Human PEG1/MEST , an imprinted gene on chromosome 7Shin Kobayashi1, Takashi Kohda1, Naoki Miyoshi1, Yoshimi Kuroiwa1, Kohzo Aisaka2, Osamu Tsutsumi3, Tomoko Kaneko-Ishino4 and Fumitoshi Ishino1,5,*
1Gene Research Center, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226, Japan, 2Department of Obstetrics and Gynecology, Teikyo University, Ichihara Hospital, 3426-3 Anesaki, Ichihara, Chiba 299-01, Japan, 3Department of Obstetrics and Gynecology, The University of Tokyo, 3-5-7 Hongo, Bunkyo-ku 113, Japan, 4Tokai University, School of Health Sciences, Bohseidai, Isehara-shi, Kanagawa 259-11, Japan and 5Precursory Research for Embryonic Science and Technology (PRESTO), Japan Science and Technology Corporation (JST), Japan
Received December 26, 1996;Revised and Accepted February 21, 1997
The mouse Peg1/Mest gene is an imprinted gene that is expressed particularly in mesodermal tissues in early embryonic stages. It was the most abundant imprinted gene among eight paternally expressed genes (Peg 1-8) isolated by a subtraction-hybridization method from a mouse embryonal cDNA library. It has been mapped to proximal mouse chromosome 6, maternal duplication of which causes early embryonic lethality. The human chromosomal region that shares syntenic homology with this is 7q21-qter, and human maternal uniparental disomy 7 (UPD 7) causes apparent growth deficiency and slight morphological abnormalities. Therefore, at least one paternally expressed imprinted gene seems to be present in this region. In this report, we demonstrate that human PEG1/MEST is an imprinted gene expressed from a paternal allele and located on chromosome 7q31-34, near D7S649. It is the first imprinted gene mapped to human chromosome 7 and a candidate for a gene responsible for primordial growth retardation including Silver-Russell syndrome (SRS).
Genomic imprinting influences mammalian development, growth and behavior (1 -3 ), and some human genetic diseases and cancers have been attributed to genomic imprinting (4 ,5 ). Genes that are expressed from only the maternal or paternal genome are thought to play essential roles in these phenomena. Therefore, isolation of novel imprinted genes is important for identifying genes that cause human diseases and for clarifying the molecular mechanisms of these diseases. Although >10 imprinted genes have been reported to date, many are yet to be identified. Recently, we developed a novel subtraction-hybridization method to isolate imprinted genes systematically and obtained eight paternally expressed genes (Peg1-8) (6 -8 ) including two known imprinted genes, Igf2 and Snrpn (9 -11 ), from an 8.5 day mouse embryonal cDNA library.
Among these Pegs, Peg1 was expressed most abundantly in early embryos and showed identity to the 121a (Mest; mesoderm-specific transcript) gene that is expressed specifically in mesodermal tissues (12 ,13 ). Mouse parthenogenetic embryos cannot survive beyond day 10 of gestation which is when Peg1/Mest is expressed most abundantly (N. Miyoshi et al., unpublished data). Peg1/Mest was mapped to the proximal region of mouse chromosome 6 (12 ). Mice that have maternal duplication of this region with a Robertsonian translocation show early embryonic lethality (2 ,3 ). These results suggest that Peg1/Mest is a candidate for a gene involved in embryonic lethality.
The proximal region of mouse chromosome 6 shares syntenic homology with human chromosome 7q21-qter (14 ). Human maternal uniparental disomy 7 (UPD 7), similar to the homologous region in mouse, shows some imprinting effect (15 -20 ). To date, human maternal UPD of the whole of chromosome 7 has been reported in eight patients with intrauterine and postnatal growth retardation with slight morphological abnormalities (16 -20 ). Three cases were found in patients that were homozygous for known recessive mutations, two cases with CF (cystic fibrosis) (16 , 17 ) and one case with COL1A2 [pro[alpha]2(I) chain of type I procollagen] (18 ). Recently, Kotzot et al. investigated 35 patients that showed primordial growth retardation, including sporadic Silver-Russell syndrome (SRS) and their parents, with microsatellite markers and found four cases of UPD 7 (20 ). Another case involved a child with maternal heterodisomy of chromosome 7. A trisomy with two maternal and one paternal chromosome 7 was observed in placental cells during the fetal stage, and a subsequent loss of paternal chromosome 7 seemed to occur in embryonal cells (19 ). All these patients showed both prenatal and postnatal growth retardation with frequent morphological abnormalities. One case with uniparental isodisomy for paternal 7p and maternal 7q also showed postnatal growth retardation and morphological abnormalities (21 ). Therefore, it is probable that loss of paternally expressed gene(s) on human chromosome 7 causes these defects. Human PEG1/MEST is a candidate for such a gene if it is proved to be imprinted and located on chromosome 7.
Human PEG1/MEST was isolated from a fetal kidney cDNA library using a mouse Peg1/Mest DNA fragment as a probe (see Materials and Methods). Nine clones were isolated, and further analysis was carried out on eight clones. Of the eight clones, seven had a 2.5 kb insert and one had a 1.6 kb insert (Fig. 1 a and b). Recently, the human MEST gene was independently isolated and analyzed by Nishita et al. The result of the nucleotide sequence of the 2.5 kb transcript is almost the same as ours (22 ). The short transcript used a different poly(A) site (1558 bp), as shown in Figure 1 . The human PEG1/MEST gene seemed to have the same reading frame (from the ATG codon at 223 bp to 1230 bp in the 2.5 kb transcript) as the mouse Peg1/Mest gene. However, we could not find this ATG in two of the clones. There was a single base pair deletion at 249 bp in one clone and the transcript had a different reading frame from the PEG1/MEST protein and stops immediately after the initiation codon. In the short transcript (Fig. 1 b) there was a small deletion (23 bp) including the first ATG at 223 bp. Although it is possible that these clones represent cloning artifacts, we speculate that the second ATG codon at 250 bp may function as an initiation codon. It will be necessary to analyze the N-terminal amino acid sequences of the PEG1/MEST protein to identify the precise reading frame.
Expression of mouse Peg1/Mest was high in early embryonic stages but decreased considerably in late embryonic and neonatal stages (N. Miyoshi et al., unpublished data). Relatively early stage human embryos (6-9 weeks of gestation) were selected to analyze the expression and imprinting status of human PEG1/MEST.To assign the parental origin of the expressed allele, genomic DNA from 19 embryonal samples and peripheral blood from their parents were analyzed and five families informative for a DNA polymorphism at an AflIII site (1922 bp in Fig. 1 ) were identified. Imprinting of human PEG1/MEST was examined in these five families using the PEG1/MEST DNA polymorphism (Fig. 2 A). Two types of pattern, `a' and `b' (uncut and cut following AflIII digestion) were observed. RT-PCR analysis was carried out on both embryos and chorions. The expression level of PEG1/MEST in the chorions was about one-thirtieth of that in the embryos. The result obtained from samples 3, 4 and 5 in Table 1 is shown in Figure 2 B, C and D, respectively. For example, in Figure 2 B, the patterns of the PEG1/MEST DNA polymorphism in genomic DNA from the mother, father, embryo and chorion were `a', `a/b', `a/b' and `a/b', respectively. The expressed allele showed the `b' pattern in the latter two samples and was apparently derived from the father. All samples except one embryo (Table 1 , sample No.1*) showed monoallelic expression, and paternal expression of human PEG1/MEST was confirmed in four families (the chromosome from which the gene was expressed could not be distinguished in sample No. 4) (Table 1 ). Analysis of a human PEG3 DNA polymorphism (T. Kohda et al., unpublished data) indicated that a large amount of maternal tissues was contained in the embryonic sample that showed biallelic expression (Table 1 , sample No.1*). Pathological examination also verified that a large part of the sample consisted of maternal undeveloped decidual tissues. The paternal expression pattern `b' was confirmed in the corresponding chorion. RT-PCR analysis showed expression pattern `a' in the maternal decidual tissues (data not shown). These data supported the theory that the apparent biallelic expression pattern of this embryonic sample was derived from both the embryo and the maternal tissues. These results demonstrated that human PEG1/MEST is paternally expressed in both embryos and chorions in early development. It should be noted that maternal allele expression less than one-twentieth of that observed from the paternal allele was observed in every sample (Fig. 2 B-D, lanes 5 and 8). The possibility of maternal tissue contamination in these samples was checked by using WT1 DNA polymorphism (25 ). The data confirmed that there were no signals of maternal tissue in the samples of B and C in Figure 2 and these samples were free from maternal tissue at the level of sensitivity of PCR (data not shown), suggesting that there is the leaky expression from the maternal allele in the human PEG1/MEST. Almost no maternal expression of Peg1/Mest was detected at the same developmental stages in the mouse (6 ).
In order to map the PEG1/MEST gene, we have identified four independent YAC clones (805e8, 858e9, 920e1, 973d7) containing the PEG1/MEST gene by screening of the Centre d'Etude du Polymorphisme Humain (CEPH) YAC library (plate 805-984). There are some pseudogenes of PEG1/MEST that are not expressed in all the tissues examined (embryos and adult blood) but could be detected by PEG1/MEST cDNA by fluorescence in situ hybridization (FISH) analysis (S. Kobayashi et al., unpublished data). Thus, we chose screening of YACs for the mapping instead of FISH with a PEG1/MEST cDNA probe. The primer set shown in Figure 1 was selected to amplify only the PEG1/MEST gene and used in the PCR-based screening of the YAC library. It has been reported that three out of the four YACs possibly contained the same microsatellite marker, D7S649 (26 ). The four PEG1/MEST-positive clones were tested to determine whether both PEG1/MEST and D7S649 could be detected in the same clone by PCR assays. PEG1/MEST and D7S649 were detected simultaneously in all four YAC clones (Fig. 3 A). The D7S649 marker is located between D7S530 and D7S500 (27 ) and these two markers have been mapped cytogenetically to 7q31-32 and 7q31-34, respectively (28 ). Thus, we concluded that human PEG1/MEST maps to 7q31-34 and is located near D7S649, probably within ~1 Mb, taking into consideration the insert size of the YAC library (Fig. 3 B).
Human PEG1/MEST is highly homologous to mouse Peg1/Mest and encodes a similar protein that belongs to the [alpha]/[beta] hydrolase fold family. Because the catalytic specificities of this class of enzymes are radically different, including haloalkans, lipids and epoxides (24 ), it is very difficult to propose a substrate for the PEG1/MEST protein. Therefore, the biological function of PEG1/MEST remains unclear. In the mouse, parthenogenetic cells that do not express any paternally expressed genes, including Peg1/Mest,were segregated out from mesodermal tissues in normal*parthenogenetic chimeras (29 ,30 ). Lack of paternally expressed gene(s) must be involved in this phenomenon. To date, Peg1/Mest, Igf2 and Peg3 are the candidate imprinted genes that show mesodermal-specific expression in early development. Thus, it is possible that the PEG1/MEST protein metabolizes some biological substance that affects the growth and maintenance of mesodermal cells via its hydrolase activity. Not only the two mammalian species mentioned above but also a variety of other animals, including the African green monkey (Cercopithecus aethiops), marsupial rat (Dasyuroides byrnei byrnei), goldfish (Carrassius auratus) and fruit fly (Drosophila melanogaster) seem to have genes homologous to Peg1/Mest on zoo blot analysis (12 ).Therefore, PEG1/MEST is evolutionally conserved in higher animals and this may point to the functional importance of PEG1/MEST protein in embryonic development. Analysis of Peg1/Mest knockout mice will shed light on its role in early mammalian development.
Human PEG1/MEST was also proved to be imprinted and paternally expressed in this study. Nishita et al. (22 ) recently reported that human MEST gene was expressed at much higher levels in hydatidiform moles than in dermoide cysts and suggested that human MEST was imprinted. It is important to test for imprinting in the tissues that are physiologically relevant. However, it is not adequate for verification of PEG1/MEST imprinting to compare the expression levels in totally different two tissues (placentae and embryos). Moreover, it is known that expression of imprinted genes such as the H19 gene in the placentae of androgenetic embryos does not necessarily reflect the expression of normal placentae (31 ). Thus, in order to prove the imprinted status of PEG1/MEST conclusively, it is necessary to analyze parental expression in the families directly.
. Summary of the verification of human PEG1/MEST imprinting
Sample no.
Maternal DNA
Paternal DNA
Embryonal DNA
PEG1/MEST expression in embryo
PEG1/MEST expression in chorion
1 (6W4D)
a/b
b
a/b
a/b*
b
2 (7W3D)
a
a/b
a/b
-
b
3 (7W5D)
a
a/b
a/b
b
b
4 (7W5D)
a/b
a/b
a/b
a
a
5 (9W5D)
a/b
b
a/b
b
b
*Due to contamination by maternal tissues (see text).Numbers in parentheses indicate gestational age of the samples, e.g. (6W4D) means 6 weeks 4 days. In sample 2, embryo was not obtained. Polymorphic patterns indicated by `a' and `b' correspond to those shown in Figure 2A. Although we used whole embryo, the embryonal tissue could not be identified as coming from any specific organ due to the early stage termination.
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*To whom correspondence should be addressed. Tel: +81 45 924 5812; Fax: +81 45 924 5814; Email: fishino@bio.titech.ac.jp
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