Domain-Specific Response of Imprinted Genes to Reduced DNMT1

Abstract

Imprinted genes are expressed in a monoallelic, parent-of-origin-specific manner. Clusters of imprinted genes are regulated by imprinting control regions (ICRs) characterized by DNA methylation of one allele. This methylation is critical for imprinting; a reduction in the DNA methyltransferase DNMT1 causes a widespread loss of imprinting. To better understand the role of DNA methylation in the regulation of imprinting, we characterized the effects of Dnmt1 mutations on the expression of a panel of imprinted genes in the embryo and placenta. We found striking differences among imprinted domains. The Igf2 and Peg3 domains showed imprinting perturbations with both null and partial loss-of-function mutations, and both domains had pairs of coordinately regulated genes with opposite responses to loss of DNMT1 function, suggesting these domains employ similar regulatory mechanisms. Genes in the Kcnq1 domain were less sensitive to the absence of DNMT1. Cdkn1c exhibited imprinting perturbations only in null mutants, while Kcnq1 and Ascl2 were largely unaffected by a loss of DNMT1 function. These results emphasize the critical role for DNA methylation in imprinting and reveal the different ways it controls gene expression.

Epigenetics and imprinting in human disease

“…Intriguingly, the extraembryonic and embryonic tissues may use different mechanisms to maintain imprinting, as demonstrated by experiments assaying imprinted gene expression in mice that are deficient for the maintenance DNA methyltransferase, DNMT1 (Lewis et al, 2004, Weaver et al, 2010. These experiments show that placenta-specific imprinted genes in the Kcnq1 cluster, including Osbpl5, Tssc4, Cd81, and Ascl2, maintain imprinting in the absence of DNMT1.…”

Nonallelic Transcriptional Roles of CTCF and Cohesins at Imprinted Loci

“…Reaction mixtures were set up in duplicates or triplicates by using Ready-To-Go PCR beads (Amersham) with a 5-min initial incubation with TaqStart antibody (Clontech), followed by the addition of 0.2 to 0.6 M primers (Table 2), 1ϫ EvaGreen (Biotium), and 1.25 to 5 mM MgCl 2 . Data analysis was performed by using Light Cycler 4.0 software using the Relative Quantification program (Monocolor) to determine the ratio of each ChIP sample relative to input or cDNA levels relative to the acidic ribosomal phosphoprotein P0 (Arpp0), ␤-actin, or GAPDH gene (64,66). For statistical analysis, the two-tail paired Student t test was used, and differences with a P value of Ͻ0.05 were considered significant.…”

“…H19 RNA assays were conducted on cDNA using the LightCycler real-time PCR system (Roche Molecular Biochemicals) as described previously (46). Igf2 and Gtl2 RNA assays were conducted on cDNA by allele-specific restriction digests as previously described (66). Dlk1 RNA assays were conducted, with some modifications, as previously described (20).…”

Long noncoding RNA-mediated maintenance of DNA methylation and transcriptional gene silencing

“…Moreover, loss of imprinted silencing of Slc22a18 and Cdkn1c genes was detected in different Dnmt1 mutant backgrounds (Lewis et al, 2004;Weaver et al, 2010). To gain further insights into the role of Kcnq1ot1 and Dnmt1 interaction in the maintenance of CpG methylation at the somatic DMRs, we assayed the enrichment of Dnmt1 and Kcnq1ot1 ncRNA at the somatic DMRs by using ChIP and ChOP techniques, respectively, in embryonic and extraembryonic tissues of the ICR 2loxER8.5 and ICR 1loxER8.5 litters.…”