Human Molecular Genetics, 2002, Vol. 11, No. 4 411-418
© 2002 Oxford University Press
A human H19 transgene exhibits impaired paternal-specific imprint acquisition and maintenance in mice
Howard Hughes Medical Institute and Department of Molecular Biology, Princeton University, Princeton, NJ 08544, USA
Received October 10, 2001; Revised and Accepted December 11, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting, the differential expression of autosomal genes based on their parent of origin, is observed in all eutherian mammals that have been examined. In most instances the genes that are imprinted in one species are imprinted in others as well, suggesting that imprinting predated eutherian radiation. For example, the RNA-coding H19 gene is repressed upon paternal inheritance in all species examined to date. Thus, it is surprising that there is remarkably little sequence conservation among the cis-acting DNA regulatory elements that are required for imprinting of H19 and the tightly linked Igf2 gene. The most conserved characteristic in the imprinting control region (ICR) is the presence of multiple binding sites for the zinc finger protein CTCF, raising the possibility that CTCF binding might be sufficient for the reciprocal imprinting of H19 and Igf2. To investigate whether a human H19 transgene, harboring seven CTCF sites, is correctly recognized and imprinted in the mouse, a 100 kb transgene containing the human H19 gene was introduced into the mouse germline. The human transgene was specifically methylated after passage through the male germline in a copy number-dependent manner, but the methylation was unstable, undergoing progressive loss during development. Consequently, the transgene was highly expressed upon both maternal and paternal inheritance. These results argue that the signals for both the acquisition and maintenance of methylation imprinting are diverging rapidly.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting results from germline-specific epigenetic modifications of the genome, which lead to differential expression of the two parental alleles of autosomal genes. One such epigenetic modification is DNA methylation, which has been shown to be an essential component of the gene silencing mechanism in the soma (1). Methylation controls the activity of regulatory elements termed imprinting control regions (ICRs), several of which have been shown to directly regulate the allele-specific expression of multiple linked genes (2–4).
The imprinting of the H19 and insulin-like growth factor 2 (Igf2) genes is under the control of an ICR that lies between –2 and –4 kb upstream of the H19 gene, and the activity of the ICR is modulated by differential methylation in this region (3). In mice, the ICR is extensively methylated in sperm (5,6), and methylation is maintained after fertilization during a period when the genome undergoes global demethylation (7). After implantation, methylation spreads through the H19 promoter and gene body, thereby silencing the gene on the paternal chromosome. The ICR is unmethylated in eggs and the maternal chromosome is maintained in this state during development (7). The unmethylated ICR acts as a chromatin insulator on the maternal allele (8,9), silencing Igf2 by blocking its interaction with enhancers located on the opposite side of the ICR and 3' of the H19 gene (Fig. 1). As this model predicts, deletions of the ICR on the maternal chromosome result in biallelic expression of Igf2 (3,10,11). The enhancer blocking activity requires the binding of CTCF, a zinc finger-containing protein that mediates enhancer-blocking activity in the vicinity of multiple genes in vertebrates (8,9,12,13).
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A more limited analysis of the human H19 gene reveals that a region in its 5' flank is also methylated in the male germline (14–16) and maintained in that state in somatic cells. On the maternal chromosome the region is largely unmethylated and contains multiple conserved CTCF binding sites (17,18). It is likely that this region also functions as an enhancer blocker, based on the observation that inappropriate methylation of the region, which is known to inhibit CTCF binding, is correlated with biallelic expression of IGF2 in patients with Beckwith–Weidemann syndrome, Wilms’ tumour and other cancers (14,19–22). With the exception of the CTCF binding sites, which are reiterated seven times in the human ICR and four times in the mouse (18), there is a striking lack of sequence conservation between the mouse and human ICRs (15). This suggests that reiterated CTCF sites may be sufficient to confer imprinted expression on Igf2.
Despite the apparent conservation of imprinting among species, no transgene derived from humans has displayed imprinted expression in mice. For example, large human-derived transgenes containing the imprinted genes SNRPN and p57KIP2 failed to exhibit imprinted expression despite the fact that murine Snrpn transgenes were imprinted (23,24), and somatic cell hybrid cell lines retaining human chromosomes 15 or 11 on a rodent background exhibit appropriate methylation and imprinted expression of multiple human genes, including SNRPN (25). It could be that the transgenes contained insufficient information for establishment or maintenance of the imprints. Alternatively, the imprinting machinery may have diverged such that the mouse germline is unable to recognize human ICRs and establish primary epigenetic modifications, but can maintain them once they are in place. To help resolve this question, we have utilized a 100 kb human H19 transgene which contained all sequences that are held in common with mouse transgenes that imprint in single copy. In contrast to results obtained with murine transgenes, we find that the male germ cells fail to methylate a single-copy human H19 transgene, but the putative human ICR region is extensively methylated in lines bearing multiple copies. However, this methylation is lost during embryonic development, demonstrating that imprint acquisition and maintenance of the human H19 ICR imprinting is defective in the mouse.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of transgenic lines
Initial transgenic mouse studies with the murine H19 gene were conducted with 14 kb transgenes that included 4 kb of sequence 5' of the gene, including a majority of the differentially methylated ICR, the H19 gene itself and 8–12 kb of 3' sequences (Fig. 1A). These transgenes were consistently imprinted, but only in multi-copy arrays (5,26,27). In contrast, larger single-copy transgenes encompassing either –95 to 36 kb or –7 to 130 kb relative to the start of transcription of the H19 gene are imprinted correctly (11,28) (Fig. 1A). More recently, a 15.7 kb mouse transgene extending from –5.7 to 10 kb was shown to be imprinted in one single-copy line (Fig. 1A), thus defining the minimal sequences required for imprinting of the mouse gene (29). To test whether the human H19 gene could be imprinted after passage through the mouse germline, we microinjected a 100 kb P1 clone containing the human H19 gene and
50 kb of 5' and 3' flanking sequences into fertilized mouse zygotes (Fig. 1). Six transgenic lines were obtained and the copy number of each insert was ascertained by Southern blot analysis with probes to the human H19 gene and ICR region (Table 1). The integrity of the transgenes was confirmed by hybridization of EcoRI-digested DNA with six probes spanning the P1 clone (Fig. 1). Two lines, 1-9 and 1-26, did not hybridize to the 3'-most probe used (probe F), whereas four lines hybridized to all probes examined. Because lines 1-9 and 1-26 hybridized to the probes covering all the known regulatory elements, these lines were included in the analyses of methylation and expression.
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Expression of the human H19 transgene
Northern blot analysis of neonatal liver RNAs was used to quantitate the expression of the human H19 gene after transmission through the maternal and paternal germlines. Following paternal transmission, human H19 RNA was detected in neonatal liver in all but the single-copy line (1-9) and RNA levels were approximately proportional to the transgene copy number (Fig. 2A). Equivalent amounts of RNA were observed upon maternal transmission in two lines examined (Fig. 2B). In the case of the single-copy line, H19 was not expressed after maternal or paternal inheritance (Fig. 2B). The human H19 gene was also expressed after paternal transmission in neonatal skeletal muscle in a copy-number-dependent manner (Fig. 2C). These data indicate that the transgene contains both endodermal and mesodermal enhancers, as expected from the location of these elements in the mouse. Lower levels of the human H19 transcript were also observed in neonatal brain (Fig. 2C).
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In humans, H19 expression is downregulated in adult tissues, although moderate levels are detected in muscle, liver and heart, and low levels in the kidney and pancreas (30; and data not shown). This differs from the mouse, where silencing is observed in all tissues except muscle, which continues to express H19 RNA at low levels. The human H19 transgene transcripts were repressed in adult skeletal muscle, tongue, brain and kidney, relative to the levels in neonatal tissues (Fig. 2D). However, expression in adult liver was approximately 10-fold higher than in neonates in both lines in which adult expression was studied (Fig. 2D).
Methylation analysis of the human H19 transgene
Consistent with the high levels of RNA, Southern blot analysis revealed that the paternally inherited human H19 ICR was completely unmethylated in somatic tissues in all lines (Fig. 3A and B), indistinguishable from maternal transmission (data not shown). In order to ask whether the transgene was methylated in gametes, we examined the methylation status of the H19 ICR in testis DNA (Fig. 3C; Table 1). This region is known to be extensively methylated in human spermatocytes (14,15,17). The human ICR was heavily methylated in the three lines with seven to 20 transgene copies (1-2, 1-7 and 1-15), less methylated in the two four-copy lines (1-6 and 1-2) and unmethylated in the single-copy line (1-9). Thus, the human ICR is recognized as a target for methylation in the male germline in response to high copy. As expected, the mouse ICR is fully methylated in these DNA samples (Fig. 3C).
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The endogenous H19 promoter region is largely unmethylated in human spermatocytes (14). Analysis of this region of the transgene in mouse testis showed that limited DNA methylation of the human H19 promoter was detectable in a 20-copy line (1-15); however, the other lines were devoid of methylation in this region (Fig. 3D). Thus, the methylation, when it occurs, is mostly restricted to the ICR region, and does not spread to the promoter even though the relative CpG density of the two regions is comparable, as shown in Figure 3A.
To further explore the specificity of the methylation, we examined whether the ICR was methylated during oogenesis. Given the limited amount of DNA available from oocytes, we developed a PCR-based assay to amplify fragments of both mouse and human ICRs containing CTCF binding sites that had previously been shown to be methylated in sperm (7,14). The PCR was performed on both intact DNA and DNA digested with BstUI, which cleaves in a methylation-sensitive manner within and adjacent to CTCF sites in both mouse and human ICRs (Fig. 4A). Phosphoimager analysis was used to determine the ratios of human:mouse PCR products before and after digestion in order to assess the degree of methylation of the human transgenes. To verify the reliability of the assay we analyzed testis DNA from a highly methylated line (85%; line 1-2) and a less well methylated line (22%; 1-26), as well as DNA prepared from Dnmt–/– homozygous ES cells that is almost completely unmethylated (Fig. 4B). The ratios of human:mouse products after BstUI digestion, normalized using the intensities of the undigested bands, indicates that the assay can readily detect differences in methylation status of the region (Fig. 4B).
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Oocytes from super-ovulated 1-2 transgenic females were collected and analyzed by PCR before and after digestion with BstUI. The failure to amplify either mouse or human ICR following BstUI digestion (Fig. 4B), demonstrates that the human ICR is completely unmethylated in oocytes, as is the endogenous mouse ICR, as reported previously. Thus, the methylation in sperm is germline specific.
The absence of methylation of the human H19 transgene in the soma argues that the paternal methylation is lost sometime in development. In the mouse, the methylation of the ICR survives a period of global demethylation of the genome (7) that begins shortly after fertilization with selective demethylation of the paternal genome (31,32), and continues during the morula and blastocyst stage to include both parental genomes (33,34). Both PCR primer sets (Fig. 4A) encompassed regions shown previously to contain methyl groups that were retained during the preimplantation stage of development (7,15). To investigate when the gametic methylation of the human H19 transgenes was lost, the PCR assay was used to examine methylation of the human ICR during development.
The methylation status of the paternally transmitted human H19 ICR was first assessed in fertilized eggs obtained from matings using transgenic males and collected 14 h post-fertilization, a time when demethylation of the paternal genome has commenced, but prior to pronuclear fusion and the first round of replication. At this stage the methylation of the transgene was stable, as indicated by the similar intensities of the bands before and after BstUI digestion. At the 16–32 cell morula stage, there was an 25% decrease in the intensity after digestion, indicating that loss of methylation had begun. Further demethylation was evident by the 128 cell blastocyst stage, and 9.5 d.p.c. embryos display a nearly complete loss of methylation. Thus, while the human ICR can assemble into a methylated state during spermatogenesis, it cannot sustain that state in the embryo, indicating that it is not being recognized as an ICR by the mouse embryo.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The pattern of expression of the human H19 transgene faithfully replicated the patterns reported for the human and mouse H19 genes, arguing that the P1 clone contains the requisite transcriptional regulatory sequences for activation and postnatal repression of the gene. The transgene was highly expressed in neonatal tissues at levels comparable to those of the mouse H19 gene. Moreover, the level of expression was independent of integration site and correlated with copy number, suggesting the presence of a locus control region-like element on the transgene.
Based on the efficient repression of the endogenous mouse H19 gene in liver after birth, the high level expression of the human transgene in adult liver in two lines studied was unexpected. While H19 is expressed in adult human liver, its level is substantially lower than in neonatal liver (30). This difference in the behavior of the human H19 transgene may reflect a species-specific difference in the response of the human promoter to postnatal repression in mouse liver. Expression in adult muscle (skeletal muscle and tongue) was variable among the two lines analyzed and may reflect either a site of integration or copy number effect.
In contrast to the sequences required for tissue-specific expression in neonates, our results suggest that elements required for imprint establishment and maintenance are poorly conserved in evolution. The human H19 transgene was able to reproducibly assemble paternal-specific methylation at the ICR only at high copy number. When methylation occurred, it was largely confined to the ICR in four of the lines, as is the case in human spermatocytes. Only in the highest copy line was slight methylation also observed within the H19 promoter. The specificity of this methylation must be conferred by sequences within the ICR, as the overall CpG density is very similar in the ICR and promoter proximal regions (Fig. 3A). Moreover, CpG residues tested within the ICR were not methylated in oocytes, suggesting germline-specific recognition of ICR. However, the methylation was not sustained in the early embryo, and consequently the gene was expressed equally upon maternal and paternal inheritance at later stages of development.
The transgene contained 50 kb of both 5' and 3' flanking sequence, and included all of the DNA held in common between the mouse transgenes that imprint in single copy. Assuming that the organization of the mouse and human loci are similar with respect to the positions of the ICR, it appears that the ability to recognize and maintain a methylation imprint has diverged within mammals. The ability of multiple copy transgenes to acquire methylation in the ICR could reflect a threshold effect, where a sufficient number of redundant weak imprinting signals allowed recognition by the methylation machinery. Such an explanation would be consistent with the behavior of 14 kb mouse transgenes that contain a portion of the ICR and imprint only in multiple copies arrays.
The methylation of the mouse ICR persists during the genome-wide demethylation of the blastocyst, a hallmark of all gametic marks that regulate imprinting. In contrast, the human H19 transgene loses most of its methylation during this period. While it is difficult to distinguish whether the loss of methylation indicates that an accurate imprint is not assembled at the ICR or that the imprint is correctly established but cannot be sustained, the failure of high copy-number transgenes that were heavily methylated in the male germline to maintain this epigenetic modification during preimplantation development suggests that maintenance of gametic marks during global demethylation may also require species-specific recognition of the mark to protect it. In combination, these results point towards imprint incompatibility between species resulting from both defects in germline acquisition of methylation and early maintenance of the mark during embryogenesis.
Given that imprinting is a form of genomic conflict, it is expected to be a rapidly evolving mechanism and could explain why no human transgene has been shown to be correctly imprinted in mice. This failure is in contrast to studies in hybrid rodent/human somatic cell lines that maintain the imprinting of the human H19 and Igf2 genes (25). This suggests that after the imprint has survived early embryogenesis, there is no species barrier to maintaining it. The hypothesis that one component of species incompatibility lies in imprint acquisition is supported by the striking lack of sequence similarity among the ICRs of the human and mouse Igf2r/IGF2R and H19 genes (15,35). The only known exceptions within the H19 ICR are the conserved CTCF sites. However, we have recently generated a mouse that is lacking all four of the CTCF sites, and find that the mutant chromosome is able to assemble and maintain a paternal imprint in a wild-type manner (C.Schoenherr and S.M.Tilghman, unpublished results), suggesting that these conserved sequences may not have a major role in acquiring methylation at the ICR. The inability to maintain methylation during preimplantation development is consistent with our finding in Peromyscus that some imprints are not sustained in F1 inter-specific hybrids (36,37).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation and analysis of transgenic mice
The 100 kb P1 (DDBJ/EMBL/GenBank accession no. AC004556) was graciously provided by Dr Glen Evans. DNA was prepared using the Qiagen maxiprep kit, digested with NotI and fractionated on a CBL4 Sepharose column. After adjusting the concentration of DNA to 2 ng/µl, it was injected into FVB fertilized zygotes that were implanted into pseudo-pregnant foster mothers. Tail DNA was prepared as described previously and founders were identified by Southern blotting using probes C and/or D. Transgene copy number was analyzed by Southern blotting equal amounts of EcoRI-digested transgenic and human DNAs, hybridization to a human H19 cDNA probe (probe D) and quantitating signal intensity using a Molecular Dynamics Phosphoimager. The derivation of probes used to assess transgene copy number and/or integrity of EcoRI-digested DNA is as follows. Probe A, 389 bp product generated using PCR primers 5'-TGCTGAATCCTCTTCTTTCTGTCTG-3' (forward) and 5'-AGCCTGGAATCTGGGGTTAGG-3' (reverse); probe B, 367 bp fragment obtained using 5'-TGGTGGAGGAGGCTGGACTG-3' (forward) and 5'-GCTGTGGGCAGGAGGGAG-3' (reverse); probe C, 1.2 kb RsaI–EagI genomic fragment spanning the 5'region of the ICR; probe D, 376 bp portion of the 3' end of the human H19 cDNA amplified using PCR primers 5'-GGAACCAGACCTCATCAGCCC-3' (forward) and 5'-GCTCACACTCACGCACACTCG-3' (reverse); probe E, 319 bp fragment generated by PCR primers 5'-GACCGATGCCCGTGTGCC-3' (forward) and 5'-ACCCCCTATCCCCCGCCAC-3' (reverse); probe F, 210 bp fragment amplified using primers 5'-GGCAGCACCGTCTTCCAG-3' (forward) and 5'-CGTGTGTTGGGGCACTGATAC-3' (reverse). All PCR reactions were carried out in PCR buffer (Perkin Elmer Cetus) containing 250 nM dNTPs, 1.5 mM MgCl2, 12% sucrose, 20 µM cresol red, 5 U Taq polymerase, 100 ng each primer and 500 ng human genomic DNA. Amplification conditions were 94°C for 30 s, 55°C for 60 s and 72°C for 60 s for 35 cycles.
RNA isolation and analysis
RNAs were prepared by homogenizing frozen tissue in Triazol (Sigma) with subsequent steps carried out according to the supplier’s recommendations. Northern blots transferred to Hybond+ were hybridized in ExpressHyb using probe D and rpL32 cDNA; relative signal intensity was established using a Molecular Dynamics Phosphoimager.
Methylation analysis
Genomic DNAs were digested with RsaI alone or with HhaI, HpaII or MspI and analyzed by gel electrophoresis and Southern blotting. Probe 1 is a 1.2 kb RsaI–EagI genomic fragment spanning the 5' region of the ICR, which hybridizes to both RsaI fragments containing CTCF sites. Probe 2 is a 293 bp PCR product obtained using primers 5'-TCTCCCTCCCAGACCACTG-3' (forward) and 5'-GCGGTGACCAGCACAAGC-3' (reverse) amplified using the conditions described above. The amount of digested versus undigested material in each sample was quantitated using the Phosphoimager to calculate the percentage methylation in each line.
PCR assay of methylation loss
DNAs were incubated in 10 µl New England Biolabs (NEB) buffer 2 containing 10 U BstUI at 60°C for 4 h followed by inactivation of the enzyme at 94°C for 10 min. PCR reactions were carried out by adding a cocktail resulting in the final reaction consisting of 50 µl PCR buffer containing 250 nM dNTPs, 1 µCi [32P-
]dATP, 2.0 mM MgCl2, 12% sucrose, 20 µM cresol red, 5 U Taq polymerase, 25 ng each human primer, 50 ng each mouse primer and 500 pg genomic DNA. Amplification conditions were 40 cycles of 94°C for 30 s, 57°C for 60 s and 72°C for 60 s. Primers used to amplify the 363 bp human ICR fragment were 5'-TGTGGATAATGCCCGACCTGA-3' (forward) and 5'-CAATGAGGTGTCCCAGTTCCA-3' (reverse). The 292 bp mouse ICR fragment was amplified using primers 5'-ATTCACACGAGCATCCAGGAGG-3' (forward) and 5'-TTGATTTGGGAGTCCGAGTCCACG-3' (reverse). PCR reactions were run on 10% acrylamide/1x TBE gels and bands quantitated using the Phosphoimager.
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ACKNOWLEDGEMENTS |
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The authors would like to thank Dr Glen Evans for the human P1 clone. This work is supported by a grant from the National Institute for General Medical Sciences.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +1 609 258 6100; Fax: +1 609 258 3345; Email: smt@princeton.edu
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REFERENCES |
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