Mol. Hum. Reprod. Advance Access originally published online on May 20, 2005
Molecular Human Reproduction 2005 11(6):413-422; doi:10.1093/molehr/gah180
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Development of a monkey model for the study of primate genomic imprinting
1Department of Obstetrics and Gynecology, Faculty of Medicine, University of Tokyo 7-3-1, Hongo, Bunkyo-ku, Tokyo 113-8655, Japan and 2Division of Reproductive Sciences, Oregon National Primate Research Center, Oregon Health and Science University, Beaverton, OR 97006, USA
3 To whom correspondence should be addressed at: Department of Reproductive Sciences, Oregon National Primate Research Center, 505 NW 185th Avenue, Beaverton, OR 97006, USA. E-mail: wolfd{at}ohsu.edu
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Abstract |
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An understanding of the role of imprinted genes in primate development requires the identification of suitable genetic markers that allow analysis of allele-specific expression and methylation status. Four genes, NDN (Necdin), H19, SNRPN and IGF2, known to be imprinted in mice and humans, were selected for study in rhesus monkeys along with two imprinting centres (ICs) associated with the regulation of H19/IGF2, NDN and SNRPN. GAPD was employed as a non-imprinted control gene. Primers designed to amplify polymorphic regions in these genes and ICs were based on human sequences. Genomic DNA was isolated from peripheral blood leukocytes of 93 rhesus macaques of Indian or Chinese-origin. Sequence analysis of amplicons resulted in the identification of 32 unique SNPs. Country-of-origin related differences in SNP distributions were evident. Since disruptions in imprinted gene expression and associated developmental abnormalities may result from in vitro embryo manipulation, we also examined imprinting in NDN, H19, SNRPN and IGF2 in rhesus monkey infants produced by natural mating or by ICSI. Muscle biopsies followed by RT–PCR and sequence analysis were performed in four heterozygous animals produced by natural mating and all four genes were expressed monoallelically supporting the conclusion that these genes are normally imprinted in monkeys. In the case of ICSI, five informative infants were selected based on parental analysis. Allele-specific studies indicated that the expected uniparental expression patterns were retained in animals produced from manipulated embryos. Moreover, methylation analysis revealed that CpG islands within H19/IGF2 and SNURF/SNRPN ICs were differentially methylated. The approach described here will allow examination of imprinting in the embryos and embryonic stem cells of the monkey.
Key words: development/imprinted genes/methylation/primate/single nucleotide polymorphisms
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Introduction |
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The majority of genes in the mammalian genome are equally expressed from both maternal and paternal alleles. A small number (
50) are imprinted; epigenetically regulated and inherited in a silent state from one of the two parents and in a fully active form from the other (Rideout et al., 2001
; Surani, 2001). With some, the epigenetic mark is not absolute but rather is either partial, tissue-specific, cell-type specific or restricted to certain developmental stages. Most imprinted genes show allelic differences in DNA methylation at so-called differentially methylated regions (DMRs) which act as epigenetic modifiers of allelic expression by recruiting proteins that specifically bind to methylated or unmethylated regions (Bell and Felsenfeld, 2000; Hark et al., 2000; Schoenherr et al., 2003). Imprinting centres (IC) are regulatory sequences that harbour DMRs.
We have been interested in imprinted gene expression in the embryos and embryonic stem (ES) cells of rhesus monkeys for several reasons. First, the monkey represents a clinically relevant species to study the early developmental events that are precluded for ethical reasons in the human. Access to preimplantation stage embryos and ES cell lines in the rhesus macaque is now routine (Wolf, 2004; Wolf et al., 2004a) and the major ethical limitations associated with human studies can be largely circumvented because of the close phylogenetic relationship between monkey and man. Second, given the potential of ES cell-based therapies to be used in human medicine, an understanding of the role(s) of imprinted genes in the function of ES cell progeny both in vitro and in vivo is essential. Questions concerning the integrity of imprinting in undifferentiated and differentiated progeny can only be addressed fully in an animal model. Third, some imprints are altered in response to environmental insults (e.g., H19 and IGF2 in the mouse) (Sasaki et al., 1995; Dean et al., 1998; Doherty et al., 2000; Khosla et al., 2001; Mann et al., 2003) or to in vitro manipulations that include nuclear transfer (Zhang et al., 2004) and ICSI. The use of ICSI for the treatment of human subfertility has been associated with increased rates of Beckwith–Wiedemann syndrome, likely reflecting aberrant imprinting (Maher et al., 2003). Furthermore, abnormal expression of the imprinted genes, NDN and SNRPN, has been implicated in the aetiology of Prader-Willi syndrome (Reis et al., 1994; Mann and Bartolomei, 1999) while IGF2 and H19 abnormalities have been associated with Beckwith–Wiedemann syndrome and Wilms' tumours (Moulton et al., 1994; Weksberg et al., 2003).
In order to evaluate the role of imprinted genes in primate development, suitable genetic markers are required. Presently, despite widespread use of rhesus macaques in biomedical research, relatively little is known about the organization or content of the genome in this species, a predicament that will change in the near future with the development of a genetic linkage map (J. Rogers; personal communication), whole genome DNA sequencing (www.hgsc.bcm.tme.edu/projects/macaque/) and the commercial availability of an Affymetrix microarray for expression analysis (www.rhesusgenechip.unomaha.edu/index.html). Regardless, we have begun an investigation of SNPs in the monkey genome that are relevant to determining the parent-specific expression and methylation status of imprinted genes; those localized to the transcribed regions of several candidate genes, including NDN, H19, SNRPN and IGF2 or within postulated ICs and positioned either upstream of H19 or in the promoter-exon 1 region of SNURF/SNRPN. Here, we report the definition of 32 SNPs useful to allele-specific expression and methylation analysis in the rhesus macaque. Evidence was obtained that these four genes are imprinted in the monkey and that the expected uniparental expression of NDN, H19, SNRPN and IGF2 was retained in infants produced from in vitro manipulated embryos. Furthermore, our studies demonstrated that CpG islands within monkey H19/IGF2 and SNURF/SNRPN ICs were differentially methylated suggesting that CpG methylation could be one of the mechanisms involved in the differential marking of parental alleles in monkey gametes. This approach lays the foundation for detailed studies on the role of imprinting in a clinical relevant primate model.
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Animals
Adult animals selected from the colony maintained at Oregon National Primate Research Centre (ONPRC) and assigned for reproductive studies were accessed. Indian-derived animals (10 males and 45 females) were descendants of animals imported in the 1970s and raised at ONPRC. Chinese-origin females (38) were imported to ONPRC in the last 3–4 years as captive-reared from south China, Vietnam, Cambodia and Laos (G. Heckman, personal communication). The number of animals tested varied across loci reflecting the frequency of the particular SNP under study. All animal procedures were approved by the Institutional Animal Care and Use Committee at the ONPRC.
Genomic DNA extraction and amplification by PCR
Genomic DNA was isolated from peripheral blood leukocytes using a QIAamp DNA Blood Midi Kit (QIAGEN, Valencia, CA) according to the manufacturer's protocol. The following primers for genomic PCR were based on human consensus sequences obtained from GenBank. The relative positions of the human primers and polymorphic sites are shown in Figure 1. In one instance, H19-1R, the original human-based primer sequence was replaced by the monkey sequence when it became available in order to improve amplification efficiency. Two of these imprinted genes, IGF2 and H19, are located on chromosome 11 in both the human and rhesus monkey. NDN and SNRPN are located on chromosome 15 in the human which corresponds to chromosome 7 in the rhesus monkey (Lawce et al., 1998).
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NDN-1F; 5'-GAGCCGCCCGAATACGAGTT-3'
NDN-1R; 5'-GCAGGAGCAGTCTACCCCAA-3'
NDN-2F; 5'-ACTGCTCCTGCAGAGTTTGGAA-3'
NDN-2R; 5'-AGGAGTAATCATGAGTAGCGATTT-3'
SNRPN-1F; 5'-AGTCCCTTGGTGAGGAAGCTAGTA-3'
SNRPN-1R; 5'-CTGGCAAACTCTTTCTTTCAGCCC-3'
SNRPN-2F; 5'-GGTATGTATCACTTTGTTGGTTTCTCCC-3'
SNRPN-2R; 5'-CCCAAATTCTTAGGCTCTCAGGCT-3'
SNRPN-3F; 5'-TGTCTGGCCCATTTCTTTAGGG-3'
SNRPN-3R; 5'-ATAGGCATCACCCTCATCCACA-3'
SNRPN-4F; 5'-AGTTACTGTGGATGAGGGTGATGC-3'
SNRPN-4R; 5'-CAACTCAAATATGCAACCTCCTGCC-3'
IGF2-1F; 5'-CCAAAGTCCCGCTAAGATTCTCCA-3'
IGF2-1R; 5'-GCAAAGATGATCCCTAGGTGTGCT-3'
H19-1F; 5'-AGCTAGAGGAACCAGACCTCATCA-3'
H19-1R; 5'-ATGGAATGCTTGAAGGTTGCCC-3'
GAPD-1F; 5'-GGGACTGGCTTTCCCATAATTTCC-3'
GAPD-1R; 5'-GCCACATACCAGGAAATGAGCTTG-3'
GAPD-2F; 5'-ATCACTGCCACCCAGAAGACT-3'
GAPD-2R; 5'-CACCCTGTTGCTGTAGCCAAATTC-3'
CTCF6-F; 5'-TAGGACATTCATGGGAGCCACATC-3'
CTCF6-R; 5'-TCCTGGACCAGAGAATAAAGCAGC-3'
CTCF6-SNP-F; 5'-CACGTGTATTTCTAGAGGCTTCCC-3'
CTCF6-SNP-R; 5'-GTTAATGTCTGGCCACTTAGGGCT-3'
SNRPN-DMR-F; 5'-CCATTGATCCCAGGTTGCTTATGG-3'
SNRPN-DMR-R; 5'-TGAACATTCCGGATCTGGTTCTCC-3'
PCR reactions were carried out in a 20 µl volume containing 2.5 mM MgCl2, 2 mM dNTP mix, 0.4 µM each primer, 40 ng template DNA and 1 U of AccuSure DNA polymerase (Bioline, Randolph, MA). PCR reactions for CTCF6-SNP-F/R were carried out using 0.2 µM each primer, 250 ng of template gDNA and 45 µl of Platinum PCR Supermix High Fidelity (Invitrogen, Carlsbad, CA) containing a final concentration of 2.16 mM MgSO4, 0.198 mM dNTPs. PCR conditions were as follows (denaturation/annealing/extension): for NDN-1F/R, 35cycles 94/60/68°C for 30/60/45 s; for NDN-2F/R, 35 cycles 94/60/68°C for 30/60/45 s; for SNRPN-1F/R, 35 cycles 94/65/68°C for 30/60/45 s; for SNRPN-2F/R, 35 cycles 94/65/68°C for 30/60/45 s; for SNRPN-3F/R, 35 cycles 94/65/68°C for 30/60/45 s; for SNRPN-4F/R, 35 cycles 94/65/68°C for 30/60/45 s; for IGF2-1F/R, 35 cycles 94/65/68°C for 30/60/45 s; for H19-1F/R, 35 cycles 95/62/68°C for 90/60/45 s; for GAPD-1F/1R, 35cycles 94/65/68°C for 30/60/45 s, for CTCF6-SNP-F/R, 35 cycles of 94/58.9/68°C for 30/60/60 s; for SNRPN-DMR–F/R, 35 cycles of 94/61/68°C for 30/60/45 s. Amplicons were electrophoresed through 1.6% TBE agarose gels stained with ethidium bromide and visualized on a UV transilluminator.
Sequence analysis
PCR products were purified (QIAquick Gel Extraction Kit, QIAGEN) and sequenced with an ABI 3100 capillary genetic analyzer (Applied Biosystems, Foster City, CA) using BigDye terminator sequencing chemistry (Wen, 2001). Sequencing results were analysed using software Sequencher (Gene Codes Corporation, Ann Arbor, MI).
Restriction enzyme digestion
To create restriction fragment length polymorphisms (RFLP), 5 µl of PCR products were digested in a 20 µl volume with 10 IU of restriction enzyme at 37°C for 16 h and digested samples were electrophoresed through agarose gels, stained with ethidium bromide and visualized on a UV transilluminator. Restriction enzymes were selected based on specificity using a Webcutter 2.0 RFLP (online software from Yale University http://www.firstmarket.com/cutter/cut2.html). XbaI and AvaI were appropriate for NDN SNP-1 and H19 SNP-3, respectively.
Allele frequency
Frequency was determined by the formula; 2xthe number of animals homozygous for the allele+the number of heterozygous animals divided by 2x the number of animals tested (Zhang et al., 2004).
Total RNA extraction from muscle biopsy tissues and RT–PCR
Monkeys were anesthetized with Propofol and approximately 20 mg of muscle tissue was removed from the quadriceps femoris using a 16-gauge biopsy needle (Jorgensen Laboratory Inc., Loveland, CO).
Total RNA was isolated by using TRIzol® (Invitrogen) according to the manufacturer's protocol. To eliminate DNA contamination, samples were treated with preamplification-grade DNase I (Invitrogen). RT was performed with a Superscript III system (Invitrogen) and PCR reactions were done for each sample with and without (negative control) RT. PCR protocols were the same as those described above for genomic DNA in NDN, H19, SNRPN and IGF2. Regarding the GAPD (glyceraldehyde-3-phosphate dehydrogenase) gene, exon primers GAPD-2F/2R were used to amplify a polymorphic region in the amplicon with intron primers, GAPD-1F/1R. PCR conditions were 35 cycles of 94/60/68°C for 30/60/45 s.
Amplicons were sequenced as described above for genomic DNA samples. Expressed alleles were determined from sequence analysis of the amplicons generated from genomic DNA of both parents and their infants and complimentary DNA from skeletal muscle tissue.
Bisulphite modification of DNA and methylation-specific PCR
Genomic DNA was extracted from muscle biopsy samples using a DNeasy Tissue Kit (QIAGEN) and approximately 2 µg of gDNA was modified by busulphite treatment using a CpG Genome Modification Kit (Chemicon International, Temecula, CA) according to the manufacturer's protocols. Primers for methylation-specific PCR (MSP) were designed using online software (http://www.urogene.org/methprimer/) as described elsewhere (Li and Dahiya, 2002). MSP primers were:
Bi-H19-DMR methyl F; 5'-GTATAAGAATTCGGAGATTTTTGC-3'
Bi-H19-DMR methyl R; 5'-GTAAACCCTACGACACCTAACGTA-3'
Bi-H19-DMR unmethyl F; 5'-TATAAGAATTTGGAGATTTTTGTGT-3'
Bi-H19-DMR unmethyl R; 5'-CATAAACCCTACAACACCTAACATA-3'
SNRPN bi methyl F; 5'-GGT ATT GGG ATT TTT GTA TTG CGG-3'
SNRPN bi methyl R; 5'-ACG CAA CTA ACC TTA CTC GC-3'
SNRPN bi unmethyl F; 5'-GGT ATT GGG ATT TTT GTA TTG TG-3'
SNRPN bi unmethyl R; 5'-ACA CAA CTA ACC TTA CTC ACT C-3'
PCR reactions were carried out in a 50 µl volume containing 1.5 mM MgCl2, 0.2 mM each dNTP, 0.25 µM each primer, 5 µl modified DNA and 2.5 U of Platinum Taq DNA Polymerase, (Invitrogen). PCR conditions were; for Bi-H19-DMR methyl F/R and Bi-H19-DMR unmethyl F/R, 35 cycles of 94/51/72°C for 30/30/45 s; for SNRPN bi methyl F/R, 35 cycles of 94/53/72°C for 30/30/45 s; for SNRPN bi unmethyl F/R, 35 cycles of 94/49/72°C for 30/30/45 s. Amplicons were electrophoresed through 1.6% TBE agarose gels stained with ethidium bromide and visualized on a UV transilluminator.
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Results |
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Detection of single nucleotide polymorphisms in NDN, SNRPN, IGF2, H19 and GAPD
In order to define SNPs in rhesus monkeys that could be exploited in allele-specific expression studies, we first selected appropriate primers based on consensus human DNA sequences. These primers were used to amplify, by PCR, the sequence of interest in genomic DNA isolated from peripheral blood leukocytes. PCR products were then purified and sequenced for individual animals. In this manner, we discovered 2 SNPs in NDN, 3 in H19, 13 in SNRPN, 4 in IGF2, and 1 in GAPD, a non-imprinted gene. The GenBank accession numbers for these monkey sequences along with sites and base substitutions for all SNPs are described in Table I. The percent homology of these monkey amplicons to the human sequence varied from 90.4 to 97.1%.
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In the case of NDN, overlapping PCR amplicons covering 914 bp that were 564 and 361 bp in length, respectively, resulted from the use of two primer pairs, 1F/1R and 2F/2R. This region contained two novel SNP sites, a G to A polymorphism at nucleotide position 135 and a T to C transition at position 795 in exon 1 (Table I).
Using primer pair H19-1F/1R, a PCR amplicon consisting of 525 bp was sequenced with the identification of three SNPs; a G to A polymorphism at position 358; a G to A transition at 439 and a T to C transition at 457 in exon 5.
The primer pair, SNRPN-1F/1R, amplified a 338 bp region containing one SNP, a G to A transition at position 132 while SNRPN-2F/2R amplified a 273 bp region containing T to C polymorphisms at positions 83 and 95 (including exon 8 in the human). Overlapping PCR amplicons covering 1042 bp resulted from the use of SNRPN-3F/3R and SNRPN-4F/4R which were 564 and 505 bp in length, respectively. Nine SNPs were identified in this sequence (Table I).
With IGF2, no evidence of a monkey SNP in the polymorphic region in exon 9 identified in Figure 1 in the human was obtained. However, the primer pair IGF2-1F/1R amplified a 471 bp sequence containing four polymorphic sites in exon 9, downstream of a CA repeat region. These sites included T to C polymorphisms at 169, 286 and 405 bp and an A to C conversion at position 371.
As a non-imprinted gene control, we used GAPD. GAPD-1F and GAPD-1R amplified a 517 bp product putatively covering the region from intron 7 to the exon 8/intron 8 boundary. A single SNP was identified as a G to A polymorphism at position 358.
Distribution of SNPs in Indian-derived versus Chinese-origin animals
Genomic DNA from up to 93 animals was screened depending on the SNP under study and the results have been summarized as a function of animal origin. Substrain differences in tested animals were seen in several SNPs (Tables II and III). For SNP NDN-1, the majority of animals tested were homozygous, either G/G (45) or A/A (7), the former presumably reflecting the wild-type. In 23 of the 75 animals, a G/A heterologous condition existed. This polymorphism showed a significant difference in allele frequency by Chi-square analysis dependent on the animal's country of origin; it was only found in Indian-origin animals. Similarly, for SNPs in H19, only Indian-origin animals showed the polymorphisms. Although SNPs 1-13 in the SNRPN tended to be expressed most often in Chinese-origin animals, allele frequency for the non-dominant allele was low. There were no examples of SNPs in IGF2 that were significantly different as a function of the substrain, although the number of tested animals was low. The SNP identified in GADP was defined in 21 animals and found to be present in both Indian- and Chinese-origin monkeys with an overall genotype distribution of 42.9/28.6/28.6 for A/A, G/G and A/G, respectively. An allele frequency of 57% A versus 43% G was statistically indistinguishable from the 50:50 ratio expected for a non-imprinted gene. The results in Tables II and III provide valuable insights required for the selection of animals to be used in allele-specific expression studies.
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The imprint status of NDN, H19, IGF2 and SNRPN in monkeys
The genes selected for study, NDN, H19, IGF2 and SNRPN are known to be imprinted in mice, cattle and humans (Bartolomei et al., 1991
; DeChiara et al., 1991; Leff et al., 1992; Giannoukakis et al., 1993; Zhang et al., 1993; Reed and Leff 1994; MacDonald and Wevrick, 1997; Zhang et al., 2004). Preliminary results from gene expression profiling of primate parthenogenetic stem cells suggest that at least NDN and SNRPN as paternally expressed genes are also imprinted in the cynomolgus macaque (Hipp et al., 2004). We examined the status of these genes in rhesus monkey somatic (skeletal muscle) tissues with the expectation that if they were imprinted, we would see differential expression of the two alleles in somatic cells. In four infants produced by natural mating, heterozygosity in their genomic DNA samples for the SNPs listed in Table I were found; two for NDN, one for H19, two for SNRPN, three for IGF2, and two for GAPD.
Using the primers NDN-1F/R, PCR and direct sequence analysis of the resulting amplicon, we confirmed G/A heterozygosity in female 24063. The cDNA isolated from muscle in this animal and amplified using the same primer set showed expression of only the G allele, the outcome expected for an imprinted gene (Figure 2A).
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Similarly, female, 24073, was heterozygous (C/T) for H19-SNP-3 and cDNA analysis from the muscle tissue biopsy showed expression of only the T allele (Figure 2D).
Female, 23985, was heterozygous (A/G) for both SNRPN-SNP-10 and IGF2-SNP-2 (C/T). The cDNA from her muscle cells expressed only the A and T alleles, respectively, again indicating that these two genes are imprinted in the monkey (Figure 2B and C).
Finally, female, 24063, who was also heterozygous (G/A) for GAPD-SNP-1, showed evidence of biallelic expression of this non-imprinted gene in the cDNA from her muscle (Figure 2E).
Expressed alleles examined in other informative animals showed the expected monoallelic expression of all four imprinted genes and biallelic expression of GAPD (Table IV). These results strongly suggest that these four genes are imprinted in muscle cells of monkeys, although it was not possible to determine which parental allele was expressed without characterizing both parents.
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Analysis of allele-specific expression in ICSI produced monkeys
We began to address the potential impact of oocyte/embryo manipulation on imprinting in the monkey by examining allele-specific expression in somatic cells. This undertaking is especially relevant to H19 that has been shown to be unstable in mice (Sasaki et al., 1995
; Dean et al., 1998; Doherty et al., 2000; Khosla et al., 2001; Mann et al., 2003) and cattle (Zhang et al., 2004). Genomic DNA was isolated from the blood cells of gamete donors for ICSI produced infants (Wolf et al., 2004b) and was screened for the SNPs in Tables I–III. Based on parental analysis, five ICSI-infants were selected and genomic DNA from their blood samples was screened for the presence of heterozygosity in possibly informative genes. As screening primers, we used NDN-1F/R, SNRPN-4F/R, IGF2-1F/R, H19-1F/R and GAPD-1F/R. With NDN and SNRPN, we selected primers based on the availability of RFLP (NDN-SNP-1) and the frequency of the SNP (SNRPN-SNP-5-13). In five ICSI produced monkeys, allele-specific expression analysis was possible; two for NDN, four for SNRPN, two for IGF2, four for H19 and four for GAPD.
Analysis of genomic DNA from female, 24089, and her parents indicated that she was informative for allele-specific expression of all four imprinted genes. Direct sequence analysis of the amplicon produced with NDN-1F/1R identified this female as heterozygous for the G to A polymorphism located at 135 bp (NDN-SNP-1), while the paternal and the maternal genomic DNA samples were homozygous for G and A, respectively, at the same polymorphic site. Examination of the expressed allele in this infant after RT–PCR using NDN-1F/R of an RNA preparation from skeletal muscle tissue and direct sequencing of the cDNA produced, indicated that only the paternal, G allele, was expressed (Figure 2F).
Using the SNRPN-4F/R primer pair for PCR and sequence analysis on genomic DNA, 24089 was heterozygous (G/A) at nucleotide 775 (SNRPN-SNP-10) while her mother and father were homozygous (G/G) and heterozygous (G/A), respectively. This indicated that the G allele of this infant was inherited from her father while the A allele originated from her mother. Subsequent RT–PCR and direct sequence analysis of cDNA samples showed that this female expressed only her father's G allele (Figure 2G).
Analysis of the amplicons produced with primer pair IGF2-1F/R indicated that this female was also heterozygous (T/C) at position 286 (IGF2-SNP-2). The paternal and the maternal genomic DNAs were heterozygous (C/T) and homozygous (C/C), respectively. Processing RNA samples as described above resulted in the detection of only paternal expression in IGF2 (Figure 2H).
Genomic PCR with direct sequencing of the amplicons produced with H19-1F/R revealed that female 24089, was heterozygous (C/T) at nucleotide position 457 (H19-SNP-3) while the paternal and maternal genomic DNA samples were homozygous for the T and C allele, respectively. RT–PCR with direct sequence analysis indicated that only the C, maternal allele was expressed (Figure 2I). Expressed alleles were examined in the skeletal muscle biopsies in all other informative infants and the results were consistent with the parent-specific expression seen in female 24089 (Table IV).
In the case of the non-imprinted gene control, heterozygous ICSI produced infants were identified as the G/A polymorphism in GAPD at nucleotide position 358 and cDNA analysis showed the expected biallelic expression of this gene (results from male, 23280, are shown in Figure 2J).
SNPs and methylation status of H19/IGF2 and SNURF/SNRPN ICs
Imprinting of IGF2 and H19 is reciprocally controlled by a common IC located approximately 2 kb upstream of H19 and CpG islands within this IC are differentially methylated in mice and humans (Zhang et al., 1993; Hark et al., 2000; Schoenherr et al., 2003; Sparago et al., 2004). We amplified a region in rhesus monkey gDNA corresponding to the human CTCF-6 binding site upstream of H19 using primers (CTCF6-1F/R) designed for the human sequence (Genbank accession #AF087017; Figure 3). Subsequent sequencing of a 989 bp monkey amplicon revealed 89% homology to the human (Genbank accession #AY725988). The corresponding monkey region also included a CTCF motif of 48 bps identical to the human CTCF-6 binding site (Bell and Felsenfeld, 2000). This region consisted of two CpG islands comprised of 55 CpG dinucleotides compared to 46 in the human DMR (Figure 3). Subsequently, we designed a new set of primers (CTCF6-SNP-1F/R) based on the monkey consensus sequence and screened animals for the presence of SNPs in this region. Four distinct polymorphic sites were revealed at positions 485, 494, 668 and 712 in the resultant 900 bp amplicon (Table I).
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Using primers SNRPN-DMR-1F/R, we also amplified the imprinting centre within promoter/exon 1 of the SNURF/SNRPN gene associated with the cis-acting regulatory region for the Prader-Willi syndrome (Nicholls and Knepper, 2001
) (Figure 4). Sequence analysis of a 463 bp amplicon (GenBank accession for the monkey #AY725989) showed 95% homology to the human sequence. In the monkey, this region contained a CpG island comprised of 27 CpG sites compared to 24 in the human. Following sequence analysis of gDNA derived from several males and females, five SNPs at nucleotide positions 145, 222, 266, 320 and 323 were detected (Table I).
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To assess the methylation status of H19/IGF2 and SNURF/SNRPN ICs, we subjected gDNA isolated from the muscle tissue of natural and ICSI produced monkeys to bisulphite modification followed by methylation-specific PCR. For each of these regions, primer sets were designed that discriminated between methylated and unmethylated alleles. Primer sequences were chosen for regions containing at least two CpG dinucleotides to provide maximum MSP specificity. Both methylated and unmethylated primer sets, Bi-H19-DMR methyl F/R and Bi-H19-DMR unmethyl F/R, for H19/IGF2 IC were expected to amplify 220 bp fragments including the CTCF-6 site (Figure 3). The results shown in Figure 5A demonstrate that muscle gDNA isolated from two natural and two ICSI produced infants was amplified when both methylated (M) and unmethylated (U) primers were used suggesting that this region is differentially methylated in the monkey. Similarly, methylated (SNRPN bi methyl F/R) and unmethylated (SNRPN bi unmethyl F/R) primers for the SNURF/SNRPN IC were each designed to amplify the same 206 bp fragments containing a CpG island as shown in Figure 4. Gel electrophoretic analysis revealed that both methylated and unmethylated alleles were readily amplified in gDNA from naturally produced (23985) and ICSI produced (24089 and 23672) monkeys (Figure 5B).
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Discussion |
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We describe a number of SNPs in the rhesus monkey that will be useful to allele-specific expression studies in the embryos and ES cells from this primate. The selected genes, NDN, H19, SNRPN and IGF2 are known to be imprinted in mice and humans (Hanel and Wevrick, 2001
; Nicholls and Knepper, 2001; Weksberg et al., 2003; Lau et al., 2004) and expression analysis studies on a parthenote-derived ES cell line suggests that at least NDN and SNPRN are paternally expressed and therefore also imprinted in the cynomolgus macaque (Hipp et al., 2004). We also have preliminary results from rhesus monkey blastocysts supporting the paternal expression of SNRPN and IGF2, maternal expression of H19 and maternal or biallelic NDN expression (Fujimoto et al., 2004). To examine allele-specific expression in monkey tissues, we designed primers based on human sequences and successfully amplified polymorphic regions in the exons of five genes and two ICs. In 30 of 32 cases, the high homology between the monkey and human genomes supported adequate amplification. In the remaining two cases, the monkey amplicon was sequenced and used to refine the primer sequence. SNP frequency varied greatly, a factor that may be important to their utility.
Additionally in the present study, we examined the presence of SNPs in a region upstream of H19 postulated to be an imprinting centre that regulates H19 and IGF2 expression reciprocally. Methylation of this site on the paternal allele blocks the expression of the paternal copy of H19 (Weksberg et al., 2003), and also prevents the binding of the multivalent 11-zinc finger protein, CTCF, to CTCF-binding motifs. The absence of CTCF allows the paternal IGF2 promoter to utilize downstream enhancers. The maternal allele is normally unmethylated and able to bind the CTCF protein. Such CTCF-bound IC acts as an enhancer-blocking sequence (insulator) and prevents access of the IGF2 promoter to downstream enhancers, thus blocking the expression of the maternal IGF2 allele. While CTCF binding sequences in the IGF2/H19 IC are highly conserved between human and mouse, the human IC differs from homologous mouse region which is organized into two units, each consisting of two types of direct repeats, and contains seven potential CTCF binding sites compared to four in the mouse (Frevel et al., 1999; Bell and Felsenfeld, 2000; Sparago et al., 2004). However, only the sixth of the seven CTCF-binding sites has been demonstrated to be differentially methylated in normal tissue. Moreover loss of IGF2 and H19 imprinting in Wilms' tumour, bladder and colon cancers and osteosarcoma has been restricted to aberrant methylation of this region (Takai et al., 2001; Ulaner et al., 2003). The corresponding monkey region studied here showed 89% homology to the human, however, the CTCF-6 binding sites were identical. This together with evidence that the monkey region is also differentially methylated suggests that a similar mechanism regulates IGF2 and H19 expression in monkeys and humans. A second region was studied which has been associated with Prader-Willi syndrome in humans. In this case, the imprinting domain on human chromosome 15 is controlled by an IC, which has been mapped to the promoter/exon 1 region of SNURF/SNPRN (El-Maarri et al., 2001; Nicholls and Knepper, 2001). This IC is also subject to parent-specific epigenetic modification of DNA believed to be established during germ cell development; the maternal allele is marked by CpG methylation, while the paternal allele is unmethylated. Here we showed that the homologous region in monkeys contains a CpG island that is also differentially methylated. These SNPs will allow discrimination between maternal and paternal alleles in subsequent methylation studies.
Rhesus macaques are the most widely used non-human primate in biomedical research. This species is endemic to India and Southeast Asia (Viray et al., 2001). Within this wide distribution, subspecies are recognized with perhaps the most obvious, at least to biomedical research, being Indian-derived versus Chinese-origin animals with unique differences in morphometry (Clarke and O'Neil, 1999), immune systems (Viray et al., 2001) and SIV disease progression (Trichel et al., 2002). Polymorphisms (simple sequence repeat loci) have been useful in the evaluation of substrains including genetic diversity, pedigree establishment, phylogenetic relationships, behavioural ecology, social structure and paternity determination (Morin et al., 1997). In the present study we noted differences in the distribution and frequencies of the defined SNPs, some were only present in Indian-origin animals while others were unique to those of Chinese-origin. For example, NDN SNP-1 and H19 SNP-1,2,3 were found only in Indian-origin monkeys and all of the SNRPN SNPs except SNPs-2 and 10 were confined to Chinese-origin animals. We also noted that one homozygous population was often over-represented, presumably reflecting the wild-type and the infrequent occurrence of the SNP. This finding carries relevance to the feasibility of defining homozygous or informative parental combinations in anticipation of allele-specific expression analysis. Parenthetically, it would be interesting to examine how these SNP variants impact amino acid coding potential. Indeed this may represent an entirely new approach to identifying genetic models that could be pursued by generating heterozygous or homozygous embryos carrying SNPs with evaluation of their developmental capacity (J. Hennebold, personal communication).
All four genes were shown to be expressed mono-allelically in all informative monkeys examined and were expressed from the expected alleles in ICSI produced monkeys. This result indicates that these four genes are also imprinted in the rhesus macaque, and that our embryo manipulations did not change imprinting status, although further and more comprehensive examination will be necessary to draw final conclusions. Nevertheless, our findings will enable an extension of allele-specific expression analysis to in vitro cultured monkey embryos and ES cells.
In summary, we have identified for the first time in rhesus macaques, several SNPs in NDN, H19, SNRPN, IGF2 and two related ICs that will be useful for allele-specific expression and methylation studies in this primate. The SNPs described herein should prove useful for studies of the ontogeny of the imprinting mark in early primate development that can not be conducted in humans for ethical reasons as well as in evaluating the integrity of the mark in ES cells and their progeny.
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Appreciation is expressed to Drs James Byrne and Jon Hennebold for their critical review of this manuscript and to J. White for administrative support. This work was supported by National Institutes of Health grants RR00163 to D. Dorsa and U54 HD18185 to R. L. Stouffer and the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan to A. Fujimoto.
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Submitted on February 16, 2005; resubmitted on April 13, 2005; accepted on April 18, 2005.
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