An intragenic methylated region in the imprinted Igf2 gene augments transcription

Abstract

DNA methylation is usually associated with transcriptional silencing, but in the imprinted mouse Igf2 gene, the paternally expressed copy is methylated in two discrete differentially methylated regions (DMRs). DMR1 is located upstream of the fetal promoters and has been shown to be a methylation sensitive silencer. Here we examine the role of the intragenic DMR2 by gene targeting. In contrast to DMR1, deletion of DMR2 on the maternal allele did not lead to activation of the silent Igf2 gene. Deletion of a 54 bp methylated core region in DMR2 on the paternal allele, however, reduced Igf2 mRNA levels and was associated with fetal growth retardation. Nuclear run-on assays showed that the core region influenced transcription initiation, and luciferase reporter assays suggested that its methylation increases transcription. These results reveal a novel mechanism of gene expression whereby intragenic methylation can increase levels of transcription.

Placental programming of anxiety in adulthood revealed by Igf2-null models

“…We first established the effects of the Igf2 manipulations on birth weight and postnatal growth. Confirming and extending the original findings of Constância et al 13,19 , in the Igf2-total KO model, lack of all main Igf2 transcripts in both placenta and foetus led to reduced birth weight, B55% of wild-type (WT) littermate controls and a reduced physical weight that continued throughout life (Fig. 2).…”

“…Production of Igf2-P0 KO and Igf2-total KO mice, which were backcrossed to a C57BL/6 background, has been described previously 13,19 . To generate the experimental animals, carrier male mice of each line (which did not express the Igf2 mutations in somatic cells because of matrilineal inheritance of the mutated allele) were paired with unmanipulated female CD1 mice (Harlan Olac, UK) producing either WT or KO offspring in which the mutations were now present on the paternal allele and therefore expressed in somatic cells.…”

“…In the mouse, there are four main Igf2 transcripts expressed during development: the placental-specific P0 transcript mapping to the labyrinthine trophoblast, the key exchange barrier for nutrients; the P2 and P3 transcripts, expressed in both placenta and foetus; and the P1 transcript expressed mainly in liver 18 . In our experiments we compared mice engineered to be null for the placenta-specific P0 transcript 13 (Igf2-P0 KO) with mice with disruptions of all four Igf2 transcripts, and therefore null for Igf2 in both placenta and foetus 19 (Igf2-total KO) (see Fig. 1 (ref.…”

“…Igf2 is imprinted with gene expression mainly from the paternal allele of mouse chromosome 7; expression from the maternal allele is attenuated (B5%) excluding the choroid plexus/leptomeninges in brain where expression is bi-allelic 57,58 . Igf2-total KO mice were created by insertion of a LacZ-targeting vector in the first coding exon abolishing expression of the four main Igf2 transcripts (P0, P1, P2 and P3) in both the placenta and foetus 19 . This leads to IUGR with the mice remaining small for life 19,59 .…”

“…Igf2-total KO mice were created by insertion of a LacZ-targeting vector in the first coding exon abolishing expression of the four main Igf2 transcripts (P0, P1, P2 and P3) in both the placenta and foetus 19 . This leads to IUGR with the mice remaining small for life 19,59 . The Igf2-P0 KO mice were created by deletion of the U2 exon in the P0 promoter region; this prevents production of the placenta-specific P0 Igf2 transcript but does not interfere with placental or foetal expression of the other main Igf2 transcripts 13 .…”