Genomics 88 (2006) 650–658 www.elsevier.com/locate/ygeno

The human HYMAI/PLAGL1 differentially methylated region acts as an imprint control region in mice ⁎ Takahiro Arima a, , Katsuhisa Yamasaki b,c, Rosalind M. John d, Kiyoko Kato a, Kunihiko Sakumi c, Yusaku Nakabeppu c, Norio Wake a, Tomohiro Kono e

a Medical Institute of Bioregulation, Division of Molecular and Cell Therapeutics, Department of Molecular Genetics, Kyusyu University, 4546, Tsurumihara, Beppu, Oita 874-0838, Japan b Medical Institute of Bioregulation, Division of Neurofunctional Genomics, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan c Specified Research Promoting Group, Institute of Microbial Chemistry, 3-4-23 Kamiosaki, Shinagawa-ku, Tokyo 141-0021, Japan d Cardiff School of Biosciences, Museum Avenue, Cardiff CF10 3US, UK e Department of BioScience, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan Received 11 March 2006; accepted 10 July 2006 Available online 22 August 2006

Abstract

Imprinting centers (IC) can be defined as cis-elements that are recognized in the germ line and are epigenetically modified to bring about the full imprinting program in a somatic cell. Two paternally expressed human , HYMAI and PLAGL1 (LOT1/ZAC), are located within human 6q24. Within this region lies a 1-kb CpG island that is differentially methylated in somatic cells, unmethylated in sperm, and methylated in mature oocytes in mice, characteristic features of an IC. Loss of methylation of the homologous region in humans is observed in patients with transient neonatal diabetes mellitus and hypermethylation is associated with a variety of cancers, suggesting that this region regulates the expression of one or more key genes in this region involved in these diseases. We now report that a transgene carrying the human HYMAI/PLAGL1 DMR was methylated in the correct parent-origin-specific manner in mice and this was sufficient to confer imprinted expression from the transgene. Therefore, we propose that this DMR functions as the IC for the HYMAI/PLAGL1 domain. © 2006 Elsevier Inc. All rights reserved.

Keywords: Genomic imprinting; DNA methylation; Imprint control region; HYMAI/PLAGL1

Imprinting is a non-Mendelian form of inheritance in which appears to be mediated by imprinting centers (IC). IC are only one of the two inherited alleles is expressed. This is responsible for the establishment of differential imprinted dependent on differential epigenetic marking of the two genomic marks in the germ line, the maintenance of these parental alleles in the germ line [1–4]. The differential marks through development, and the ultimate execution of expression of imprinted genes accounts for the requirement differential expression programs for the two parental alleles for both maternal and paternal genomes in normal develop- [10]. The most likely candidate for the gametic mark that ment [5,6]. Many imprinted genes play significant roles in distinguishes the parental alleles is DNA methylation [1]. regulating embryo growth, placental function, and neurobeha- DNA methylation is a both heritable and reversible epigenetic vioral processes (summarized in http://www.mgu.har.mrc.ac. modification that is stably propagated after DNA replication uk/research/imprinting/function.html). Studies of a number of and influences expression [11]. Allele-specific DNA chromosomal domains have demonstrated that imprinted genes methylation has been observed within all well-characterized are predominantly located in clusters, where they exhibit a imprinted domains. In some instances, the methylation is complex sharing of control elements [7–9]. This regulation present at the promoter of the inactive gene, suggesting a direct role for DNA methylation in silencing of the gene. ⁎ Corresponding author. Fax: +81 957 271661. We previously identified the human imprinted region at E-mail address: [email protected] (T. Arima). chromosome 6q24, syntenic to mouse chromosome 10A, in a

0888-7543/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.ygeno.2006.07.005 T. Arima et al. / Genomics 88 (2006) 650–658 651 screen for imprinted genes [12,13]. Two paternally expressed island overlapping exon 1 of PLAGL1/HYMAI is differentially human genes are located in this domain, HYMAI and PLAGL1 methylated in somatic cells [17]. The region is unmethylated in (LOT1/ZAC) [12,14,15]. HYMAI generates an untranslated sperm but methylated in growing oocytes, a difference that mRNA of unknown function, while PLAGL1 encodes a zinc persists between parental alleles throughout pre-and postim- finger that localizes to the nuclear compartment and plantation development. A region within this differentially functions as a transcription factor [16]. We showed that a CpG methylated CpG island exhibits a high degree of homology

Fig. 1. Generation and methylation analysis of the human HYMAI/PLAGL1 DMR transgenic mice. (A) Structure of the transgene human HYMAI/PLAGL1 DMR (938 bp) region and pIRES-eGFP expression vector (5.3 kb) (Clontech). The human HYMAI/PLAGL1 DMR includes a promoter (at −477 on the transcriptional start site) and exon 1 of HYMAI. PCR primers for bisulfate-treated genomic DNA of the human DMR are indicated. 17 CpG sites within the human HYMAI/PLAGL1 DMR were analyzed (the vertical bars). The restriction sites are BamHI (B), XhoI (X), SmaI (S), and NotI (N). (B) Genomic DNA from the tails of F1 transgenic animals was subjected to Southern blotting. The DNA was digested with BamHI and XhoI and the methylation-sensitive restriction enzyme NotI and hybridized with a human DMR probe. Bottom: Methylation status of the human HYMAI/PLAGL1 DMR in lines 12 and 21 analyzed by bisulfite sequencing. Each row represents a unique methylation profile within the pool of clones sequenced. Each circle within a row represents a single CpG site (open circles, unmethylated cytosines; filled circles, methylated cytosines). (C) Genomic DNA from the tails of six F2 transgenic animals from line 68 (from maternal transmission) and seven F2 transgenic animals from line 12 (from paternal transmission) was subjected to Southern blotting. The DNA was digested as described for (B). (D) Southern analysis of DNA from F3 individuals inheriting the transgenes from either a male or a female from line 12 or 68. Both lines show the maternal-specific methylation and paternal-specific unmethylated patterns. C, control DNA (12-4-1) digested with BamHI and XhoI without the methylation-sensitive restriction enzyme NotI. Bottom: Analysis of the transgenic human HYMAI/PLAGL1 DMR by bisulfite PCR methylation assay in F3 embryos after paternal or maternal transmission for line 12. Individual sequenced clones are shown. Black circles indicate methylation; white circles represent unmethylated residues in individual clones. The sequenced clones after maternal transmission are methylated in the transgenic human DMR. In contrast, the clones after paternal transmission are almost all unmethylated. 652 T. Arima et al. / Genomics 88 (2006) 650–658

Fig. 1 (continued). between mouse and human [17,18]. We and others have Evidence supporting a role for this CpG island in recently shown that acquisition of oocyte methylation at the regulating gene expression comes from studies on human mouse Hymai/Plagl1 CpG island is completed in early growing cancers. Hypermethylation of this region leading to oocytes at the same time as the primary imprinting of Plagl1 transcriptional silencing of PLAGL1 has been reported in a expression [19,20]. These features led us to suggest that this variety of human cancers, including ovarian cancer [21,22]. CpG island marks the position of the IC for this domain. Furthermore, patients with transient neonatal diabetes T. Arima et al. / Genomics 88 (2006) 650–658 653 mellitus consistently show loss of methylation at this CpG appropriate mark, and 1 line demonstrated the unmethylated island [23]. pattern (Fig. 1B). These results were confirmed by bisulfite Previously, IC have been defined by genetic analysis using analysis. After bisulfite treatment of the DNA samples, the deletions to map the elements that may be necessary for this human HYMAI/PLAGL1 DMR was amplified by PCR, process or by using transgenes in mice to determine the minimal fragments were subcloned and sequenced, and methylation sequence information sufficient to carry out the full imprinting status was determined at the 17 CpG sites of the human HY- program [24–41]. There is evidence that an IC lies within a 175- MAI/PLAGL1 DMR. Results are shown in Fig. 1B and Table 1 kb region spanning the human PLAGL1/HYMAI locus since a and were consistent with the data obtained by Southern PAC clone carrying this sequence is apparently able to direct blotting. imprinted expression of PLAGL1 in mice [42]. In this study, we For an IC to function, not only must the epigenetic mark be examined the imprinting capabilities of a much smaller region, made in the appropriate germ line but also it must be erased the human HYMAI/PLAGL1 differentially methylated CpG so that the appropriate, sex-specific mark can be reestablished island, in transgenic mice. We found that the human HYMAI/ for the next generation and to switch back and forth from the PLAGL1 CpG island can acquire parental-origin-specific male to the female imprint. We therefore chose to examine the methylation in the maternal germ line and that this modulates ability of our transgene to switch imprint in line 68, whose expression of a linked reporter. This supports our hypothesis founder was originally male, and in line 12, whose founder that this CpG island is the IC for the HYMAI/PLAGL1 imprinted was female. For line 68, we found that the F1 female, who domain. had inherited an unmethylated DMR from her male parent, passed on a methylated DMR to all six of her offspring (Fig. Results 1C). Therefore in this line, the imprint switched from unmethylated (male) to methylated (female). For line 12, the Production of transgenic mice male F1 animal that had inherited a methylated DMR from his female parent passed on an unmethylated DMR to six of his To test whether the human HYMAI/PLAGL1 DMR might offspring, again showing appropriate switching. Lines 8, 21, be sufficient to promote a parent-origin-specific methylation 25, 29, 55, 62, 69, and 81 showed similar switching (data not pattern and imprinted expression in the mouse, we shown). constructed a human HYMAI/PLAGL1 DMR reporter Finally, we examined whether the methylation imprint transgene by cloning the DMR into the pIRES-eGFP vector persisted. We examined methylation after both paternal and (Fig. 1A). We generated 28 founder animals by pronuclear maternal transmission in the F2 generation by Southern injection, which were initially identified in a PCR screen blotting and bisulfite-modified genomic sequencing (Fig. (28/200). To determine the copy number of the integrated 1D). The appropriate switch was again observed. transgenes, we performed Southern blotting using the same amount of DNA in human placenta and compared it to the DNA methylation of the human HYMAI/PLAGL1 DMR density of the band using the densitometer (Table 1). We modulates expression of the linked reporter then crossed transgenic founders with the wild-type C57BL/ 6J mice to establish independent lines. We obtained germ- DNA methylation influences gene expression. In some line transmission of the transgene in 13 independent lines, 6 instances, the methylation is present on the inactive gene, lines from male founders and 7 lines from female founders. suggesting a role for DNA methylation in silencing of the We also made transgenic mice with only the pIRES-eGFP vector as a control. Table 1 Methylation profiles of human HYMAI/PLAGL1 DMR F1 transgenic mice Allele-specific methylation of the human HYMAI/PLAGL1 Line Copy number % methylation DMR in transgenic mice Female founder 8 6 92.7 12 22 89.4 The endogenous human HYMAI/PLAGL1 DMR has a 17 20 63.5 maternal-origin-specific methylation pattern [17,19]. We exam- 25 18 83.5 ined the methylation pattern of the transgene inherited from the 49 20 56.5 29 16 87.1 male and female founders in F1 individuals. Tail DNA from 14 5 21.2 one transgenic F1 animal was analyzed for each line. DNA was Male founder 21 6 15.3 digested with XhoI and BamHI and the methylation-sensitive 55 4 15.9 restriction enzymes NotI and/or SmaI. We performed Southern 62 18 20.0 blot using the human HYMAI/PLAGL1 DMR probe (shown in 81 16 13.5 68 6 13.5 Fig. 1A). We found that the six lines for which the founder was 69 3 11.6 male showed the unmethylated pattern as would be predicted The copy number and methylation pattern of the human HYMAI/PLAGL1 DMR for paternal inheritance of this DMR. Six of the lines for which transgene for paternal and maternal transmission from founder animals are the founder was female showed either full or partial methyla- shown. % methylation of the human HYMAI/PLAGL1 DMR was obtained by tion, again consistent with this region acquiring the germ-line- bisulfite-PCR sequencing. 654 T. Arima et al. / Genomics 88 (2006) 650–658 gene. We therefore examined whether the methylation status of demonstrating that the imprint-linked expression was preserved the transgene had an affect on eGFP reporter gene expression. into adulthood and in extraembryonic tissues (Fig. 2B). We performed Northern blotting of the mRNA from F3 neonate liver after male or female transmission for line 12 and Silencing of the HYMAI/PLAGL1 DMR transgene in the line 68 (Fig. 2A). For line 12, we found that the eGFP mRNA nongrowing oocytes was expressed more highly after paternal transmission (unmethylated transgene) than after maternal transmission The establishment of methylation imprints proceeds in a (methylated transgenes). Some expression from the methylated gene-specific manner while oocytes are arrested at prophase I transgene was detectable, which suggests that the DNA and transitioned from primordial to antral follicle [43–45].We methylation was not able to suppress all transcription previously reported that the acquisition of the methylation completely. This may be because the hybrid sequence is not imprint of the mouse Hymai/Plagl1 DMR begins around 5 days able to respond exactly as an imprinted gene or perhaps be due postpartum (dpp) in the early nongrowing oocyte and the to copy number. Nonetheless, there is still a distinct difference primary imprinting of Plagl1 is completed during the 5-to 10- in the level of expression after maternal and paternal dpp oocyte growth phase [20]. We examined whether imprint- transmission suggesting that this DNA region can act to ing of our transgene followed this time scale. eGFP expression regulate allele-specific gene expression. A similar pattern was was determined in 5-, 10-, and 15-dpp ovaries from juvenile obtained for line 68 (Fig. 2A). mice using immunofluorescence microscopy (Fig. 3). Almost Next, we analyzed whether appropriate allele-specific all oocytes from the primordial follicle in 5-dpp ovaries showed expression from the transgene was also present in the placenta strong expression of eGFP (percentage of highly expressed and whether it persisted in adult tissues. Expression of eGFP oocytes, 82.8%, 92/111 (n=5)). However, the 10-dpp early was detected strongly in brain, kidney, and placenta after male growing oocytes showed a mixture of high expression (45.7%, transmission (Fig. 2B), with weaker expression in the liver, 32/70 (n=5)) and low expression (38.5%, 27/70 (n=5)). lung, and spleen. After female transmission, a similar but much Almost all of the germ cells from 15-dpp oocytes derived lower level of expression was observed in these tissues, from early antral follicles showed no expression of eGFP

Fig. 2. Methylation of the transgenic human HYMAI/PLAGL1 DMR suppresses expression of a linked reporter. (A) Northern bolt analysis of RNA isolated from F3 neonate liver carrying the maternally inherited (lanes 12-1-1, 12-1-2, 12-1-4) and paternally inherited (lanes 12-4-1, 12-4-2, 12-4-3) transgene hybridized sequentially with eGFP and Gapdh cDNAs. There is high expression of eGFP after paternal transmission but not after maternal transmission. The relative intensity (eGFP)is shown under the lanes (eGFP/Gapdh ratio). (B) eGFP expression in adult brain, liver, lung, spleen, placenta, and kidney. Maternally inherited transgene (left) and paternal transgene (right). Gene expression was quantified on the BAS 2000 system (Fuji Film) and normalized for the total RNA by rehybridizing with Gapdh. T. Arima et al. / Genomics 88 (2006) 650–658 655

Fig. 3. The timing of imprinting of the human HYMAI/PLAGL1 DMR during postnatal oocyte growth. eGFP expression was examined by immunofluorescence microscopy analysis in oocytes at different stages of development. Scale bar, 300 μm. (A) Low-(left image) and high-(middle image) magnification H+E section of 5-dpp ovary is shown with black arrow indicating sample primordial follicle stage oocyte. Image on right shows eGFP signal obtained from this follicle. (B) Low- (left image) and high-(middle image) magnification H+E section of 10-dpp ovary in which there is a mixture of oocytes with low expression of eGFP (shown in right-hand image, top row) and high expression (right-hand image, bottom row). (C) Low-(left image) and high-(middle image) magnification H+E section of 15- dpp ovary. Early antral follicle stage oocyte (right image) shows low expression of eGFP.

(12.2%, 4/33 (n=5)). These data suggest that the human DMR DNA methylation is able, at least in part, to silence expression acquires the maternal silencing imprint during the 5-to 10-dpp of a linked reporter gene on our construct, suggesting that this oocyte growth stage in a fashion similar to that of the mouse sequence forms part of the IC from the human HYMAI/PLAGL1 DMR. imprinted domain. In conclusion, we have shown that a 936-bp region of the human PLAGL1 DMR can be recognized by the imprinting Discussion machinery of the mouse germ line and correctly methylated in the female germ line at the time point at which the endogenous Despite the apparent conservation of imprinting among mouse DMR is known to acquire an imprint. Furthermore, the mammals, there are few examples of human transgenes 656 T. Arima et al. / Genomics 88 (2006) 650–658 imprinting appropriately in mice [38,39,46–48]. Furthermore, base by base to identify the sequence motif recognized in the there are few data available to show that small regions can female germ line that establishes DNA methylation or, reliably direct imprinting on transgenes, thus making the precise conversely, a sequence motif that blocks DNA methylation dissection of these regions quite challenging. Here we have in the male germ line. shown that a small (936 bp) region from the human HYMAI/ PLAGL1 DMR can acquire the appropriate maternal-specific Materials and methods methylation in the mouse germ line and that this robust methylation imprint can switch back and forth between the Production and analysis of human HYMAI/PLAGL1 DMR transgenic germ lines for three generations (Fig. 4). This region was mice previously shown to contain maternal-specific methylation in humans [12,14] as well as in mice [17]. This demonstrates first The eGFP-tagged HYMAI/PLAGL1 DMR mammalian expression vector was constructed by cloning a 936-bp fragment spanning the human HYMAI/ that, at least for this DMR, there is no species barrier between PLAGL1 DMR into the XhoI and BamHI sites upstream of the IRES of the human and mouse in the imprinting mechanism. In contrast, pIRES-eGFP vector (Clontech, Palo Alto, CA, USA). DNAwas purified using a imprint acquisition of human H19 DMR does not appear to be midiprep kit (Qiagen, Tokyo, Japan) and linearized by XhoI digestion prior to conserved in transgenic mice [38]. Although we cannot exclude microinjection into F1 (C57BL/6J×CBA) embryos. Embryos were then the possibility that the human DMR acts only in the context of transferred to C57BL/6J pseudopregnant females, all according to standard procedures [49]. Genomic DNA was isolated from the tail biopsy of newborn the IRES-eGFP transgene, this work provides further evidence mice as described previously [14]. The transgenic event was initially examined that this region is the major IC for the HYMAI/PLAGL1 domain. using two kinds of PCR to detect the human HYMAI/PLAGL1 DMR and the The basic principles that govern the establishment and eGFP fragment, respectively. Primers used for the HYMAI/PLAGL1 DMR maintenance of domain-wide imprinted gene expression were 5″-AGTATGTCTGATACAGTCTG-3″ and 5″-TTTGCGCGCCGCC- ″ ″ ″ patterns have been elucidated in a general manner and seem TACGTG-3 and for eGFP were 5 -CGCACCATCTTCTTCAAGGACG-3 and 5″-TGCTCAGGTAGTGGTTGTCG-3′. The amplification consisted of a to be mediated at the molecular level by DMRs. The order of total of 30 cycles at 95°C for 30 s, 65°C for 30 s, and 72°C for 30 s in a Perkin– events and all the players are still to be elucidated. Our Elmer GeneAmp PCR System 2400. After PCR screening, we performed findings will make this process easier as we can focus all our Southern blotting to determine the copy number of the transgenic integrations by efforts on a much smaller region, identifying that comparison with a known quantity of human placental genomic DNA. Genomic μ bind to this sequence in extracts from different material, such DNAs (5 g) were digested with XhoI and BamHI. Filters were probed with a [32P]dCTP-labeled 936-bp HYMAI/PLAGL1 DMR fragment. Hybridization as mouse oocytes, at different stages of development and signals were analyzed using a Fuji Phosphoimager and on Kodak film (Tokyo, establishment of the imprint. Having defined an IC within a Japan). Twenty-eight transgenic animals were identified. Subsequently, 936-bp sequence, we can now proceed to analyze this region founders were crossed with wild-type C57BL/6J mice to generate 13

Fig. 4. Family tree of transgenic lines showing switch from maternal to paternal to maternal imprint in line 12 and from paternal to maternal to paternal in line 68. T. Arima et al. / Genomics 88 (2006) 650–658 657 independent transgenic lines. All further analysis was performed on the C57BL/ origin effects in humans and animals, Hum. Mol. Genet. 10 (1998) 6J strain background. 1599–1609. [9] R.A. Drewell, J.D. Brenton, J.F.-K. Ainscough, S.C. Barton, K.J. Hilton, Methylation analysis K.L. Arney, L. Dandolo, M.A. Surani, Deletion of a silencer element disrupts H19 imprinting independently of a DNA methylation epigenetic – Genomic DNAs of the transgenic mice were digested with XhoI and BamHI switch, Development 127 (2000) 3419 3428. and a methylation-sensitive endonuclease, NotI and/or SmaI, followed by [10] I. Ben-Porath, H. Cedar, Imprinting: focusing on the center, Curr. Opin. – electrophoresis on 1% agarose gels. Southern blots were performed using a 936- Genet. Dev. 10 (2000) 550 554. bp fragment from the human HYMAI/PLAGL1 DMR. [11] E. Li, Chromatin modification and epigenetic reprogramming in – Bisulfite treatment was carried using the EZ DNA methylation kit (Zymo mammalian development, Nat. Rev. Genet. 6 (2002) 662 673. Research, CA, USA) according to the manufacturer's instructions. Different [12] M. Kamiya, H. Judson, Y. Okazaki, M. Kusakabe, M. Muramatsu, S. samples of bisulfite-treated DNA were amplified by single PCR of the human Takada, N. Takagi, T. Arima, N. Wake, K. Kamimura, K. Satomura, R. DMR. The primer sequences and PCR conditions for the HYMAI/PLAGL1 Hermann, D.T. Bonthron, Y. Hayashizaki, The cell cycle control gene — DMR were as described previously [19]. The amplified fragments were cloned ZAC/PLAGL1 is imprinted A strong candidate gene for transient – into the TOPO TA vector (Invitrogen, Tokyo, Japan) and individual clones were neonatal diabetes, Hum. Mol. Genet. 9 (2000) 453 460. sequenced using a T7 primer and/or M13 reverse primer. [13] A. El Kharroubi, G. Piras, C.L. Stewart, DNA demethylation reactivates a subset of imprinted genes in uniparental mouse embryonic fibroblasts, J. Biol. Chem. 276 (2001) 8674–8680. Northern blot analysis [14] T. Arima, R.A. Drewell, M. Oshimura, N. Wake, M.A. Surani, A novel imprinted gene, HYMAI, is located within an imprinted domain on human Total RNA was isolated from neonate liver and adult organs using Isogen containing ZAC, Genomics 67 (2000) 248–255. (Nippon Gene, Tokyo, Japan). Northern blot analysis was carried out as [15] G. Piras, A. El Kharroubi, S. Kozlov, D. Escalante-Alcalde, L. – previously described [17]. eGFP cDNA (321 bp; pIRES2-eGFP vector 1542 Hernandez, N.G. Copeland, D.J. Gilbert, N.A. Jenkins, C.L. Stewart, 1863) was used as a probe. Gene expression was quantified on the BAS 2000 Zac1 (Lot1), a potential tumor suppressor gene, and the gene for epsilon- system (Fuji Film) and normalized for total RNA by rehybridizing with Gapdh sarcoglycan are maternally imprinted genes identified by a subtractive (Clontech). The relative intensity of eGFP is presented as an eGFP/Gapdh ratio. screen of novel uniparental fibroblast lines, Mol. Cell. Biol. 20 (2000) 3308–3315. GFP analysis [16] D. Spengler, M. Villalba, A. Hoffmann, C. Pantaloni, S. Houssami, J. Bockaert, L. Journot, Regulation of apoptosis and cell cycle arrest by Zac1, Sections were observed by immunofluorescence microscopy (LSM510- a novel zinc finger protein expressed in the pituitary gland and the brain, META, Zeiss, Germany, and SZY9, Olympus, Japan). The ovaries of F3 EMBO J. 16 (1997) 2814–2825. transgenic mice at 5, 10, and 15 dpp were used in the analysis of eGFP expression [17] T. Arima, R.A. Drewell, K.L. Arney, J. Inoue, Y. Makita, A. Hata, M. in their oocytes. Oshimura, N. Wake, M.A. Surani, A conserved imprinting control region at the HYMAI/ZAC domain is implicated in transient neonatal diabetes mellitus, Hum. Mol. Genet. 10 (2001) 1475–1483. Acknowledgments [18] A. Varrault, B. Bilanges, D.J. Mackay, E. Basyuk, B. Ahr, C. Fernandez, D.O. Robinson, J. Bockaert, L. Journot, Characterization of the We thank Y. Yoshikawa and T. Nakashima for technical methylation-sensitive promoter of the imprinted ZAC gene supports its role in transient neonatal diabetes mellitus, J. Biol. Chem. 22 (2001) assistance and the company Macrogen, Inc. (Seoul, Korea), for 18653–18656. making transgenic mice by the method of microinjection, and [19] R.J. Smith, P. Arnaud, G. Konfortova, W.L. Dean, C.V. Beechey, G. all our lab members for their support and valuable suggestions. Kelsey, The mouse Zac1 locus: basis for imprinting and comparison with This work was supported by a grant from the Ministry of Health human ZAC, Gene 292 (2002) 101–112. and Welfare of Japan. [20] T. Arima, N. Wake, Establishment of the primary imprint of the HYMAI/ PLAGL1 imprint control region during oogenesis, Cytogenet. Genome Res. 113 (2006) 247–252. [21] A. Abdollahi, D. Pisarcik, D. Roberts, J. Weinstein, P. Cairns, T.C. References Hamilton, LOT1 (PLAGL1/ZAC1), the candidate tumor suppressor gene at chromosome 6q24–25, is epigenetically regulated in cancer, J. Biol. [1] M.A. Surani, Imprinting and the initiation of gene silencing in the germ Chem. 278 (2003) 6041–6049. line, Cell 93 (1998) 309–312. [22] T. Kamikihara, T. Arima, K. Kato, T. Matsuda, H. Kato, T. Douchi, Y. [2] W. Reik, J. Walter, Imprinting mechanisms in mammals, Curr. Opin. Nagata, M. Nakao, N. Wake, Epigenetic silencing of the imprinted gene Genet. Dev. 8 (1998) 154–164. ZAC by DNA methylation is an early event in the progression of human [3] R. Ohlsson, B. Tycko, C. Sapienza, Monoallelic expression: ‘there can ovarian cancer, Int. J. Cancer 115 (2005) 690–700. only be one’, Trends Genet. 14 (1998) 435–438. [23] I.K. Temple, J.P. Shield, Transient neonatal diabetes, a disorder of [4] S.M. Tilghman, The sins of the fathers and mothers: genomic imprinting in imprinting, J. Med. Genet. 12 (2002) 872–875. mammalian development, Cell 96 (1999) 185–193. [24] A. Wutz, O.W. Smrzka, N. Schweifer, K. Schellander, E.F. Wagner, D.P. [5] J. McGrath, D. Solter, Completion of mouse embryogenesis requires both Barlow, Imprinted expression of the Igf2r gene depends on an intronic the maternal and paternal genomes, Cell 37 (1984) 179–183. CpG island, Nature 389 (1997) 745–749. [6] M.A. Surani, S.C. Barton, M.L. Norris, Development of reconstituted [25] P.A. Leighton, R.S. Ingram, J. Eggenschwiler, A. Efstratiadis, S.M. mouse eggs suggests imprinting of the genome during gametogenesis, Tilghman, Disruption of imprinting caused by deletion of the H19 gene Nature 308 (1984) 548–550. region in mice, Nature 375 (1995) 34–39. [7] T. Moore, M. Constancia, M. Zubair, B. Bailleul, R. Feil, H. Sasaki, [26] P.A. Leighton, J.R. Saam, R.S. Ingram, C.L. Stewart, S.M. Tilghman, An W. Reik, Multiple imprinted sense and antisense transcripts, differential enhancer deletion affects both H19 and Igf2 expression, Genes Dev. 9 methylation and tandem repeats in a putative imprinting control region (1995) 2079–2089. upstream of mouse Igf2, Proc. Natl. Acad. Sci. USA 23 (1997) [27] J.L. Thorvaldsen, K.L. Duran, M.S. Bartolomei, Deletion of the H19 12509–12514. differentially methylated domain results in loss of imprinted expression of [8] I.M. Morison, A.E. Reeve, A catalogue of imprinted genes and parent-of- H19 and Igf2, Genes Dev. 12 (1998) 3693–3702. 658 T. Arima et al. / Genomics 88 (2006) 650–658

[28] R.D. Nicholls, S. Saitoh, B. Horsthemke, Imprinting in Prader–Willi and impaired paternal-specific imprint acquisition and maintenance in mice, Angelman syndromes, Trends Genet. 14 (1998) 194–200. Hum. Mol. Genet. 11 (2002) 411–418. [29] M.R. Mann, M.S. Bartolomei, Towards a molecular understanding of [39] R. Shemer, A.Y. Hershko, J. Perk, R. Mostoslavsky, B. Tsuberi, H. Cedar, Prader–Willi and Angelman syndromes, Hum. Mol. Genet. 8 (1999) K. Buiting, A. Razin, The imprinting box of the Prader–Willi/Angelman 1867–1873. syndrome domain, Nat. Genet. 26 (2000) 440–443. [30] T. Ohta, K. Buiting, H. Kokkonen, S. McCandless, S. Heeger, H. Leisti, [40] B. Kantor, Y. Kaufman, K. Makedonski, A. Razin, R. Shemer, Establishing D.J. Driscoll, S.B. Cassidy, B. Horsthemke, R.D. Nicholls, Molecular the epigenetic status of the Prader–Willi/Angelman imprinting center in mechanism of Angelman syndrome in two large families involves an the gametes and embryo, Hum. Mol. Genet. 22 (2004) 2767–2779. imprinting mutation, Am. J. Hum. Genet. 64 (1999) 385–396. [41] B. Reinhart, M. Eljanne, J.R. Chaillet, Shared role for differentially [31] T. Ohta, T.A. Gray, P.K. Rogan, K. Buiting, J.M. Gabriel, S. Saitoh, B. methylated domains of imprinted genes, Mol. Cell. Biol. (2002) Muralidhar, B. Bilienska, M.W. Krajewska, D.J. Driscoll, B. Horsthemke, 2089–2098. M.G. Butler, R.D. Nicholls, Imprinting-mutation mechanisms in Prader– [42] D. Ma, J.P. Shield, W. Dean, I. Leclerc, C. Knauf, R.R. Burcelin, G.A. Willi syndrome, Am. J. Hum. Genet. 64 (1999) 397–413. Rutter, G. Kelsey, Impaired glucose homeostasis in transgenic mice [32] K. Buiting, C. Lich, S. Cottrell, A. Barnicoat, B. Horsthemke, A 5-kb IC expressing the human transient neonatal diabetes mellitus locus, TNDM, J. deletion in a family with Angelman syndrome reduces the shortest region Clin. Invest. 114 (2004) 339–348. of deletion overlap to 880 bp, Hum. Genet. 105 (1999) 665–666. [43] Y. Obata, T. Kono, Maternal primary imprinting is established at a specific [33] B. Dittrich, K. Buiting, B. Korn, S. Rickard, J. Buxton, S. Saitoh, R.D. time for each gene throughout oocyte growth, J. Biol. Chem. 277 (2002) Nicholls, A. Poustka, A. Winterpacht, B. Zabel, B. Horsthemke, Imprint 5285–5289. switching on human chromosome 15 may involve alternative transcripts of [44] D. Lucifero, C. Mertineit, H.J. Clarke, T.H. Bestor, J.M. Trasler, the SNRPN gene, Nat. Genet. 14 (1996) 163–170. Methylation dynamics of imprinted genes in mouse germ cells, Genomics [34] T. Yang, T.E. Adamson, J.L. Resnick, S. Leff, R. Wevrick, U. Francke, N.A. 79 (2002) 530–538. Jenkins, N.G. Copeland, C.I. Brannan, A mouse model for Prader–Willi [45] D. Lucifero, E.R.W. Mann, M.S. Bartolomei, J.M. Trasler, Gene-specific syndrome imprinting-centre mutations, Nat. Genet. 19 (1998) 25–31. timing and epigenetic memory in oocyte imprinting, Hum. Mol. Genet. 13 [35] J.M. Gabriel, M. Merchant, T. Ohta, Y. Ji, R.G. Caldwell, M.J. Ramsey, (2004) 839–849. J.D. Tucker, R. Longnecker, R.D. Nicholls, A transgene insertion [46] S.M. Blaydes, M. Elmore, T. Yang, C.I. Brannan, Analysis of murine creating a heritable chromosome deletion mouse model of Prader–Willi Snrpn and human SNRPN gene imprinting in transgenic mice, Mamm. and Angelman syndromes, Proc. Natl. Acad. Sci. USA 96 (1999) Genome. 10 (1999) 549–555. 9258–9263. [47] R.M. John, M. Hodges, P. Little, S.C. Barton, M.A. Surani, A human p57 [36] T.F. Tsai, Y.H. Jiang, J. Bressler, D. Armstrong, A.L. Beaudet, Paternal (KIP2) transgene is not activated by passage through the maternal mouse deletion from Snrpn to Ube3a in the mouse causes hypotonia, growth germline, Hum. Mol. Genet. 12 (1999) 2211–2219. retardation and partial lethality and provides evidence for a gene [48] J.M. Gabriel, M.J. Higgins, T.C. Gebuhr, T.B. Shows, S. Saitoh, R.D. contributing to Prader–Willi syndrome, Hum. Mol. Genet. 8 (1999) Nicholls, A model system to study genomic imprinting of human genes, 1357–1364. Proc. Natl. Acad. Sci. USA 95 (1998) 14857–14862. [37] F. Sleutels, D.P. Barlow, Investigation of elements sufficient to imprint the [49] B. Hogan, R. Beddington, F. Constantini, E. Lucy, Manipulating the mouse air promoter, 21 (2001) 5008-5017. Mouse Embryo—A Laboratory manual, 2nd ed., Cold Spring Harbor [38] B.K. Jones, J. Levorse, S.M. Tilghman, A human H19 transgene exhibits Laboratory, Cold Spring Harbor, NY, (1999).