Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region

Abstract

The DNA methylation state of the H19/Igf2 imprinting control region (ICR) is differentially set during gametogenesis. To identify factors responsible for the paternally specific DNA methylation of the ICR, germ line and somatic extracts were screened for proteins that bind to the ICR in a germ line-specific manner. A specific DNA binding activity that was restricted to the male germ line and enriched in neonatal testis was identified. Its three binding sites within the ICR are very similar to the consensus sequence for nuclear receptor extended half sites. To determine if these binding sites are required for establishment of the paternal epigenetic state, a mouse strain in which the three sites were mutated was generated. The mutated ICR was able to establish a male-specific epigenetic state in sperm that was indistinguishable from that established by the wild-type ICR, indicating that these sequences are either redundant or have no function. An analysis of the methylated state of the mutant ICR in the soma revealed no differences from the wild-type ICR but did uncover in both mutant and wild-type chromosomes a significant relaxation in the stringency of the methylated state of the paternal allele and the unmethylated state of the maternal allele in neonatal and adult tissues.

FIG. 1.

Characterization of the AB binding sites by EMSA. (A) Illustration of the relative locations of AB and CTCF binding sites within the ICR. (B to E) Positions of AB binding activity (•). (B) AB-2 binding activity in protein extracts from various tissues. NT, neonatal testis; L, adult liver; NO, neonatal ovary; B, adult brain; T, adult testis; E, 16-dpc embryo. (C) Binding of AB-1, AB-2, and AB-3 in neonatal testis extract in the absence (−) or presence of AB-2 or a nonspecific probe. (D and E) AB binding activity with wild-type (WT) and mutant probes (see Table 1).

FIG. 2.

Mutating the AB binding sites in ES cells and mice. (A) Illustration of the targeting strategy, with (from top to bottom) the structure of the wild type chromosome (WT), the targeting construct, the modified chromosome in ES cells, and the vector-excised chromosome (F₁). The location of the external probe used in panel B is indicated, along with the SpeI sites. Rectangles, H19 structural gene and ICR. (B) Southern analysis of SpeI-digested DNA isolated from ES cell colonies. T, correctly targeted ES cells used to generate chimeric animals; *, incorrectly targeted ES cells. (C) PCR genotyping of F₁ offspring. PCR discriminates between the animals in which the selectable marker has not been excised (flox'ed allele) and those that have undergone its deletion (Δ Neo · Cre). (D) PCR genotyping of the mutant allele in the progeny of an F₁ intercross. The mutant product was digested with SpeI.

FIG. 3.

Methylation analysis of neonatal AB binding site mutant animals. Shown are bisulfite methylation sequence data obtained from 6-dpp liver DNA of mutant alleles (P^M and M^M, reflecting whether the chromosome was inherited from a father or mother, respectively) or wild-type alleles (P^WT and M^WT). The region contains 16 CpG residues (open [unmethylated] and solid [methylated] circles). Numbers indicate the numbers of individual chromosomes (when more than one) with identical CpG methylation patterns. Vertical line between CpG 9 and 10, position of the AB-1 mutation. CpG residues 4 and 5 are within CTCF site 1; CpG residues 11 to 13 are within CTCF site 2. (A) Paternal grandmaternal mutant chromosomes. (B) Inferred paternal wild-type chromosomes, based on >50% methylation. (C) Paternal mutant chromosomes of progeny from a paternal homozygote (P^M/M). (D) Paternal wild-type chromosomes of animals inheriting a maternal mutation.

FIG. 4.

Bisulfite methylation analysis of adult AB binding site mutant animals. Bisulfite sequencing was performed on 6-dpp (A and B) or 35-dpp (C and D) liver DNA. (A) Maternal grandmaternal mutant chromosomes. (B) Maternal wild-type chromosomes from animals inheriting mutant paternal chromosomes. (C) Paternal grandmaternal mutant chromosomes. (D) Inferred paternal chromosomes of wild-type littermates of animals in panel C. All nomenclature is described in the Fig. 3 legend.