Shared role for differentially methylated domains of imprinted genes

Abstract

For most imprinted genes, a difference in expression between the maternal and paternal alleles is associated with a corresponding difference in DNA methylation that is localized to a differentially methylated domain (DMD). Removal of a gene's DMD leads to a loss of imprinting. These observations suggest that DMDs have a determinative role in genomic imprinting. To examine this possibility, we introduced sequences from the DMDs of the imprinted Igf2r, H19, and Snrpn genes into a nonimprinted derivative of the normally imprinted RSVIgmyc transgene, created by excising its own DMD. Hybrid transgenes with sequences from the Igf2r DMD2 were consistently imprinted, with the maternal allele being more methylated than the paternal allele. Only the repeated sequences within DMD2 were required for imprinting these transgenes. Hybrid transgenes containing H19 and Snrpn DMD sequences and ones containing sequences from the long terminal repeat of a murine intracisternal A particle retrotransposon were not imprinted. The Igf2r hybrid transgenes are comprised entirely of mouse genomic DNA and behave as endogenous imprinted genes in inbred wild-type and mutant mouse strains. These types of hybrid transgenes can be used to elucidate the functions of DMD sequences in genomic imprinting.

FIG. 1.

The DMD of RSVIgmyc. (A) Schematic of the RSVIgmyc transgene linearized at the unique KpnI restriction site. Cα and RSV hybridization probes for Southern blots are shown as thick black lines. MspI, HhaI, and BstUI methylation-sensitive restriction sites are shown as vertical lines. The boxed numbers 1, 2, and 3 designate the exons of the c-myc gene, and 3′ myc indicates the noncoding genomic sequence of c-myc. pBR/RSV refers to pBR322 vector sequences and RSV LTR sequences. IgA indicates coding sequences and switch recombination sequences of IgA. E, EcoRI; B, BglII; X, XbaI. (B) Southern blot of DNA samples from maternal (M) and paternal (P) hemizygous carriers hybridized with the RSV probe. DNA samples were digested with EcoRI to isolate the pBR/RSV region, followed by digestion with either HpaII, HhaI, BstUI, or MspI. A 2.5-kb hybridization band indicates methylation of all of the relevant methylation-sensitive restriction sites. (C) Southern blot of DNA samples from maternal (M) and paternal (P) hemizygous carriers hybridized with the Cα probe. DNAs were digested with EcoRI and BglII to isolate the IgA region immediately adjacent to the pBR/RSV region, followed by digestion with HpaII, HhaI, BstUI, or MspI. The Cα hybridization probe recognizes a methylated 3.3-kb EcoRI-BglII transgene fragment and an 8-kb endogenous band. All DNA samples were obtained from tail biopsies performed at the time of weaning (3 to 4 weeks).

FIG. 2.

Requirement of the RSVIgmyc DMD for transgene imprinting. (A) Schematic of transgenes derived from RSVIgmyc. Black lines indicate the probes used in the Southern blots below. Ig/myc retains all of the RSVIgmyc transgene sequence, except the pBR/RSV region between the two EcoRI (E) sites. pBR/RSV is composed entirely of the sequence delineated by the two EcoRI sites. (B to D) Southern blots of DNAs from maternal (M) or paternal (P) hemizygous carriers of the Ig/myc transgene digested with either HpaII, HhaI, BstUI, or MspI and hybridized with probes to various regions of the transgene (B, probe a, 3′ c-myc region; C, Cα probe, IgA region; D, probe b, exon 3 region of c-myc). (E) The Southern blot was performed as described above and hybridized with a probe to the RSV region of the transgene. All DNA samples were obtained from tail biopsies performed at the time of weaning (3 to 4 weeks).

FIG. 3.

RSVIgmyc and derivative transgenes. (A) Transgenes created by replacement of the pBR322 and RSV region (pBR/RSV) of the RSVIgmyc transgene, bordered by EcoRI sites (E), with fragments containing DMDs from endogenous imprinted genes or the LTR of an IAP element (solid boxes). (B) Sources of DNA fragments (solid boxes) used to replace pBR/RSV. Arrows indicate start sites of transcription, and open boxes define DMDs. The drawing is not to scale. The following accession numbers and sequences were used: H19SIgmyc and H19LIgmyc, U19619 (30), nt 1434 to 1726 (292 nt) and nt 942 to 3174 (2,232 nt), respectively; Igf2rIgmyc, L06446 (27), nt 741 to 1408 (667 nt); SnrpnIgmyc, AF130843 (13), nt 3237 to 3745 (508 nt); and IAPIgmyc, reference (Fig. 2B).

FIG. 4.

Restoration of transgene imprinting. (A) Sequence of the Igf2r DMD2 region used to replace pBR/RSV. The entire depicted sequence (nt 741 to 1408) was used to construct Igf2rIgmyc. The nt 741 to 950, including the TR1 repeats (gray boxes), were used to construct TR1Igmyc. The nt 950 to 1408, composed of TR2+3 repeats (underlined sequences), were used to create the TR2+3Igmyc transgene. Sequences are from accession no. L06446 (27). (B) Southern blot of DNAs from maternal (M) or paternal (P) hemizygous carriers of the Igf2rIgmyc, TR1Igmyc, or TR2+3Igmyc transgenes, digested with HpaII or MspI, and probed with the Cα probe to the IgA region of the transgene. All DNA samples were obtained from tail biopsies performed at the time of weaning (3 to 4 weeks). (C) Igf2rIgmyc transgenic mice were crossed to C57BL/6 mice for three generations, and Southern blots were performed as described above.

FIG. 5.

Loss of transgene imprinting on a Dnmt1o-deficient background. Dnmt1^Δ1o females carrying the Igf2rIgmyc transgene in an FVB/N background (left panel) or wild-type females carrying the Igf2rIgmyc transgene in an FVB/N background (right panel) were mated to FVB/N males. DNA was extracted from entire transgenic D10.5 embryos and digested with HpaII, and Southern blots were performed with the Cα probe to the IgA region of the transgene.

FIG. 6.

Other hybrid transgenes. Representative Southern blots of DNAs from maternal (M) or paternal (P) hemizygous carriers of the H19SIgmyc, H19LIgmyc, SnrpnIgmyc, and IAPIgmyc transgenes. DNA was digested with HpaII, and Southern blots were hybridized with the Cα probe. All DNA samples were obtained from tail biopsies performed at the time of weaning (3 to 4 weeks).

FIG. 7.

Role of the DMD in genomic imprinting. (A) Diagrams of the imprinted Igf2r and Snrpn genes and the imprinted RSVIgmyc transgene. Exons are shown as black boxes, and the DMD of each gene is represented by an open box. The region of each DMD containing tandem repeats is enlarged, and the organization and sizes of the repeats are shown by arrows (indirect repeats within the pBR322 sequence are not pictured). The locations of the sequences present in the Igf2rIgmyc and the SnrpnIgmyc transgenes are indicated by gray lines. (B) Sequences of tandem repeats. The TR2+3 repeats from the Igf2r DMD2, all repeats from the Snrpn DMD, two sets of repeats from pBR322, and all repeats from RSV are shown. Nucleotide positions are from accession no. L06446 (Igf2r) and AF130843 (Snrpn). (C) Model for the role of the DMD in establishing the allele-specific methylation of a maternally imprinted gene. The DMD sequences (gray) interact with the methylation machinery to acquire a default state of methylation (highly methylated in this example), and the flanking genomic sequences are required to prevent this methylation in the male germ line.