H19 imprinting control region methylation requires an imprinted environment only in the male germ line

Abstract

The 2.4-kb H19 imprinting control region (H19ICR) is required to establish parent-of-origin-specific epigenetic marks and expression patterns at the Igf2/H19 locus. H19ICR activity is regulated by DNA methylation. The ICR is methylated in sperm but not in oocytes, and this paternal chromosome-specific methylation is maintained throughout development. We recently showed that the H19ICR can work as an ICR even when inserted into the normally nonimprinted alpha fetoprotein locus. Paternal but not maternal copies of the ICR become methylated in somatic tissue. However, the ectopic ICR remains unmethylated in sperm. To extend these findings and investigate the mechanisms that lead to methylation of the H19ICR in the male germ line, we characterized novel mouse knock-in lines. Our data confirm that the 2.4-kb element is an autonomously acting ICR whose function is not dependent on germ line methylation. Ectopic ICRs become methylated in the male germ line, but the timing of methylation is influenced by the insertion site and by additional genetic information. Our results support the idea that DNA methylation is not the primary genomic imprint and that the H19ICR insertion is sufficient to transmit parent-of-origin-dependent DNA methylation patterns independent of its methylation status in sperm.

FIG. 1.

Schematic depictions of the wild-type Igf2/H19, Afp, and CD3 loci, the knock-in mouse models, and the BAC transgene described in this study. Transcriptional start sites are indicated by the horizontal arrows above the chromosomes. (A) Wild-type and mutant alleles at the Igf2/H19 locus. Open and closed circles downstream of the H19 gene indicate the shared endodermal and mesodermal enhancers, respectively. A CpG island (***), defined by 54% cytosine-guanine dinucleotides and an observed versus expected CpG ratio of 0.64 within 500 bp, covers the H19 promoter and was annotated by a computer search using the EMBL-EB1 CpG Plot computer program (http://www.ebi.ac.uk/Tools/emboss/cpgplot/). An approximately 450-bp region rich in guanine nucleotides (boxed G) is located between the H19ICR and the CpG island. The H19-BAC1 transgene (26) carries the H19 sequence from kb −7 to +140. The H19R chromosome (26) carries the H19ICR inserted as a 2.4-kb BglII fragment at the +10-kb EcoRI site. All numbering is relative to the H19 transcriptional start site. (B) Wild-type and H19ICR insertion alleles at the Afp locus. Enhancers (gray circles) are located upstream of Afp (49). H19ICR insertions are at the XbaI site at −0.9 kb relative to the Afp transcriptional start. Afp-A (45) carries the H19 sequence from kb−4.4 to −2, Afp-D (45) carries the H19 sequence from kb −9.7 to −0.8, and Afp-DCK (this study) carries the H19 sequence from kb −4.4 to +3.0. (C) Wild-type and H19ICR insertion alleles at the CD3 locus. CD3-CMG (this study) carries the H19 sequence from kb −4.4 to −2.0, inserted at the BpuAI site between CD3 gamma and CD3 delta.

FIG. 2.

DNA methylation at the endogenous and ectopic H19ICRs in somatic tissues and in testis. DNAs isolated from kidney or testis were digested with SacI (−) or SacI plus AciI (+) and then analyzed by Southern blotting. The identity of the ICR insertion and its parental origin are indicated above the lanes. (A) The 3.8- and 6.7-kb SacI fragments carrying the H19ICR (thickened line) at the endogenous Igf2/H19 locus (top line) and at the CD3-CMG chromosome (bottom line) are depicted. The arrowhead above the top line indicates the polymorphic SacI site that distinguishes wild-type domesticus (Dom) and castaneus (Cas) H19 alleles, cleaving castaneus H19 alleles into 2.5-kb (Cas1) and 1.3-kb (Cas2) SacI fragments. AciI restriction sites within the SacI fragments are indicated by vertical lines. The 1.6-kb KpnI-HindIII probe used to identify the ICR is indicated. (B) By crossing CD3-CMG animals with DIS7CAS mice (19) carrying the endogenous H19ICR on a castaneus allele, we were able to use SacI digestion to distinguish between the three H19ICRs present in these progeny, i.e., the ICR inserted at CD3 and the maternal and paternal endogenous H19ICRs. The ICR insertion is always of the same parental origin as the endogenous domesticus (Dom) allele. (C) Methylation of the H19ICR insertion in Afp-DCK and H19-BAC1 mice was analyzed in animals homozygous for the H19^Δ13 mutation, which deletes the entire H19 gene and 10 kb of upstream sequence, including the endogenous H19ICR. Thus, the insertion is the only H19ICR in these animals.

FIG. 3.

DNA methylation of individual CpG dinucleotides within the ectopic H19ICR at CD3 in sperm (Sp) and testis (T). Genomic DNAs from animals carrying only the ectopic H19ICR in their genomes were analyzed by bisulfite sequencing. Numbers 1 to 5 indicate regions within the ICR that were individually amplified by PCR. Region 1 shows the most proximal and region 5 shows the most distal region relative to the endogenous H19 promoter. Each lane of circles represents one individual clone. Lanes drawn for different regions do not originate from the same chromosome. Open circles represent unmethylated CpGs, and closed circles represent methylated CpGs. Arrows beneath individual CpGs indicate AciI restriction sites harboring the given CpG within their sequence. The Snrpn and Rasgrf1 DMRs were used as positive controls to test the accuracy of the bisulfite assay. In sperm, the Snrpn DMR (36) is unmethylated and the Rasgrf1 DMR (33) is methylated.

FIG. 4.

DNA methylation patterns of the exogenous H19ICR at CD3 in zygotes and in E3.5 morulae and blastocysts. The DNA region analyzed by bisulfite sequencing in this study covers 15 CpGs within the H19ICR, from kb −4.0 to −3.5, and includes two CTCF sites. This region corresponds to the promoter-distal region 5, whose methylation patterns in sperm and testis are shown in Fig. 3. Zygotes, morulae, and blastocysts had paternal alleles inherited from CD3-CMG fathers. Morulae and blastocysts were homozygous for the H19^Δ13 allele. Thus, the ectopic ICR was the only H19ICR in these genomes. Zygotes were in an H19⁺ (cas)/H19^Δ13 background. Clones associated with the ectopic H19ICR were identified using single nucleotide polymorphisms that distinguish castaneus and domesticus H19ICRs. Each circle represents one CpG, and each lane represents one clone. Unmethylated CpGs and methylated CpGs are depicted as open circles and filled circles, respectively.