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. 2001 Dec;21(23):8189-96.
doi: 10.1128/MCB.21.23.8189-8196.2001.

Regulatory mechanisms at the mouse Igf2/H19 locus

C R Kaffer  1 , A Grinberg, K Pfeifer
Affiliations

Regulatory mechanisms at the mouse Igf2/H19 locus

C R Kaffer et al. Mol Cell Biol. 2001 Dec.

Abstract

The closely linked H19 and Igf2 genes show highly similar patterns of gene expression but are reciprocally imprinted. H19 is expressed almost exclusively from the maternally inherited chromosome, while Igf2 expression is mostly from the paternal chromosome. In humans, loss of imprinting at this locus is associated with tumors and with developmental disorders. Monoallelic expression at the imprinted Igf2/H19 locus occurs by at least two distinct mechanisms: a developmentally regulated silencing of the paternal H19 promoter, and transcriptional insulation of the maternal Igf2 promoters. Both mechanisms of allele-specific silencing are ultimately dependent on a common cis-acting element located just upstream of the H19 promoter. The coordinated expression patterns and some experimental data support the idea that positive regulatory elements are also shared by the two genes. To clarify the organization and function of positive and negative regulatory elements at the H19/Igf2 locus, we analyzed two mouse mutations. First, we generated a deletion allele to localize enhancers used in vivo for expression of both H19 and Igf2 in mesodermal tissues to sequences downstream of the H19 gene. Coincidentally, we demonstrated that some expression of Igf2 is independent of the shared enhancer element. Second, we used this new information to further characterize an ectopic H19 differentially regulated region and the associated insulator. We demonstrated that its activity is parent-of-origin dependent. In contrast to recent results from Drosophila model systems; we showed that this duplication of a mammalian insulator does not interfere with its normal function. Implications of these findings for current models for monoallelic gene expression at this locus are discussed.

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Figures

FIG. 1
FIG. 1
Diagram of the wild-type, CRK10, and VM3 chromosomes in the Igf2/H19 region. (a) Summary of the wild-type (i), CRK10 (ii), and VM3 (iii) alleles, depicting the relative organization of the Igf2, H19, Nctc1, L23mrp, and Tnnt3 genes (open rectangles). Endodermal and skeletal-muscle-specific enhancers for H19 and Igf2 expression are represented by closed and hatched circles, respectively. The mapped locations of these enhancers are based on in vivo analyses of chromosomal deletions (reference and this study) and also on in vitro transfection experiments (20, 46). The ICE, for imprinting control element, is defined genetically as the minimal upstream sequence required to imprint single-copy H19 transgenes (20) and as a sequence whose deletion results in the loss of imprinting of H19 and Igf2 (20, 36, 42). Within the ICE is the 2-kb DMR (for differentially methylated region) (oval) that is hypermethylated specifically on the paternal chromosome (4, 8, 14, 29, 43, 44). Mapping to the DMR are domains of maternal-chromosome-specific nuclease hypersensitivity. The CRK10 allele (ii) carries a 9.2-kb insertion at kb +10.7 (relative to the H19 transcriptional start site). The inserted DNA includes the H19DMR, all the maternal-chromosome-specific nuclease-hypersensitive domains, and additional 5′ sequences that encompass all of the 5′ sequence elements required to imprint single-copy H19 transgenes (20). The inserted element is the minimal DNA sequence that is known to carry a parent-of-origin-specific transcriptional insulator (reference and this study). The VM3 allele (iii) deletes sequences showing muscle-specific enhancer activity in vitro and in transgenic mice (18, 20). This deletion encompasses the Nctc1 gene body. (b) Strategy for construction of the VM3 allele by deleting from kb +10.7 to +34.7 downstream of the H19 gene. The wild-type chromosome (i), the targeted VM3neo chromosome (ii), and the targeted VM3 chromosome after Cre recombinase-mediated excision of the neomycin-selectable marker (iii) are depicted. The endodermal enhancers, as defined by in vitro transfection studies, are represented by a closed circle (46). The deletion confirming the role of these enhancers for in vivo expression of both H19 and Igf2 is indicated by the closed rectangle below line i (26). Also depicted below line i are sequences conserved between human and mouse (C) (18) as well as sequences showing enhancer activity specifically in myoblast cell lines (hatched rectangle) (20). The flanking sequences used to direct homologous recombination are represented by the thickened lines. Probes used to detect the correctly targeted clones are depicted above line i. B, BamHI; S, SalI; R, EcoRI; H, HindIII; X, XbaI.
FIG. 2
FIG. 2
Northern analysis demonstrating the effect of the VM3 enhancer deletion on H19 and Igf2 expression. (a) H19 RNA levels were examined in p2 pups inheriting the VM3 mutation via the maternal chromosome (−/+). (b) Igf2 RNA levels were examined in p2 pups inheriting the VM3 mutation via the paternal chromosome (+/−). In both cases, mutants were compared to wild-type littermates (+/+). Blots were probed for EF2a to verify equal loading. For each tissue, RNA levels of at least two wild-type and two experimental animals from multiple independent litters were quantitated.
FIG. 3
FIG. 3
Effect of the CRK10 mutation on H19 and Igf2 expression. (a) H19 RNA levels in embryonic day 18.5 pups inheriting the CRK10 mutation via the maternal chromosome (CRK10/+) were analyzed by Northern blotting. (b) Igf2 RNA levels in skeletal muscle of pups inheriting the mutation via the paternal chromosome (+/CRK10) were also examined by Northern blotting. Expression of wild type littermates (+/+) was also quantitated. Blots were probed with EF2c to verify equal loading. For each tissue, RNA levels of at least two wild-type and two experimental animals from multiple independent litters were quantitated. (c) In situ analysis of H19 expression patterns in embryonic day 14 embryos. Wt, wild type; H, heart; G, gut; K, kidney.
FIG. 4
FIG. 4
Methylation analysis of sequences downstream of the CRK10 insertion mutation. DNAs from heterozygous pups inheriting the CRK10 mutation via the maternal or the paternal chromosome were digested with BamHI and hybridized with an 2.4-kb EcoRI-BamHI fragment from sequences immediately 3′ of the CRK10 insertion. This hybridization reveals an 11-kb band specific to the wild-type chromosome and a 3.4-kb band specific to the CRK10 chromosome. The 3.4-kb band is thus a junction fragment including 1 kb of sequences from the H19DMR (thin line on the diagram) and 2.4 kb of 3′-flanking sequences (thick line on the diagram). Topologically, these flanking sequences are in the same position relative to the inserted H19DMR as the endogenous H19 promoter and gene body are to the endogenous H19DMR. To determine whether these flanking sequences are differentially methylated in a parent-of-origin-specific manner, aliquots of the BamHI-digested DNA were also digested with methylation-sensitive enzymes, including TaiI (Tai) and HpaII (Hpa). A restriction map of the 3.4-kb BamHI fragment specific to the CRK10 chromosome is depicted, with the probe shown as a thickened line. The thin line represents the sequences that are part of the H19DMR that was inserted at the EcoRI (RI) site.
FIG. 5
FIG. 5
Effect of maternal-chromosome-based inheritance of the CRK10 mutation on Igf2 expression. Skeletal muscle and liver RNA samples were treated with DNase I, amplified for Igf2 by reverse transcription-PCR, and analyzed for allele-specific expression by SNuPE. Expression was analyzed in neonates inheriting the CRK10 mutation with an M. domesticus Igf2 allele via the maternal chromosome and inheriting a wild-type chromosome with an M. castaneous Igf2 allele via the paternal chromosome (CRK10/+). Their control littermates likewise have maternal-chromosome-derived domesticus and paternal-chromosome-derived M. castaneous alleles of Igf2, but both chromosomes are wild type (+/+). Lane F1 is a control representing a 50:50 mix of M. castaneous and M. domesticus substrates. M. domesticus (or maternal) RNA results in incorporation of cytosine (C), and M. casteneous (or paternal) RNA results in incorporation of thymidine (T).
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