Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster

Abstract

Genomic imprinting is an epigenetic process that results in the preferential silencing of one of the two parental copies of a gene. Although the precise mechanisms by which genomic imprinting occurs are unknown, the tendency of imprinted genes to exist in chromosomal clusters suggests long-range regulation through shared regulatory elements. We characterize a 800-kb region on the distal end of mouse chromosome 7 that contains a cluster of four maternally expressed genes, H19, Mash2, Kvlqt1, and p57(Kip2), as well as two paternally expressed genes, Igf2 and Ins2, and assess the expression and imprinting of Mash2, Kvlqt1, and p57(Kip2) during development in embryonic and extraembryonic tissues. Unlike Igf2 and Ins2, which depend on H19 for their imprinting, Mash2, p57(Kip2), and Kvlqt1 are unaffected by a deletion of the H19 gene region, suggesting that these more telomeric genes are not regulated by the mechanism that controls H19, Igf2, and Ins2. Mutations in human p57(Kip2) have been implicated in Beckwith-Wiedemann syndrome, a disease that has also been associated with loss of imprinting of IGF2. We find, however, that a deletion of the gene has no effect on imprinting within the cluster. Surprisingly, the three maternally expressed genes are regulated very differently by DNA methylation; p57(Kip2) is activated, Kvlqt1 is silenced, and Mash2 is unaffected in mice lacking DNA methyltransferase. We conclude that H19 is not a global regulator of imprinting on distal chromosome 7 and that the telomeric genes are imprinted by a separate mechanism(s).

FIG. 1

Genetic and physical mapping of genes on distal chromosome 7. (A) Haplotype analysis showing the segregation patterns of all loci in the interspecific backcross. Each column represents the haplotype of a chromosome that was inherited from the (BTBR × M. spretus)F₁ parent. Shaded boxes represent BTBR alleles, whereas open boxes represent M. spretus alleles. The number of offspring inheriting each type of chromosome is shown at the bottom of each column. The percent recombination (R) between adjacent loci with the standard error (SE) is shown at the right of the grid. On the far right is shown the orientation of the imprinted genes relative to the centromere (cent) and telomere (tel). (B) Physical mapping of the imprinted genes on distal chromosome 7. The YAC contig is represented by thin lines, and the BAC contig is represented by thicker lines. The placement of the genes along the contig is indicated at the bottom. Arrows beneath the genes represent their transcriptional orientation. The asterisk indicates that the orientation of CD81 is based on that of the human. The 5′ and 3′ ends of Kvlqt1 cDNA are at least 250 kb apart, but its intron-exon structure remains unknown.

FIG. 2

Temporal regulation of Kvlqt1, p57^Kip2, CD81, and Mash2 imprinting during embryogenesis. (A) For Kvlqt1, p57^Kip2, and CD81, RT-PCR analysis was performed on F₁ progeny from a reciprocal interspecific cross between 129/Sv M. domesticus (D) and BTBR(SPR H19-p57) (S) mice dissected at e6.5, e7.5, e8.5, e9.5, and e12.5 (labeled 6, 7, 8, 9, and 12, respectively). The e6.5 embryo and extraembryonic tissues were dissected out together (lane 3); otherwise, the embryo was separated from the ectoplacental cone or placenta for analysis (lanes 4 to 10 and 13 to 19). 129/Sv or BTBR(SPR H19-p57) embryo (lanes 1 and 2) and placenta (lanes 11 and 12) at e12.5 was analyzed to show the parental alleles. (B) Kvlqt1 expression in e13.5 head and in neonatal tissues. RT-PCR analysis of e13.5 head RNA and postnatal day 4 tissues from F₁ progeny of a reciprocal cross between 129/Sv M. domesticus (D) and B6(CAST H19-p57) (C). (C) Mash2 RT-PCR analysis of F₁ progeny from a reciprocal interspecific cross between BTBR (D) and BTBR(SPR H19-p57) (S) mice. Dissections were of the whole decidua at e6.5, e7.5, and e8.5, of extraembryonic tissue at e9.5, and of placenta alone at e12.5.

FIG. 3

Effect of an H19 deletion on the imprinting of Mash2, p57^Kip2, and Kvlqt1: RT-PCR analysis of e12.5 placenta (Mash2 and Kvlqt1) and e12.5 embryo (Kvlqt1 and p57^Kip2). Females heterozygous for the H19 gene body deletion (H19^Δ13) (31) were crossed to BTBR(SPR H19-p57) males, and the F₁ wild-type (+/+) and heterozygous (+/−) littermates (mat) were subjected to allele-specific assays (lanes 3 and 4). The reciprocal cross resulting in paternal inheritance of H19^Δ13 (pat) was also analyzed (lanes 5 and 6). 129/Sv (D) or BTBR(SPR H19-p57) (S) embryo or placenta (lanes 1 and 2) at e12.5 was analyzed to show the parental alleles.

FIG. 4

Effect of a p57^Kip2 mutation on the imprinting of H19, Igf2, and Kvlqt1. (A) Females heterozygous for the p57^Kip2 deletion (mat) (62) were crossed to B6(CAST H19-p57) males, and the F₁ wild-type (wt) and heterozygous (mut) littermates were analyzed by use of allele-specific RNase protection assays of H19 and Igf2 RNAs in head, trunk, and placenta (plac) RNAs. The reciprocal cross resulting in paternal inheritance of the deletion allele (pat) was also analyzed. C57BL/6 (D) or B6(CAST H19-p57) (C) embryo or placenta at e12.5 was analyzed to show the parental alleles. (B) The same placental samples from the p57^Kip2 deletion progeny were analyzed by RT-PCR for Kvlqt1 expression.

FIG. 4

Effect of a p57^Kip2 mutation on the imprinting of H19, Igf2, and Kvlqt1. (A) Females heterozygous for the p57^Kip2 deletion (mat) (62) were crossed to B6(CAST H19-p57) males, and the F₁ wild-type (wt) and heterozygous (mut) littermates were analyzed by use of allele-specific RNase protection assays of H19 and Igf2 RNAs in head, trunk, and placenta (plac) RNAs. The reciprocal cross resulting in paternal inheritance of the deletion allele (pat) was also analyzed. C57BL/6 (D) or B6(CAST H19-p57) (C) embryo or placenta at e12.5 was analyzed to show the parental alleles. (B) The same placental samples from the p57^Kip2 deletion progeny were analyzed by RT-PCR for Kvlqt1 expression.

FIG. 5

Effect of a Dnmt mutation on the imprinting of p57^Kip2, Kvlqt1, Mash2, and H19. Heterozygous Dnmt^s mice (33) were crossed to BTBR(SPR H19-p57) mice that were also heterozygous for Dnmt^s and wild-type (+/+) or mutant (−/−) progeny were dissected into embryonic, ectoplacental cone (E.P.C.), and yolk sac samples at e9.5. Yolk sac DNA was used to genotype the samples. Wild-type or mutant embryonic or ectoplacental cone samples were pooled for RT-PCR imprinting analyses. Abbreviations are the same as in Fig. 2.