Loss of imprinting at the Dlk1-Gtl2 locus caused by insertional mutagenesis in the Gtl2 5' region

Abstract

Background: The Dlk1 and Gtl2 genes define a region of mouse chromosome 12 that is subject to genomic imprinting, the parental allele-specific expression of a gene. Although imprinted genes play important roles in growth and development, the mechanisms by which imprinting is established and maintained are poorly understood. Differentially methylated regions (DMRs), which carry methylation on only one parental allele, are involved in imprinting control at many loci. The Dlk1-Gtl2 region contains three known DMRs, the Dlk1 DMR in the 3' region of Dlk1, the intergenic DMR 15 kb upstream of Gtl2, and the Gtl2 DMR at the Gtl2 promoter. Three mouse models are analyzed here that provide new information about the regulation of Dlk1-Gtl2 imprinting.

Results: A previously existing insertional mutation (Gtl2lacZ), and a targeted deletion in which the Gtl2 upstream region was replaced by a Neo cassette (Gtl2Delta5'Neo), display partial lethality and dwarfism upon paternal inheritance. Molecular characterization shows that both mutations cause loss of imprinting and changes in expression of the Dlk1, Gtl2 and Meg8/Rian genes. Dlk1 levels are decreased upon paternal inheritance of either mutation, suggesting Dlk1 may be causative for the lethality and dwarfism. Loss of imprinting on the paternal chromosome in both Gtl2lacZ and Gtl2Delta5'Neo mice is accompanied by the loss of paternal-specific Gtl2 DMR methylation, while maternal loss of imprinting suggests a previously unknown regulatory role for the maternal Gtl2 DMR. Unexpectedly, when the Neo gene is excised, Gtl2Delta5' animals are of normal size, imprinting is unchanged and the Gtl2 DMR is properly methylated. The exogenous DNA sequences integrated upstream of Gtl2 are therefore responsible for the growth and imprinting effects.

Conclusion: These data provide further evidence for the coregulation of the imprinted Dlk1 and Gtl2 genes, and support a role for Dlk1 as an important neonatal growth factor. The ability of the Gtl2lacZ and Gtl2Delta5'Neo mutations to cause long-range changes in imprinting and gene expression suggest that regional imprinting regulatory elements may lie in proximity to the integration site.

Figure 1

Dlk1-Gtl2 domain and Gtl2lacZ integration. A) Schematic of the Dlk1-Gtl2 imprinted domain. Maternally expressed genes are shown in pink and paternally expressed genes in blue; the direction of their transcription is indicated. Black lollipops indicate the positions of the three known Dlk1-Gtl2 DMRs, which have been designated with individual letters, D, Dlk1 DMR, I, IG DMR, and G, Gtl2 DMR. The dotted arrow indicates a genomic region that produces multiple miRNAs and snoRNAs; emerging evidence suggests contiguous transcription through this region. B) Diagram of the Gtl2lacZ integration. The blue arrows represent the lacZ genes and the green arrows represent the Neo genes. The broken line represents an internal rearrangement that has been refractory to analysis; its size is estimated by Southern blotting. The gold box represents the Gtl2 DMR. C) Growth curves of Gtl2lacZPat¹²⁹ mice in comparison to wild type littermates (WT, n = 11, Gtl2lacZPat¹²⁹, n = 3). D) Growth curves of Gtl2lacZPat^B6 mice in comparison to wild type littermates (WT, n = 6, Gtl2lacZPat^B6, n = 7). E) Growth curves of Gtl2lacZMat¹²⁹ mice in comparison to wild type littermates (WT, n = 7, Gtl2lacZMat¹²⁹, n = 8). F) Growth curves of Gtl2lacZMat^B6 mice in comparison to wild type littermates (WT, n = 7, Gtl2lacZMat^B6, n = 8). In all graphs the solid line represents wild type animals and the dashed line represents Gtl2lacZ animals.

Figure 9

Breeding strategies for the Gtl2lacZ and Gtl2Δ5'Neo mice. The crosses generated for gene expression and imprinting analysis are detailed. In each cross, the final animals examined for expression/imprinting are indicated at the far right.

Figure 2

Expression levels of Dlk1 and Gtl2 mRNAs in Gtl2lacZ embryos. A) Representative Northern blots for Dlk1 and Gtl2 RNA levels, in comparison to βactin, in Gtl2lacZPat¹²⁹ (left) and Gtl2lacZPat^B6 (right) embryo and placenta. B) Graphical representation of quantitative expression data for Gtl2lacZPat¹²⁹ (top) and Gtl2lacZPat^B6 (bottom) embryos. In each graph, the black bars represent wild type (the mean of these values was set to 100), and the gray bars represent Gtl2lacZPat, E indicates embryo samples and P indicates placental samples; quantitative data is given for embryo only. P-values are indicated below each graph. (Top graph, Dlk1, WT, n = 3, Gtl2lacZPat¹²⁹, n = 4; Gtl2, WT, n = 3, Gtl2lacZPat¹²⁹, n = 4. Bottom graph, Dlk1, WT, n = 4, Gtl2lacZPat^B6, n = 4; Gtl2, WT, n = 4, Gtl2lacZPat^B6, n = 4). C) Representative Northern blots for Dlk1 and Gtl2 RNA levels, in comparison to β-actin, in Gtl2lacZMat¹²⁹ (left) and Gtl2lacZMat^B6 (right) embryos. D) Graphical representation of quantitative expression data for Gtl2lacZMat¹²⁹ (top) and Gtl2lacZMat^B6 (bottom) embryos. In each graph, the black bars represent wild type (the mean of these values was set to 100), and the gray bars represent Gtl2lacZMat. P-values are indicated below each graph. Differences in β-actin levels are the result of analyzing different midgestation embryonic stages. (Top graph, Dlk1, WT, n = 4, Gtl2lacZMat¹²⁹, n = 4; Gtl2, WT, n = 4, Gtl2lacZMat¹²⁹, n = 4. Bottom graph, Dlk1, WT, n = 4, Gtl2lacZMat^B6, n = 4; Gtl2, WT, n = 4, Gtl2lacZMat^B6, n = 4). All expression data includes embryos from at least two litters.

Figure 3

Dlk1-Gtl2 imprinting analysis in Gtl2lacZ embryos. A) Imprinting assay for Dlk1 in wild type (left), Gtl2lacZPat¹²⁹ (center) and Gtl2lacZPat^B6 (right) embryos. B) Imprinting assay for Dlk1 in wild type (left), Gtl2lacZMat¹²⁹ (center) and Gtl2lacZMat^B6 (right) embryos. The Dlk1 polymorphism is an "A" in D embryos and a "G" in C embryos; the polymorphic base is boxed on each chromatogram. C) Imprinting assay for Gtl2 in Gtl2lacZPat¹²⁹ and Gtl2lacZPat^B6 embryos. D) Imprinting assay for Gtl2 in Gtl2lacZMat¹²⁹ and Gtl2lacZMat^B6 embryos. The Gtl2 polymorphism alters an SfcI restriction enzyme site, with the site present in D and absent in C; the D and C bands are indicated at the right.

Figure 4

Gene targeting of the Gtl2 upstream region. A) Schematic representation of the Gtl2 upstream region and the Gtl2Δ5'Neo and Gtl2Δ5' targeted loci. The targeting event replaces a 2.8-kb region of Gtl2 upstream sequence with the Neo gene (Gtl2Δ5'Neo allele). Cre-mediated excision removes the Neo gene, leaving a single loxP site in the locus (Gtl2Δ5' allele). The black boxes represent the Gtl2 exons (only exons 1–5 are shown), the gold box represents the Gtl2 DMR, the green box represents the Neo gene, the purple boxes represent highly conserved regions across species and the vertical arrow indicates the position of the Gtl2lacZ integration. B) Growth curves of Gtl2Δ5'NeoPat¹²⁹ mice in comparison to wild type littermates (WT, n = 4, Gtl2Δ5'NeoPat¹²⁹, n = 8). C) Growth curves of Gtl2Δ5'NeoPat^B6 mice in comparison to wild type littermates (WT, n = 11, Gtl2Δ5'NeoPat^B6, n = 5). In all graphs the solid line represents wild type animals and the dashed line represents Gtl2Δ5'Neo animals.

Figure 5

Expression analysis for Dlk1-Gtl2 mRNA in Gtl2Δ5'Neo embryos. A) Representative Northern blots for Dlk1 and Gtl2 RNA levels, in comparison to β-actin, in Gtl2Δ5'NeoPat¹²⁹ (left) and Gtl2Δ5'NeoPat^B6 (right) embryos. B) Graphical representation of quantitative expression data for Gtl2Δ5'NeoPat¹²⁹ (top) and Gtl2Δ5'NeoPat^B6 (bottom) embryos. In each graph, the black bars represent wild type (the mean of these values was set to 100), and the gray bars represent Gtl2Δ5'NeoPat. P-values are indicated below each graph. (Top graph, Dlk1, WT, n = 4, Gtl2Δ5'NeoPat¹²⁹, n = 6; Gtl2, WT, n = 4, Gtl2Δ5'NeoPat¹²⁹, n = 6. Bottom graph, Dlk1, WT, n = 6, Gtl2Δ5'NeoPat^B6, n = 10; Gtl2, WT, n = 6, Gtl2Δ5'NeoPat^B6, n = 10). C) Representative Northern blots for Dlk1 and Gtl2 RNA levels, in comparison to β-actin, in Gtl2Δ5'NeoMat¹²⁹ (left) and Gtl2Δ5'NeoMat^B6 (right) embryos. D) Graphical representation of quantitative expression data for Gtl2Δ5'NeoMat¹²⁹ (top) and Gtl2Δ5'NeoMat^B6 (bottom) embryos. In each graph, the black bars represent wild type (the mean of these values was set to 100), and the gray bars represent Gtl2Δ5'NeoMat. P-values are indicated below each graph. (Top graph, Dlk1, WT, n = 4, Gtl2Δ5'NeoMat¹²⁹, n = 6; Gtl2, WT, n = 4, Gtl2Δ5'NeoMat¹²⁹, n = 6. Bottom graph, Dlk1, WT, n = 4, Gtl2Δ5'NeoMat^B6, n = 4; Gtl2, WT, n = 4, Gtl2Δ5'NeoMat^B6, n = 4).

Figure 6

Dlk1-Gtl2 imprinting analysis in Gtl2Δ5'Neo embryos. A) Imprinting assay for Dlk1 in wild type (left) and Gtl2Δ5'NeoPat (right) embryos. B) Imprinting assay for Dlk1 in wild type (left), Gtl2Δ5'NeoMat¹²⁹ (center) and Gtl2Δ5'NeoMat^B6 (right) embryos. The Dlk1 polymorphism is an "A" in D embryos and a "G" in C embryos; the polymorphic base is boxed on each chromatogram. C) Imprinting assay for Gtl2 in Gtl2Δ5'NeoPat embryos. D) Imprinting assay for Gtl2 in Gtl2Δ5'NeoMat¹²⁹ and Gtl2Δ5'NeoMat^B6 embryos. The Gtl2 polymorphism alters an SfcI restriction enzyme site, with the site present in D and absent in C; the D and C bands are indicated at the right.

Figure 7

Methylation analysis of the Gtl2 DMR in Gtl2Δ5'NeoPat and Gtl2Δ5'Pat embryos. A) Schematic of the Gtl2 DMR region in wild type, Gtl2Δ5'Neo and Gtl2Δ5' alleles. The grey box represents the Gtl2Δ5' deletion region, the green box represents the Neo gene and the black box represents Gtl2 exon 1. The vertical arrows represent recognition sites for the MspI/HpaII restriction enzymes, and the loxP sites are indicated by black triangles. The methylation assay detects a 2.2-kb endogenous HincII fragment that spans the Gtl2 DMR. Modifications in the Gtl2Δ5'Neo and Gtl2Δ5' alleles generate 3.8-kb and 3.5-kb fragments, respectively. B) Methylation assay for the Gtl2 DMR in Gtl2Δ5'NeoPat¹²⁹ and Gtl2Δ5'Pat¹²⁹ embryos. C) Methylation assay for the Gtl2 DMR in Gtl2Δ5'NeoMat¹²⁹ and Gtl2Δ5'Mat¹²⁹ embryos. Restriction sites are, H, HincII, Hp, HpaII and M, MspI.

Figure 8

Meg8/Rian imprinting analysis in Gtl2Δ5'Neo embryos. A) Imprinting assay for the Meg8 (left) and Rian (right) genes in Gtl2Δ5'NeoPat¹²⁹ and Gtl2Δ5'Pat¹²⁹ embryos. B) Imprinting assay for the Meg8 (left) and Rian (right) genes in Gtl2Δ5'NeoMat¹²⁹ and Gtl2Δ5'Mat¹²⁹ embryos. The Meg8 imprinting assay detects an NlaIII polymorphism, with the site present in C mice and absent in D mice. The Rian imprinting assay detects a HinfI polymorphism, with the site present in D mice, and absent in C mice.