic changes in DNA methylation and 3D chromatin topology of the entire Dlk1-Dio3 locus. Although the targeted repression of either IG-DMR or Gtl2 promoter is sufficient to cause LOI, the stability of LOI phenotype depends on the IG-DMR status, suggesting a functional hierarchy. These findings establish the IG-DMR as a novel type of bipartite control element and provide mechanistic insights into the control of Dlk1-Dio3 imprinting by allele-specific restriction of the active DNA (de)methylation machinery. Overall design: Experiments were performed in duplicate to generate two technical replicates per sample. JF1/B6 iPSC's (2 × 106) were crosslinked in 1% formaldehyde at room temperature for 10 min and quenched with 125 mM glycine for 5 min at room temperature. The cell pellets were washed twice in PBS and resuspended in 300 µl lysis buffer (10mM Tris-HCl (pH 8.0), 10mM NaCl, 0.2% Igepal CA630 (Sigma I8896), on ice for 20 min. Following centrifugation at 2,500g for 5 min at 4°C, the pellet was resuspended in in 50uL of 0.5% SDS and incubated for 10 min at 65°C. SDS was quenched with 145uL water and 25uL of 10% TritonX-100. 25ul of Cutsmart buffer was added with DpnII enzyme (10 µl; NEB, R0543M) and the samples were incubated overnight at 37 °C with 700rpm rotation. The samples were then diluted with 663µl Milli-Q water, 120 µl T4 ligation buffer (NEB B0202), 60ul ATP 10mM, 120 µl Triton X-100, 12ul BSA 10mg/ml and incubated with Ligase (5 µl, 400U/µl ; NEB M0202L) for 3h on a rotor at room temperature. The samples were then treated with proteinase K and reverse crosslinked overnight. Following RNAse treatment, phenol/chloroform extraction and DNA precipitation, the pellets were dissolved in 100 µl of 10 mM Tris pH 7.5 and digested overnight at 37°C by adding 20 µl Cutsmart buffer (NEB), 10ul µl NlaIII (NEB, R0125) and 70 µl Milli-Q water. Following enzyme inactivation, the samples were diluted in 2345 ul Milli-Q water, 300 ul 10×ligation buffer (NEB), 150ul ATP 10mM and incubated with, 5 µl T4 DNA ligase 2M U/ul (NEB, M0202M) overnight at 16°C. The DNA was purified by phenol/chloroform extraction, ethanol precipitation and Zymo columns (D4014). For allele-specific 4C-seq library preparation, primers were designed upstream of a SNP within IG-DMR. Due to lack of suitable restriction fragments on ?MatTRE samples, non-allele specific libraries were prepared with the Gtl2 DMR as viewpoint. Library preparation was further performed by using a PCR strategy as previously described (Krijger et al. 2019). Briefly, 4x200 ng of 4C-template DNA was used to PCR amplify the libraries using the Roche Expand long template PCR system (Roche, 11681842001). Primers were removed using Ampure XP beads (Beckman Coulter, A63880). A second round of PCR was performed using the initial PCR library as a template, with overlapping primers to add the full adapters. The libraries were sequenced on a miSeq platform in SE150 mode.