A bipartite element with allele-specific functions safeguards DNA methylation imprints at the Dlk1-Dio3 locus

Abstract

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 noncanonical 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 DNA (de)methylation machinery..

Epigenome editing reveals core DNA methylation for imprinting control in the Dlk1-Dio3 imprinted domain

“…These observations, in addition to our results, suggest that changes in the binding state of ZFP57 at IG-DMR-Rep can be a starting point for shifting the epigenetic state of the entire IG-DMR. On the other hand, the mechanism by which DNA methylation state at IG-DMR affects Meg3 -DMR would be associated with chromatin loop formation, as proposed previously ( 16 , 19 ), and factors associated with loop formation (CTCF, YY1, SMC1A, STAG1/2, RAD21 and ZFP143) bind IG- and Meg3 -DMRs ( 52 ) ( Supplementary Figure S6 ).…”

“…IG-DMR can be divided into two subdomains, i.e. IG CGI and IG TRE comprising the 5′- and 3′-halves of IG-DMR, respectively ( 16 ) (Figure 1D ). Two gRNAs (gR1 and gR2) corresponded to sequences in IG CGI in which histone H3-K4 trimethylation (H3K4me3) is enriched.…”

“…The molecular mechanism by which the DNA methylation state at IG-DMR-Rep spreads throughout the entire IG-DMR is also intriguing. As IG-DMR-Rep contains a tandem repeat of ZFP57 binding sequences, ZFP57, which binds to methylated DNA, would be a key player in this mechanism ( 16 , 45–47 ). ZFP57 can act as a scaffold for the KAP1 complex, which contains KAP1 (also known as TRIM28), CBX1/3/5 and SUMO1/2.…”

“…Knockout mice lacking the entire IG-DMR demonstrated its critical role in imprinting regulation of the whole Dlk1-Dio3 domain; in other words, IG-DMR functions as the ICR of the domain ( 15 ). Several recent studies have shown that IG-DMR is a bipartite element that consists of 5′ IG CGI (CGI: CpG island) and 3′ IG TRE (TRE: transcriptional regulatory element) ( 4 , 16 , 17 ). IG CGI contains a 216-bp tandem repeat called IG-DMR-Rep, and its deletion in mice revealed an essential role of IG-DMR-Rep in the maintenance of DNA hypermethylation on the paternal allele after fertilisation ( 4 , 18 ).…”

“…IG CGI contains a 216-bp tandem repeat called IG-DMR-Rep, and its deletion in mice revealed an essential role of IG-DMR-Rep in the maintenance of DNA hypermethylation on the paternal allele after fertilisation ( 4 , 18 ). On the other hand, unmethylated IG TRE of the maternal allele functions as an enhancer of Meg3 ( 16 , 19–21 ). Meg3 -DMR, which is located in the Meg3 promoter, acquires DNA methylation on the paternal allele at the post-implantation stage, whereas the maternal allele remains hypomethylated ( 22 ).…”