• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. Sep 2009; 29(17): 4595–4603.
Published online Jun 22, 2009. doi:  10.1128/MCB.00275-09
PMCID: PMC2725707

A Randomly Integrated Transgenic H19 Imprinting Control Region Acquires Methylation Imprinting Independently of Its Establishment in Germ Cells[down-pointing small open triangle]

Abstract

The imprinted expression of the mouse Igf2/H19 locus is governed by the differential methylation of the imprinting control region (ICR), which is established initially in germ cells and subsequently maintained in somatic cells, depending on its parental origin. By grafting a 2.9-kbp H19 ICR fragment into a human β-globin yeast artificial chromosome in transgenic mice, we previously showed that the ICR could recapitulate imprinted methylation and expression at a heterologous locus, suggesting that the H19 ICR in the β-globin locus contained sufficient information to maintain the methylation mark (K. Tanimoto, M. Shimotsuma, H. Matsuzaki, A. Omori, J. Bungert, J. D. Engel, and A. Fukamizu, Proc. Natl. Acad. Sci. USA 102:10250-10255, 2005). Curiously, however, the transgenic H19 ICR was not methylated in sperm, which was distinct from that seen in the endogenous locus. Here, we reevaluated the ability of the H19 ICR to mark the parental origin using more rigid criteria. In the testis, the methylation levels of the solitary 2.9-kbp transgenic ICR fragment varied significantly between six transgenic mouse lines. However, in somatic cells, the paternally inherited ICR fragment exhibited consistently higher methylation levels at five out of six randomly integrated sites in the mouse genome. These results clearly demonstrated that the H19 ICR could acquire parent-of-origin-dependent methylation after fertilization independently of the chromosomal integration site or the prerequisite methylation acquisition in male germ cells.

A subset of mammalian genes is subject to genomic imprinting that restricts their expression to one allele, which depends on whether they are paternally or maternally inherited (24). This monoallelic expression is achieved through multiple processes. First, an epigenetic mark is set differently on both alleles. This mark is thought to be acquired during gametogenesis, since this is the only period in life when these genomes are in distinct compartments and therefore can be differentially modified. This differential epigenetic mark is maintained after fertilization and then is recognized and converted into the imprinted expression pattern by the transcriptional machinery.

The mouse Igf2 and H19 genes on distal chromosome 7 are imprinted and reciprocally expressed. The Igf2 gene is activated only when paternally inherited, while the H19 gene is maternally transcribed (2, 8). The 2-kbp region located −2 to −4 kbp relative to the transcription initiation site of the H19 gene has been identified as the imprinting control region (ICR; also called the differentially methylated domain [DMD]) (Fig. (Fig.1A),1A), and this region is differentially methylated depending on its parental origin (20, 36, 37). Genetic evidence has demonstrated that the H19 ICR is necessary to regulate the imprinted expression at this locus. The targeted deletions of all or most of this ICR in the endogenous locus caused a loss of imprinted expression of both the Igf2 and H19 genes (15, 27, 33, 35). Furthermore, H19 gene fragments could recapitulate monoallelic H19 expression in a single-copy transgene when the entire ICR was included in the construct (7). In addition, mutating DNA methyltransferase 1 (Dnmt1) resulted in the loss of the imprinting of the Igf2 and H19 genes (17), supporting a role of methylation in the regulation of imprinted gene expression. Collectively, the H19 ICR and its differential methylation appear to be crucial for the manifestation of allele-specific expression by transcriptional regulators.

FIG. 1.
Generation and characterization of H19 ICR TgM. (A) Genomic structure of the mouse Igf2/H19 locus. The H19 ICR (DMD) is located within a 2.9-kbp SacI (Sa; at −4.7 kbp relative to the transcription initiation site of the H19 gene)-BamHI (B; at ...

According to a recent model, a hypomethylated ICR on the maternal allele functions as an insulator by binding the CCCTC-binding factor (CTCF) protein, which prevents the activation of the distal Igf2 gene from the shared enhancer located 3′ to H19 and allows exclusive H19 expression. On the other hand, a hypermethylated paternal ICR represses H19 gene transcription by inducing epigenetic changes at the H19 promoter and prevents CTCF from binding to the ICR, thereby allowing Igf2 expression (3, 9, 14, 15).

The H19 ICR is methylated in sperm but not in oocytes, and the paternal ICR is methylated exclusively throughout development (20, 36-38). Therefore, the differential DNA methylation of the ICR is the best candidate for the primary mark that designates the parental origin. Since DNA methyltransferase activities are found in both gametes, it is presumed that there are cis DNA sequences within the H19 ICR that induce methylation in sperm and/or prohibit methylation in oocytes. Studies of postimplantation embryos or neonates have shown that mutations in the CTCF binding sites of the H19 ICR caused the aberrant methylation of the maternal ICR (11, 21, 22, 26, 31), while mutations in the CpG motifs at the CTCF binding sites resulted in the decreased methylation of the paternal ICR (12). However, these mutations did not change the methylation pattern in germ cells (11, 12, 26, 31). Furthermore, deleting all or most of the H19 ICR from the endogenous locus had little impact on the methylation of the remaining H19 upstream region in male germ cells or in preimplantation embryos, although differential methylation was lost afterwards (33-35). Accordingly, while the ICR is indispensable for maintaining paternal allele-specific methylation, it may not be required to control methylation acquisition at the H19 locus in male germ cells. Thus, the DNA sequences that control the initial step that establishes the parental mark and its position within the locus are still unknown.

In a previous study, we tested the function of the H19 ICR in a heterologous locus (32). We inserted a 2.9-kbp DNA fragment encompassing the H19 ICR into a 150-kbp human β-globin yeast artificial chromosome (YAC) between the locus control region hypersensitive site 1 (HS1) and the epsilon-globin gene (Fig. (Fig.1B,1B, top), and we generated YAC transgenic mice (TgM; ICR/β-globin; previously referred to as ICR). In erythroid cells of both yolk sac and adult spleen, the expression levels of β-like globin genes were higher when the transgene was paternally inherited. In addition, the paternally inherited transgenic ICR was more heavily methylated than the maternally inherited ICR, and CTCF was recruited preferentially to the maternal transgenic ICR in adult erythroid cells (32). The methylation of the paternally inherited transgenic ICR was also observed in extraembryonic yolk sac tissues (H. Matsuzaki and K. Tanimoto, unpublished observations). These results suggested that the 2.9-kbp ICR fragment contains sufficient information to autonomously recapitulate imprinted expression as well as imprinted methylation at the normally nonimprinted β-globin locus. Consistently with this, Park et al. (23) reported that a 2.4-kbp H19 ICR fragment knocked in at the alpha-fetoprotein (Afp) locus also was methylated in somatic cells, but only when it was paternally inherited. An unexpected observation, however, was that the ICR fragments in the β-globin or Afp loci were not methylated in male germ cells (23, 32). These results raised an intriguing possibility that the differential methylation of the H19 ICR could be acquired after fertilization by deciphering a primary mark other than DNA methylation, which likely is set on the ICR in germ cells to designate the parental origin.

To fully understand the genomic imprinting mechanism in the Igf2/H19 locus, it is essential to identify minimal requirements within the H19 ICR that are necessary and dominant to establish parent-of-origin-specific methylation. In this sense, our previous finding that the H19 ICR embedded in the 150-kbp human β-globin YAC transgenes could establish methylation imprinting at random chromosomal sites made an important contribution (32). However, since it is formally possible that the activity of the H19 ICR in the 150-kbp YAC is protected from the effect of the surrounding chromatin environment (13), the dominance of the H19 ICR activity might not have been evaluated precisely in the YAC TgM system. In addition, it also is possible that the differential methylation of the ICR at heterologous loci in the postfertilization period is acquired by a novel imprinting mechanism that occurs only within specific chromosomal contexts, such as in the β-globin or Afp gene loci, rather than being caused by an intrinsic activity.

In the current work, we generated TgM lines carrying the 2.9-kbp H19 ICR fragments that were randomly inserted into the mouse genome to determine whether the H19 ICR fragment possesses autonomous activity that acquires imprinted methylation irrespective of its integration site. In all but one of the TgM lines, the paternally inherited transgenic ICRs exhibited higher methylation levels than maternally inherited ICRs in somatic cells. However, the methylation status in male germ cells was not consistent with that in somatic cells, i.e., methylation acquisition was observed in a few of the lines but not in all lines. The results support the idea that the H19 ICR fragment contained sufficient information to mark its parental origin, and that the ICR could be methylated on the paternal allele after fertilization by a mechanism that is distinct from methylation acquisition in male germ cells.

MATERIALS AND METHODS

TgM.

The HS1/ICR transgene was constructed in pHS1/loxPw+/ICR as previously described (32). In this plasmid, a 2.9-kbp SacI-BamHI fragment from the upstream region of the H19 gene (the SacI site in the fragment was converted into a BamHI site) was flanked by loxP sites and sequences from the human β-globin gene (~500 bp) to discriminate the transgenic from endogenous ICRs in Southern blot and PCR analyses. The HS1/ICR fragment was excised using XhoI and XbaI. Purified DNA was diluted with injection buffer (10 mM Tris-HCl, pH 7.4, 0.1 mM EDTA, 100 mM NaCl) to 2 to 5 ng/μl and microinjected into fertilized mouse eggs from ICR mice (Charles River Laboratories). The founder offspring (F0) were identified by PCR and the Southern blot analysis of tail DNA and were bred to ICR mice to derive F1 progeny. Six TgM lines were established, and the copy numbers of the transgenes were analyzed by Southern blotting. The chromosomal integration sites of the transgenes were identified by inverse PCR. The location of the transgenes in lines 362 and 378 also was determined by chromosomal fluorescence in situ hybridization. To decrease the copy number of the transgenic ICR at the same chromosomal position, the TgM of line 378, carrying five copies of the transgene, was mated with a TgM ubiquitously expressing Cre recombinase. After screening the offspring by PCR and Southern blotting using tail DNA, animals that had undergone Cre-mediated recombination were mated with wild-type animals to remove the Cre recombinase allele, and low-copy-number sublines were established. Animal experiments were carried out in a humane manner and were approved by the Institutional Animal Experiment Committee of the University of Tsukuba. Experiments were conducted in accordance with the Regulation of Animal Experiments of the University of Tsukuba and the Fundamental Guidelines for the Proper Conduct of Animal Experiments and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science, and Technology of Japan.

Southern blotting.

Genomic DNA was prepared from tail tips of approximately 7-day-old TgM or from the erythroid cells of anemic spleens or whole testes from 6- to 8-week-old TgM using standard procedures.

To determine the copy number of the HS1/ICR TgM (Fig. (Fig.1C1C and and3C),3C), DNA was digested with BamHI, separated on an agarose gel, and transferred to a nylon membrane. The membrane was hybridized with the α-32P-labeled BS probe (Fig. (Fig.2A),2A), which was prepared from the pHS1/loxPw+/ICR plasmid excised by BamHI-ScaI, and subjected to PhosphorImager quantification (Typhoon; Amersham) and X-ray film autoradiography. After normalization to the endogenous ICR signals, transgenic ICR signals were compared to that of ICR/β-globin YAC TgM (line 1048; single copy) (32).

FIG. 2.
Methylation of the ICR in erythroid cells. (A) Restriction map of the endogenous H19 (top) and the HS1/ICR transgene (bottom) loci. Methylation-sensitive HhaI sites in the BamHI fragments (horizontal lines beneath each map) within the locus are displayed ...
FIG. 3.
Methylation of the ICR in testis. (A) Genomic DNA was prepared from the testis of TgM that inherited the transgenes either paternally (P) or maternally (M) or from nontransgenic (non-Tg) mice. DNA was digested with BamHI (−) and then HhaI (Hh) ...

For DNA methylation analysis, DNA was digested with BamHI followed by methylation-sensitive HhaI. Blots were hybridized with the BS probe (Fig. (Fig.2A2A).

Bisulfite sequencing.

Genomic DNA was extracted from erythroid cells of anemic spleens or whole testes, digested with XbaI, and then treated with sodium bisulfite using the EZ DNA methylation kit according to the manufacturer's instructions (Zymo Research, Orange, CA). Transgenic ICR-specific nested PCR, PCR product cloning, and sequence analysis were performed as described previously (32). Two subregions of the transgenic ICR (region I, nucleotides 1245 to 1995; region II, nucleotides 3050 to 3432; GenBank accession no. AF049091) were amplified by nested PCR (Fig. (Fig.2C).2C). The first-round PCR primers used are the following: for the 5′ transgenic ICR region, BGLB-3A1 (a) (5′-TCTCGTCAAACCACCTTCATTAAC-3′) and ICR-5S1 (b) (5′-GAATTTGAGGATTATGTTTAGTGG-3′); for the 3′ transgenic ICR region, ICR-3A6 (c) (5′-ATATACACCTCTAAAATAATTCCC-3′) and LCR-5S1 (d) (5′-TATAGATGTTTTAGTTTTAATAAG-3′). The sequences of second-round PCR primers are the following: for the 5′ ICR subregion (region I), ICR-3A1 (e) (5′-AACATAACAATACTATAACCATAC-3′) and ICR-5S2 (f) (5′-TTAAGGATTAGTATGAATTTTTGG-3′); for the 3′ ICR subregion (region II), ICR-3A5 (g) (5′-AACTTAACTCATTCCCTACACAAC-3′) and ICR-5S4 (h) (5′-GAATTTGGGGTATTTAAAGTTTTG-3′).

The methylation of oocyte DNA was analyzed as follows. Oocytes were collected from the oviducts of superovulated hemizygous female TgM. The cumulus cells were removed by treatment with hyaluronidase and then were washed repeatedly with phosphate-buffered saline (PBS). Oocytes in 2 μl of PBS were mixed with 8 μl of molten 2% low-melt agarose, covered with mineral oil, and then allowed to solidify on ice. Seventeen to 20 and 10 to 20 oocytes per agarose bead were used for single- and four-copy sublines from TgM line 378, respectively. The agarose beads were incubated with 800 μl of lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM EDTA, 1% sodium dodecyl sulfate, 50 μg/ml proteinase K) overnight at 50°C. After being washed three times for 15 min with 1 ml of TE (10 mM Tris-HCl and 1 mM EDTA), the beads were treated with sodium bisulfite using the reagents in the EZ DNA methylation kit. Briefly, the beads were incubated with M-dilution buffer for 30 min at 42°C and then incubated with CT-conversion reagent for 9 h at 50°C in the dark. The beads were washed once with water and five times with TE and then treated with M-desulfonation buffer for 15 min at room temperature. After being washed once with TE and twice with water, the beads were separately and directly used for the nested PCR amplification of transgenic ICR region II. Each PCR product was individually subcloned, and the DNA sequences were determined.

Combined bisulfite restriction analysis (COBRA).

The HS1/ICR fragment was excised using XhoI and XbaI from the pHS1/loxPw+/ICR plasmid, which was propagated in Dam Dcm Escherichia coli. The purified fragment was injected into the male or female pronuclei of fertilized eggs, and the injected eggs were transferred into the oviduct of foster mothers. Embryos were collected at 10.5 days postcoitum, and individual genomic DNA was extracted. After screening the genomic DNA carrying the injected HS1/ICR fragment by PCR, DNA was treated with sodium bisulfite. The transgenic ICRs were amplified by nested PCR, and the aliquots of PCR products were digested with restriction enzymes and subjected to polyacrylamide gel electrophoresis. After the ethidium bromide staining of the gels, photos were taken and the white and black aspects of the image were reversed to facilitate DNA band recognition.

RESULTS

Generation of TgM.

To reevaluate whether the ICR fragment alone could mark parent-of-origin-dependent DNA methylation regardless of its chromosomal integration site, we generated TgM using the 2.9-kbp H19 ICR fragment with flanking loxP sites and sequences from the human β-globin gene (~500 bp each) (Fig. (Fig.1B,1B, HS1/ICR). The β-globin sequences used in this transgene construct were the same as those adjacent to the ICR fragment in the ICR/β-globin YAC (32). We generated six HS1/ICR TgM lines, each of which carried from one to five copies of the transgene (Fig. (Fig.1C).1C). For some of the lines, we identified the chromosomal integration sites of the transgene by inverse PCR. The transgenes were inserted on chromosomes 1B, 2C1, 2C3, and 6B3 in lines 362, 370, 316, and 378, respectively (data not shown), suggesting that the transgene in these lines was not placed in or adjacent to the endogenous Igf2/H19 locus or other known imprinted genes (http://www.har.mrc.ac.uk/research/genomic_imprinting/maps.html and http://igc.otago.ac.nz/home.html). For lines 362 and 378, chromosomal fluorescence in situ hybridization also was used to determine the location of the transgene, and these results were consistent with the inverse PCR analysis (data not shown). The results confirmed that the generated TgM lines had different transgene copy numbers that were randomly integrated into the mouse genome.

DNA methylation analysis of the transgenic ICR in somatic cells.

We first examined the methylation status of the ICR in erythroid cells from anemic adult spleens. Because the transgene contains a unique BamHI site, the transgenic and endogenous ICRs could be distinguished by their size in Southern blotting (Fig. (Fig.2A).2A). We analyzed cytosine methylation in the recognition sequences of the methylation-sensitive HhaI enzyme using the BS probe, which detected the 5′ portion of the ICR fragment (Fig. (Fig.2A).2A). In five of six HS1/ICR TgM lines, the paternally inherited transgenic ICR was more heavily methylated than the maternally inherited one, which was consistent with the methylation pattern of the endogenous H19 ICR and the transgenic ICR inserted in the human β-globin locus (Fig. (Fig.2B).2B). These results were confirmed using EcoRI and HpaII digestion in combination with a probe recognizing the ICR around CTCF binding sites 3 and 4 (data not shown). In one of the six TgM lines, line 379, the transgenic ICR was heavily methylated regardless of its parental origin (Fig. (Fig.2B2B and data not shown), which we considered an exceptional case. To more accurately evaluate the methylation status of the transgenic ICR, we performed the bisulfite sequencing of erythroid cell-derived DNA from the TgM of line 362, which carried a single copy of the transgene. Two distinct regions of the transgenic ICR, regions I and II, were amplified by transgenic allele-specific nested PCR (Fig. (Fig.2C).2C). Although individual DNA molecules showed a wide range of methylation, the paternally inherited transgenic ICR was more heavily methylated than the maternally inherited ICR (Fig. (Fig.2C).2C). These results demonstrated that the transgenic ICR in somatic cells was differentially methylated depending on its parental origin at discrete chromosomal loci, suggesting that the 2.9-kbp ICR fragment itself contains sufficient information that designates its parental origin irrespective of its chromosomal integration site.

DNA methylation analysis of the transgenic ICR in the testis.

To determine if the HS1/ICR transgene that integrated at discrete chromosomal positions was methylated in male germ cells, we extracted DNA from the testis of the TgM and analyzed it by Southern blotting (Fig. (Fig.3A)3A) or bisulfite sequencing (Fig. (Fig.3B).3B). As shown in Fig. Fig.3A,3A, the transgenic ICR was not methylated in the testis of the single-copy lines 362 and 370. The methylation status in line 362 also was examined by bisulfite sequencing, and the two regions (I and II) in the transgenic ICR were almost completely devoid of methylation (Fig. (Fig.3B).3B). In contrast, mice with two or five transgene copies (lines 316, 346, and 378) showed moderate to high methylation levels of the transgenic ICR (Fig. (Fig.3A).3A). These results demonstrated that the methylation levels of the transgenic ICR in male germ cells of testis were different between the TgM lines, while in somatic cells of both single- and multiple-copy lines the paternally inherited transgenic ICR had consistently higher methylation levels than the maternally inherited ICR (Fig. (Fig.2B).2B). When collectively judged from the methylation status of the ICR inserted into the β-globin (Fig. (Fig.3A)3A) (32) and Afp (23) loci, these results demonstrate that the methylation of the ICR in male germ cells was not a prerequisite for the acquisition of the paternal allele-specific methylation in somatic cells.

The distinct methylation pattern of the HS1/ICR transgene in the testis described above (Fig. (Fig.3A)3A) implied an additional possibility, that transgenic ICR methylation in male germ cells was copy number dependent. We therefore investigated the methylation status in male germ cells of the testis carrying different copy numbers of the transgene at the same chromosomal position. For this purpose, we used loxP sites that were located on either side of the ICR in the HS1/ICR transgene. We crossed the TgM of line 378, which carried five copies of the transgene, with the Cre recombinase TgM to obtain lower-copy-number TgM by in vivo Cre-loxP recombination. After establishing TgM sublines carrying either one or four copies of the transgenic ICR (Fig. (Fig.3C),3C), we assessed the methylation status of testis DNA by Southern blotting. In both sublines (one or four copies), the transgenic ICR was hypermethylated irrespective of its parental origin (Fig. (Fig.3D),3D), which mirrored the results from the parental five-copy line. These results suggested that the methylation of the transgenic ICR in male germ cells was not determined solely by its copy number. Rather, methylation may be determined by the environment surrounding the chromosomal integration site.

Reprogramming of DNA methylation at the transgenic ICR.

An important feature of genomic imprinting is the reversibility of the epigenetic modification when passing through germ cells. We therefore examined transgenic ICR methylation in somatic cells of the single- and four-copy TgM sublines (line 378) at the same chromosomal position over several generations. In the single-copy line, the paternally inherited transgenic ICR was hypermethylated (Fig. (Fig.4A,4A, lanes 1 to 8). This hypermethylated transgenic ICR became completely hypomethylated in one generation when transmitted through a female (Fig. (Fig.4A,4A, lanes 9 to 17). However, the hypomethylated transgenic ICR became hypermethylated again, in one generation, when transmitted through a male (Fig. (Fig.4A,4A, lanes 18 to 23). In the four-copy line, the transgenic ICR was methylated properly following transmission through a male (Fig. (Fig.4B,4B, lanes 3 to 6), which also was true for the single-copy line. In contrast, the hypermethylated transgenic ICR became only partially hypomethylated when transmitted through a female (Fig. (Fig.4B,4B, lanes 11 to 18), which was different from the observation for the single-copy line. When passing through females over several generations, the methylation level of the four-copy ICR gradually decreased (data not shown), while the hypomethylated ICR remained persistently hypomethylated (Fig. (Fig.4B,4B, lanes 2, 7 to 10, and 19 to 26).

FIG. 4.
Reprogramming of DNA methylation in the Cre-deleted transgenic ICR. (A and B, top) The pedigrees of the single-copy (A) or the four-copy (B) sublines from line 378. Hemizygous male and female TgM are represented by filled squares and open circles, respectively. ...

To elucidate the cause for these differences in the methylation status between the single- and four-copy TgM lines after passage through females, we analyzed DNA methylation in oocytes by bisulfite sequencing. In the single-copy line, the transgenic ICR was almost completely hypomethylated in oocytes irrespective of whether it was inherited paternally or maternally (Fig. (Fig.4C).4C). These results suggested that the erasure of methylation was complete, thus the transgenic ICR was reprogrammed properly in the female germ line of the single-copy TgM. On the other hand, the paternally, but not maternally, inherited four-copy ICR retained moderate levels of methylation in oocytes (Fig. (Fig.4D).4D). These methylation levels in oocytes were compatible with those in somatic cells of offspring (Fig. (Fig.4B).4B). Therefore, variation in the methylation levels in progeny from mothers with hypermethylated ICRs was not likely due to de novo methylation during oogenesis or early embryogenesis. Rather, it seems that the incomplete erasure of methylation in female primordial germ cells and resistance to global demethylation after fertilization contributed to this variability. A similar phenomenon was reported for a multicopy H19 transgene including the H19 upstream region and the gene body (7). Therefore, the resistance to demethylation activity in female primordial germ cells may be due to the presence of multiple copies of the ICR.

Taking these results together, the reprogramming of HS1/ICR in male germ cells is complete in one generation. After maternal transmission, however, the multicopy ICR became hypomethylated slowly over generations, probably because of the incomplete erasure of methylation in the female germ line, while it was complete in one generation in the single-copy ICR.

Requirement of germ line passage for acquisition of paternal allele-specific methylation of the H19 ICR.

TgM experiments described above demonstrated that the 2.9-kbp H19 ICR fragment contained sufficient information to establish and maintain paternal allele-specific methylation, which could be acquired after the fertilization independently of methylation levels in male germ cells. Birger et al. (4) showed that the DNA fragment from differentially methylated region 2 of the Igf2r gene locus, which normally is methylated on the maternal allele, was de novo methylated after injection into the female pronucleus but not after injection into the male pronucleus, suggesting that the activity to introduce the differential methylation into the sequence was present in the pronucleus. Therefore, we examined whether the H19 ICR could be differentially methylated when injected into the male or female pronucleus. The HS1/ICR fragment was prepared from a plasmid propagated in Dam Dcm E. coli to exclude the effect of DNA methylation that is absent in mammalian cells, and it was injected into the male or female pronucleus. The methylation levels of the transgenic ICR fragments in embryonic day 10.5 embryos were analyzed by COBRA. The ICR regions were substantially methylated in some embryos, while others were almost devoid of methylation irrespective of whether they were injected into male or female pronucleus (Fig. (Fig.5).5). We also analyzed the methylation status of the transgenic ICR in F0 animals of the HS1/ICR TgM described above, which were generated by injecting DNA fragments into the male pronucleus, and we found a wide range of methylation levels of the transgenic ICR (data not shown). These results suggest that, in contrast to region 2 of the Igf2r gene, germ line passage is necessary for the H19 ICR to acquire paternal allele-specific, postfertilization methylation. However, it is formally possible that the injected H19 ICR fragment was not properly methylated because it was integrated into the genome after syngamy, where putative allele-specific marks might already have been lost.

FIG. 5.
Methylation of injected ICR fragments in embryos. The methylation-free HS1/ICR DNA fragment was injected (inj.) into the male or female pronuclei of fertilized eggs. Eggs were transferred into the oviducts of foster mothers. At embryonic day 10.5, embryos ...

DISCUSSION

To understand cis-acting sequences required for the imprinting of the Igf2/H19 locus, several studies have been conducted using TgM (1, 6, 7, 10, 15, 25). The minimal sequence reported so far to recapitulate differential methylation as a randomly integrated, low-copy-number transgene was a 7.7-kbp fragment, which consisted of a 5.5-kbp H19 gene upstream region and the truncated gene body (7). In the current study, we generated six HS1/ICR TgM lines and examined whether the 2.9-kbp H19 ICR fragment, as a randomly integrated transgene, was paternally methylated by its intrinsic activity regardless of its genomic context. In all but one line examined, the paternal allele-specific methylation of the transgenic ICR was detected in somatic cells (Fig. (Fig.2).2). This result strongly demonstrated that the 2.9-kbp fragment, which is located −1.8 to −4.7 kbp relative to the H19 transcription initiation site, contains sufficient information to mark its parental origin. Interestingly, however, the methylation levels of transgenic ICR in germ cells of the testis varied between lines (Fig. (Fig.3).3). This result suggested that methylation in male germ cells is not a prerequisite for the paternal allele-specific methylation of the H19 ICR in somatic cells. Nevertheless, germ line transmission seemed to be essential for the imprinted methylation of the ICR in somatic cells, since the ICR fragment injected into the pronucleus did not exhibit allele-specific methylation until it passed through the germ line (Fig. (Fig.55 and data not shown).

Acquisition of differential methylation of the ICR in somatic cells.

Our findings that the methylation of the transgenic ICR in male germ cells was not necessary for methylation acquisition in somatic cells have interesting implications regarding the mechanism of imprinting. Paternal and/or maternal H19 ICRs may be marked by an epigenetic modification other than DNA methylation in germ cells, and allele-specific methylation can be acquired after fertilization by referring to the so-called primary mark. Recent studies provided evidence that supports the existence of such epigenetic modifications for the Snrpn locus (18). The Snrpn differentially methylated region (DMR), which usually is methylated maternally, was not methylated in oocytes from mice homozygous for a mutant form of Zfp57, a KRAB zinc finger protein. However, the maternal allele-specific methylation of the Snrpn DMR was acquired in postimplantation embryos derived from homozygous mutant mice only when the zygotic Zfp57 was present. This phenomenon can be explained by a DNA methylation-independent marking, although it also is possible that an unidentified methylation mark around the locus acts as the parental mark.

Although CTCF sites of the H19 ICR in the endogenous locus are shown to be required for its methylation maintenance in somatic cells, these sites are thought to be dispensable for the establishment of methylation in male germ cells (11, 12, 21, 22, 26, 31). Since no cis elements responsible for methylation establishment in germ cells have been identified so far (5, 16, 28, 29), it is possible that allele-restricted histone modifications or the deposition of histone variants are used as epigenetic marks. According to our results, however, the methylation of the transgenic ICR was acquired after fertilization independently of methylation in the germ line. Therefore, it is conceivable that CTCF binding to the sites in oocytes and/or a lack of its binding in sperm is indeed an epigenetic signal that governs differential methylation acquisition after fertilization. It thus is intriguing to determine whether CTCF is involved in the postfertilization methylation acquisition in the transgenic ICR.

The endogenous H19 ICR is methylated in sperm and, after fertilization, the paternal ICR remains methylated, despite the general demethylation and epigenetic reprogramming of the whole genome (19). Although the underlying mechanism that confers this resistance is not fully understood, the transgenic H19 ICR may use this mechanism to acquire paternal allele-specific, postfertilization methylation. In addition, a fixed border between the DMD and non-DMD within the H19 locus (at least the one in the 5′ region of the H19 ICR) has been shown to form after fertilization (20, 38). We hypothesize that this phenomenon is explained by the ability of the H19 ICR to mark imprinted, postfertilization methylation.

Methylation of the ICR in male germ cells.

Curiously, the methylation levels of the transgenic H19 ICR in the testis varied between the HS1/ICR TgM lines, while those in somatic cells ended up with paternal hypermethylation in all but one TgM line. It therefore is conceivable that H19 ICR methylation in male germ cells and on the paternal allele in somatic cells are independent events controlled by distinct mechanisms. In male germ cells, the deletions of the H19 ICR from the endogenous locus had little effect on the methylation acquisition of the H19 upstream region (33-35), implying that the surrounding sequences, rather than the sequences within the ICR, contribute to the regulation of ICR methylation in the germ line. However, the chicken β-globin insulator sequence (ChβGI)2 replaced with the H19 ICR at the endogenous mouse locus was not methylated in germ cells (30), indicating that the surrounding sequences of the ICR are not sufficient to introduce methylation in (ChβGI)2. Thus, taken together with our results, in which the transgenes were integrated into distinct chromosomal positions, the combination of both internal and external H19 ICR sequences may determine whether the ICR is methylated in gametes. This is in sharp contrast to the methylation acquisition of the ICR in somatic cells, where ICR sequence alone was sufficient. According to our results, the ICR copy number does not appear to affect methylation in the testis, at least at the integration site in line 378 TgM (Fig. (Fig.3C3C).

In summary, the H19 ICR fragment itself contained sufficient information to mark its parental origin during gametogenesis and to maintain methylation on the paternal allele in somatic cells. On the other hand, the methylation of the ICR in male germ cells, which appeared to be dispensable for subsequent somatic methylation, may be controlled by a mechanism distinct from that in somatic cells.

Acknowledgments

We thank Jörg Bungert (University of Florida) for critically reading the manuscript. We also thank Y. Tanimoto for technical assistance in generating TgM.

H.M. is a research fellow of the Japan Society for the Promotion of Science. This work was supported partially by research grants from the Kato Memorial Bioscience Foundation (to H.M.), the Novartis Foundation (Japan) for the Promotion of Science (to H.M.), and a Grant-in-Aid for Young Scientists (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.T.).

Footnotes

[down-pointing small open triangle]Published ahead of print on 22 June 2009.

REFERENCES

1. Ainscough, J. F., T. Koide, M. Tada, S. Barton, and M. A. Surani. 1997. Imprinting of Igf2 and H19 from a 130 kb YAC transgene. Development 1243621-3632. [PubMed]
2. Bartolomei, M. S., S. Zemel, and S. M. Tilghman. 1991. Parental imprinting of the mouse H19 gene. Nature 351153-155. [PubMed]
3. Bell, A. C., and G. Felsenfeld. 2000. Methylation of a CTCF-dependent boundary controls imprinted expression of the Igf2 gene. Nature 405482-485. [PubMed]
4. Birger, Y., R. Shemer, J. Perk, and A. Razin. 1999. The imprinting box of the mouse Igf2r gene. Nature 39784-88. [PubMed]
5. Bowman, A. B., J. M. Levorse, R. S. Ingram, and S. M. Tilghman. 2003. Functional characterization of a testis-specific DNA binding activity at the H19/Igf2 imprinting control region. Mol. Cell. Biol. 238345-8351. [PMC free article] [PubMed]
6. Brenton, J. D., R. A. Drewell, S. Viville, K. J. Hilton, S. C. Barton, J. F. Ainscough, and M. A. Surani. 1999. A silencer element identified in Drosophila is required for imprinting of H19 reporter transgenes in mice. Proc. Natl. Acad. Sci. USA 969242-9247. [PMC free article] [PubMed]
7. Cranston, M. J., T. L. Spinka, D. A. Elson, and M. S. Bartolomei. 2001. Elucidation of the minimal sequence required to imprint H19 transgenes. Genomics 7398-107. [PubMed]
8. DeChiara, T. M., E. J. Robertson, and A. Efstratiadis. 1991. Parental imprinting of the mouse insulin-like growth factor II gene. Cell 64849-859. [PubMed]
9. Drewell, R. A., C. J. Goddard, J. O. Thomas, and M. A. Surani. 2002. Methylation-dependent silencing at the H19 imprinting control region by MeCP2. Nucleic Acids Res. 301139-1144. [PMC free article] [PubMed]
10. Elson, D. A., and M. S. Bartolomei. 1997. A 5′ differentially methylated sequence and the 3′-flanking region are necessary for H19 transgene imprinting. Mol. Cell. Biol. 17309-317. [PMC free article] [PubMed]
11. Engel, N., J. L. Thorvaldsen, and M. S. Bartolomei. 2006. CTCF binding sites promote transcription initiation and prevent DNA methylation on the maternal allele at the imprinted H19/Igf2 locus. Hum. Mol. Genet. 152945-2954. [PubMed]
12. Engel, N., A. G. West, G. Felsenfeld, and M. S. Bartolomei. 2004. Antagonism between DNA hypermethylation and enhancer-blocking activity at the H19 DMD is uncovered by CpG mutations. Nat. Genet. 36883-888. [PubMed]
13. Gaensler, K. M., M. Kitamura, and Y. W. Kan. 1993. Germ-line transmission and developmental regulation of a 150-kb yeast artificial chromosome containing the human beta-globin locus in transgenic mice. Proc. Natl. Acad. Sci. USA 9011381-11385. [PMC free article] [PubMed]
14. Hark, A. T., C. J. Schoenherr, D. J. Katz, R. S. Ingram, J. M. Levorse, and S. M. Tilghman. 2000. CTCF mediates methylation-sensitive enhancer-blocking activity at the H19/Igf2 locus. Nature 405486-489. [PubMed]
15. Kaffer, C. R., M. Srivastava, K. Y. Park, E. Ives, S. Hsieh, J. Batlle, A. Grinberg, S. P. Huang, and K. Pfeifer. 2000. A transcriptional insulator at the imprinted H19/Igf2 locus. Genes Dev. 141908-1919. [PMC free article] [PubMed]
16. Katz, D. J., M. A. Beer, J. M. Levorse, and S. M. Tilghman. 2005. Functional characterization of a novel Ku70/80 pause site at the H19/Igf2 imprinting control region. Mol. Cell. Biol. 253855-3863. [PMC free article] [PubMed]
17. Li, E., C. Beard, and R. Jaenisch. 1993. Role for DNA methylation in genomic imprinting. Nature 366362-365. [PubMed]
18. Li, X., M. Ito, F. Zhou, N. Youngson, X. Zuo, P. Leder, and A. C. Ferguson-Smith. 2008. A maternal-zygotic effect gene, Zfp57, maintains both maternal and paternal imprints. Dev. Cell 15547-557. [PMC free article] [PubMed]
19. Morgan, H. D., F. Santos, K. Green, W. Dean, and W. Reik. 2005. Epigenetic reprogramming in mammals. Hum. Mol. Genet. 14R47-R58. [PubMed]
20. Olek, A., and J. Walter. 1997. The pre-implantation ontogeny of the H19 methylation imprint. Nat. Genet. 17275-276. [PubMed]
21. Pant, V., S. Kurukuti, E. Pugacheva, S. Shamsuddin, P. Mariano, R. Renkawitz, E. Klenova, V. Lobanenkov, and R. Ohlsson. 2004. Mutation of a single CTCF target site within the H19 imprinting control region leads to loss of Igf2 imprinting and complex patterns of de novo methylation upon maternal inheritance. Mol. Cell. Biol. 243497-3504. [PMC free article] [PubMed]
22. Pant, V., P. Mariano, C. Kanduri, A. Mattsson, V. Lobanenkov, R. Heuchel, and R. Ohlsson. 2003. The nucleotides responsible for the direct physical contact between the chromatin insulator protein CTCF and the H19 imprinting control region manifest parent of origin-specific long-distance insulation and methylation-free domains. Genes Dev. 17586-590. [PMC free article] [PubMed]
23. Park, K. Y., E. A. Sellars, A. Grinberg, S. P. Huang, and K. Pfeifer. 2004. The H19 differentially methylated region marks the parental origin of a heterologous locus without gametic DNA methylation. Mol. Cell. Biol. 243588-3595. [PMC free article] [PubMed]
24. Reik, W., and J. Walter. 2001. Genomic imprinting: parental influence on the genome. Nat. Rev. Genet. 221-32. [PubMed]
25. Reinhart, B., M. Eljanne, and J. R. Chaillet. 2002. Shared role for differentially methylated domains of imprinted genes. Mol. Cell. Biol. 222089-2098. [PMC free article] [PubMed]
26. Schoenherr, C. J., J. M. Levorse, and S. M. Tilghman. 2003. CTCF maintains differential methylation at the Igf2/H19 locus. Nat. Genet. 3366-69. [PubMed]
27. Srivastava, M., S. Hsieh, A. Grinberg, L. Williams-Simons, S. P. Huang, and K. Pfeifer. 2000. H19 and Igf2 monoallelic expression is regulated in two distinct ways by a shared cis acting regulatory region upstream of H19. Genes Dev. 141186-1195. [PMC free article] [PubMed]
28. Szabó, P. E., L. Han, J. Hyo-Jung, and J. R. Mann. 2006. Mutagenesis in mice of nuclear hormone receptor binding sites in the Igf2/H19 imprinting control region. Cytogenet. Genome Res. 113238-246. [PubMed]
29. Szabó, P. E., G. P. Pfeifer, and J. R. Mann. 2004. Parent-of-origin-specific binding of nuclear hormone receptor complexes in the H19-Igf2 imprinting control region. Mol. Cell. Biol. 244858-4868. [PMC free article] [PubMed]
30. Szabó, P. E., S. H. Tang, M. R. Reed, F. J. Silva, W. M. Tsark, and J. R. Mann. 2002. The chicken beta-globin insulator element conveys chromatin boundary activity but not imprinting at the mouse Igf2/H19 domain. Development 129897-904. [PubMed]
31. Szabó, P. E., S. H. Tang, F. J. Silva, W. M. Tsark, and J. R. Mann. 2004. Role of CTCF binding sites in the Igf2/H19 imprinting control region. Mol. Cell. Biol. 244791-4800. [PMC free article] [PubMed]
32. Tanimoto, K., M. Shimotsuma, H. Matsuzaki, A. Omori, J. Bungert, J. D. Engel, and A. Fukamizu. 2005. Genomic imprinting recapitulated in the human beta-globin locus. Proc. Natl. Acad. Sci. USA 10210250-10255. [PMC free article] [PubMed]
33. Thorvaldsen, J. L., K. L. Duran, and M. S. Bartolomei. 1998. Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2. Genes Dev. 123693-3702. [PMC free article] [PubMed]
34. Thorvaldsen, J. L., A. M. Fedoriw, S. Nguyen, and M. S. Bartolomei. 2006. Developmental profile of H19 differentially methylated domain (DMD) deletion alleles reveals multiple roles of the DMD in regulating allelic expression and DNA methylation at the imprinted H19/Igf2 locus. Mol. Cell. Biol. 261245-1258. [PMC free article] [PubMed]
35. Thorvaldsen, J. L., M. R. Mann, O. Nwoko, K. L. Duran, and M. S. Bartolomei. 2002. Analysis of sequence upstream of the endogenous H19 gene reveals elements both essential and dispensable for imprinting. Mol. Cell. Biol. 222450-2462. [PMC free article] [PubMed]
36. Tremblay, K. D., K. L. Duran, and M. S. Bartolomei. 1997. A 5′ 2-kilobase-pair region of the imprinted mouse H19 gene exhibits exclusive paternal methylation throughout development. Mol. Cell. Biol. 174322-4329. [PMC free article] [PubMed]
37. Tremblay, K. D., J. R. Saam, R. S. Ingram, S. M. Tilghman, and M. S. Bartolomei. 1995. A paternal-specific methylation imprint marks the alleles of the mouse H19 gene. Nat. Genet. 9407-413. [PubMed]
38. Warnecke, P. M., J. R. Mann, M. Frommer, and S. J. Clark. 1998. Bisulfite sequencing in preimplantation embryos: DNA methylation profile of the upstream region of the mouse imprinted H19 gene. Genomics 51182-190. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...