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Hitoshi Hiura,
Masashi Toyoda,
Hiroaki Okae,
Masahiro Sakurai,
Naoko Miyauchi,
Akiko Sato,
Nobutaka Kiyokawa,
Hajime Okita,
Yoshitaka Miyagawa,
Hidenori Akutsu,
Koichiro Nishino,
Akihiro Umezawa, Takahiro Arima
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ABSTRACT: BACKGROUND: hiPSCs are generated through epigenetic reprogramming of somatic tissue. Genomic imprinting is an epigenetic phenomenon through which monoallelic gene expression is regulated in a parent-of-origin-specific manner. Reprogramming relies on the successful erasure of marks of differentiation while maintaining those required for genomic imprinting. Loss of imprinting (LOI), which occurs in many types of malignant tumors, would hinder the clinical application of hiPSCs. RESULTS: We examined the imprinting status, expression levels and DNA methylation status of eight imprinted genes in five independently generated hiPSCs. We found a low frequency of LOI in some lines. Where LOI was identified in an early passage cell line, we found that this was maintained through subsequent passages of the cells. Just as normal imprints are maintained in long-term culture, this work suggests that abnormal imprints are also stable in culture. CONCLUSIONS: Analysis of genomic imprints in hiPSCs is a necessary safety step in regenerative medicine, with relevance both to the differentiation potential of these stem cells and also their potential tumorigenic properties.
BMC Genetics 04/2013; 14(1):32. · 2.47 Impact Factor
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Hitoshi Hiura,
Hiroaki Okae,
Naoko Miyauchi,
Fumi Sato,
Akiko Sato,
Mathew Van De Pette,
Rosalind M John,
Masayo Kagami,
Kunihiko Nakai,
Hidenobu Soejima,
Tsutomu Ogata, Takahiro Arima
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ABSTRACT: There is an increased incidence of rare imprinting disorders associated with assisted reproduction technologies (ARTs). The identification of epigenetic changes at imprinted loci in ART infants has led to the suggestion that the techniques themselves may predispose embryos to acquire imprinting errors and diseases. However, it is still unknown at what point(s) these imprinting errors arise, or the risk factors.
In 2009 we conducted a Japanese nationwide epidemiological study of four well-known imprinting diseases to determine any association with ART. Using bisulfite sequencing, we examine the DNA methylation status of 22 gametic differentially methylated regions (gDMRs) located within the known imprinted loci in patients with Beckwith-Wiedemann syndrome (BWS, n=1) and also Silver-Russell syndrome (SRS, n= 5) born after ART, and compared these with patients conceived naturally.
We found a 10-fold increased frequency of BWS and SRS associated with ART. The majority of ART cases showed aberrant DNA methylation patterns at multiple imprinted loci both maternal and paternal gDMRs (5/6), with both hyper- and hypomethylation events (5/6) and also mosaic methylation errors (5/6). Although our study may have been limited by a small sample number, the fact that many of the changes were mosaic suggested that they occurred after fertilization. In contrast, few of the patients who were conceived naturally exhibited a similar pattern of mosaic alterations. The differences in methylation patterns between the patients who were conceived naturally or after ART did not manifest due to the differences in the disease phenotypes in these imprinting disorders.
A possible association between ART and BWS/SRS was found, and we observed a more widespread disruption of genomic imprints after ART. The increased frequency of imprinting disorders after ART is perhaps not surprising given the major epigenetic events that take place during early development at a time when the epigenome is most vulnerable.
Human Reproduction 06/2012; 27(8):2541-8. · 4.47 Impact Factor
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Hitoshi Hiura,
Hiroaki Okae,
Hisato Kobayash,
Naoko Miyauchi,
Fumi Sato,
Akiko Sato,
Fumihiko Suzuki,
Satoru Nagase,
Junichi Sugawara,
Kunihiko Nakai,
Nobuo Yaegashi, Takahiro Arima
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ABSTRACT: Aberrant DNA methylation leads to loss of heterozygosity (LOH) or loss of imprinting (LOI) as the first hit during human carcinogenesis. Recently we developed a new high-throughput, high-resolution DNA methylation analysis method, bisulphite PCR-Luminex (BPL), using sperm DNA and demonstrated the effectiveness of this novel approach in rapidly identifying methylation errors.
In the current study, we applied the BPL method to the analysis of DNA methylation for identification of prognostic panels of DNA methylation cancer biomarkers of imprinted genes. We found that the BPL method precisely quantified the methylation status of specific DNA regions in somatic cells. We found a higher frequency of LOI than LOH. LOI at IGF2, PEG1 and H19 were frequent alterations, with a tendency to show a more hypermethylated state. We detected changes in DNA methylation as an early event in ovarian cancer. The degree of LOI (LOH) was associated with altered DNA methylation at IGF2/H19 and PEG1.
The relative ease of BPL method provides a practical method for use within a clinical setting. We suggest that DNA methylation of H19 and PEG1 differentially methylated regions (DMRs) may provide novel biomarkers useful for screening, diagnosis and, potentially, for improving the clinical management of women with human ovarian cancer.
BMC Medical Genomics 03/2012; 5:8. · 3.69 Impact Factor
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03/2012; , ISBN: 978-953-51-0320-2
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ABSTRACT: Within the vertebrate groups, only mammals are subject to a specialized epigenetic process termed genomic imprinting in which genes are preferentially expressed from one parental allele. Imprinted expression has been reported for >100 mouse genes and, for approximately one-quarter of these genes, the imprinted expression is specific to the placenta (or extraembryonic tissues). This seemingly placenta-specific imprinted expression has garnered much attention, as has the apparent lack of conserved imprinting between the human and mouse placenta. In this study, we used a novel approach to re-investigate the placenta-specific expression using embryo transfer and trophoblast stem cells. We analyzed 20 genes previously reported to show maternal allele-specific expression in the placenta, and only 8 genes were confirmed to be imprinted. Other genes were likely to be falsely identified as imprinted due to their relatively high expression in contaminating maternal cells. Next, we performed a genome-wide transcriptome assay and identified 133 and 955 candidate imprinted genes with paternal allele- and maternal allele-specific expression. Of those we analyzed in detail, 1/6 (Gab1) of the candidates for paternal allele-specific expression and only 1/269 (Ano1) candidates for maternal allele-specific expression were authentically imprinted genes. Imprinting of Ano1 and Gab1 was specific to the placenta and neither gene displayed allele-specific promoter DNA methylation. Imprinting of ANO1, but not GAB1, was conserved in the human placenta. Our findings impose a considerable revision of the current views of placental imprinting.
Human Molecular Genetics 02/2012; 21(3):548-58. · 7.64 Impact Factor
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ABSTRACT: Genomic imprinting is an epigenetic marking and a stable transmission of monoallelic gene expression patterns in a parent of- origin-specific manner. Aberrant imprinting has been linked to a number of human genetic disorders, including congenital abnormalities, childhood cancer, behavior disorders, and cancer in adults. Imprinted genes play roles in carcinogenesis. Recently, progress in researched on epigenetic mechanisms of imprinted genes, in edition to analysis of the pathology of the oncogenetic mechanisms, has begun to be clinically applied to diagnostic methods, prevention, and cancer drug development.
Gan to kagaku ryoho. Cancer & chemotherapy 11/2011; 38(11):1745-9.
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Toshiaki Watanabe,
Shin-ichi Tomizawa,
Kohzoh Mitsuya,
Yasushi Totoki,
Yasuhiro Yamamoto,
Satomi Kuramochi-Miyagawa,
Naoko Iida,
Yuko Hoki,
Patrick J Murphy,
Atsushi Toyoda, [......],
Hitoshi Hiura, Takahiro Arima,
Asao Fujiyama,
Takashi Sado,
Tatsuhiro Shibata,
Toru Nakano,
Haifan Lin,
Kenji Ichiyanagi,
Paul D Soloway,
Hiroyuki Sasaki
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ABSTRACT: Genomic imprinting causes parental origin-specific monoallelic gene expression through differential DNA methylation established in the parental germ line. However, the mechanisms underlying how specific sequences are selectively methylated are not fully understood. We have found that the components of the PIWI-interacting RNA (piRNA) pathway are required for de novo methylation of the differentially methylated region (DMR) of the imprinted mouse Rasgrf1 locus, but not other paternally imprinted loci. A retrotransposon sequence within a noncoding RNA spanning the DMR was targeted by piRNAs generated from a different locus. A direct repeat in the DMR, which is required for the methylation and imprinting of Rasgrf1, served as a promoter for this RNA. We propose a model in which piRNAs and a target RNA direct the sequence-specific methylation of Rasgrf1.
Science 05/2011; 332(6031):848-52. · 31.20 Impact Factor
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ABSTRACT: The telomere length and subtelomeric methylated status of peripheral blood leukocytes has been reported to be correlated with many kinds of pathophysiological conditions. However, the correlation between the telomeric parameters and clinical laboratory data in metabolic disorders is not well known. This study investigated the correlation between the telomere length and subtelomeric methylated status in peripheral leukocytes and the laboratory data of male outpatients with combined metabolic disorders and no clinical history of cardiovascular or cerebrovascular event were assessed, to find good clinical laboratory markers reflecting the biological aging. The laboratory data were collected in 26 Japanese male outpatients with diabetes mellitus and hyperlipidemia, and no history of cardiovascular or cerebrovascular event, and the telomeric parameters in their peripheral leukocytes were determined by Southern blot with methylation-sensitive and insensitive isoschizomers. Any correlations between the laboratory data and the telomeric parameters were assessed. The patients showed a significant negative correlation among the bilirubin and creatine phosphokinase with the aging-associate change of the telomeric and subtelomeric parameters. Lowered serum bilirubin and creatinine phosphokinase level correlated to genomic aging represented by telomere attrition of patients with metabolic disorders.
The Aging Male 03/2011; 14(1):21-6. · 1.52 Impact Factor
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ABSTRACT: The telomere length of peripheral blood leukocytes has been reported to be inversely correlated with many kinds of pathophysiological conditions. However, correlations between telomere length in peripheral blood leukocytes and patients' physical ability are not known.
To address this problem, the physical ability of patients with cerebrovascular disease admitted to the chronic disease ward of Kyushu University Hospital was assessed with the Barthel index (BI) and the telomere length of their peripheral blood leukocytes was determined.
Women exhibited a significant correlation between the Barthel score and the expression of long telomeres (>9.4 Kb), in contrast with men who revealed no such correlation. The physical ability of older women was positively correlated with the lengths of their somatic telomeres. Among the BI items, the scores of more difficult physical performances tended to correlate with the presence of terminal restriction fragments longer than 9.4 Kb.
Aging clinical and experimental research 02/2011; 23(1):22-8. · 1.55 Impact Factor
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ABSTRACT: To assess the clinical value of bisulfite polymerase chain reaction Luminex (BPL), an automated, high-throughput procedure for the detection of alterations in DNA methylation.
Experimental prospective study.
University research laboratory and private in vitro fertilization (IVF) clinic.
A total of 337 men, 61 with severe oligozoospermia, 67 with moderate oligozoospermia, and 209 with microscopically normozoospermia.
The ejaculated sperm samples after the routine semen analysis with patients' consent.
Examination of the methylation patterns of eight imprinted loci in sperm DNA, and confirmation with combined bisulfite PCR restriction analysis (COBRA).
A total of 47 cases (13.9%) showed abnormal methylation at one or more imprinted loci (18 paternal, 18 maternal, and 11 cases with alterations of both maternal and paternal imprints).
The relative ease of the BPL method provides a practical method within a clinical setting to reduce the likelihood of abnormal samples being used in assisted reproduction treatments.
Fertility and sterility 01/2011; 95(1):129-34, 134.e1-4. · 3.97 Impact Factor
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ABSTRACT: The telomere length and subtelomeric methylated status of peripheral blood leukocytes have been reported to be correlated with many kinds of pathophysiological conditions. However, the correlation between the telomeric parameters and patients' physical ability is not known.
This study aims to study how telomeric parameters, including telomere length and the subtelomeric methylation status of peripheral blood leukocytes, are associated with the physical inability of patients with cerebrovascular disease and its improvement by inpatient rehabilitation.
The physical ability of female patients with cerebrovascular disease admitted in the chronic disease ward of Kyushu University Hospital was assessed using the Barthel index, and the telomeric parameters in their peripheral blood leukocytes were determined by Southern blotting with methylation-sensitive and -insensitive isoschizomers.
The patients revealed a significant correlation between Barthel score and the mean telomere length and expression of long telomeres (> 9.4 kb). Improvement of the Barthel index of patients during admission was correlated not to telomere length, but to subtelomeric hypermethylation of long telomeres.
The physical ability of patients was positively correlated with the lengths of their somatic telomeres, and the recovery potential of physical ability was associated with the subtelomeric hypermethylated status stabilizing long telomeric structure.
Gerontology 05/2010; 57(2):137-43. · 2.78 Impact Factor
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Hitoshi Hiura,
Atsushi Sugawara,
Hidehiko Ogawa,
Rosalind M John,
Naoko Miyauchi,
Yusuke Miyanari,
Tokumasa Horiike,
Yufeng Li,
Nobuo Yaegashi,
Hiroyuki Sasaki,
Tomohiro Kono, Takahiro Arima
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ABSTRACT: The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs.
Nucleic Acids Research 04/2010; 38(15):4929-45. · 8.03 Impact Factor
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ABSTRACT: Parent-of-origin specific expression of imprinted genes relies on the differential DNA methylation of specific genomic regions. Differentially methylated regions (DMRs) acquire DNA methylation either during gametogenesis (primary DMR) or after fertilisation when allele-specific expression is established (secondary DMR). Little is known about the function of these secondary DMRs. We investigated the DMR spanning Cdkn1c in mouse embryonic stem cells, androgenetic stem cells and embryonic germ stem cells. In all cases, expression of Cdkn1c was appropriately repressed in in vitro differentiated cells. However, stem cells failed to de novo methylate the silenced gene even after sustained differentiation. In the absence of maintained DNA methylation (Dnmt1(-/-)), Cdkn1c escapes silencing demonstrating the requirement for DNA methylation in long term silencing in vivo. We propose that postfertilisation differential methylation reflects the importance of retaining single gene dosage of a subset of imprinted loci in the adult.
Epigenetics: official journal of the DNA Methylation Society 04/2010; 5(3). · 4.58 Impact Factor
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Hisato Kobayashi,
Hitoshi Hiura,
Rosalind M John,
Akiko Sato,
Eiko Otsu,
Naoko Kobayashi,
Rei Suzuki,
Fumihiko Suzuki,
Chika Hayashi,
Takafumi Utsunomiya,
Nobuo Yaegashi, Takahiro Arima
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ABSTRACT: There is an increased prevalence of imprinting disorders, such as Beckwith-Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.
European journal of human genetics: EJHG 06/2009; 17(12):1582-91. · 3.56 Impact Factor
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Journal of Mammalian Ova Research 01/2009;
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ABSTRACT: Recent studies suggest that assisted reproductive technologies (ART), which involve the isolation, handling and culture of gametes and early embryos, are associated with an increased incidence of rare imprinting disorders. Major epigenetic events take place during this time and the process of ART may expose the epigenome to external influences, preventing the proper establishment and maintenance of genomic imprints. However, the risks of ART cannot be simply evaluated because the patients who receive ART may differ both demographically and genetically from the general population at reproductive age. In this study, we examined the DNA methylation status of seven imprinted genes using a combined bisulphite-PCR restriction analysis and sequencing technique on sperm DNA obtained from 97 infertile men. We found an abnormal paternal methylation imprint in 14 patients (14.4%) and abnormal maternal imprint in 20 patients (20.6%). The majority of these doubly defective samples were in men with moderate or severe oligospermia. These abnormalities were specific to imprinted loci as we found that global DNA methylation was normal in these samples. The outcome of ART with sperm shown to have an abnormal DNA methylation pattern was generally poor. However, one sample of sperm with both paternal and maternal methylation errors used in ICSI produced a child of normal appearance without any abnormalities in their imprinted methylation pattern. Our data suggest that sperm from infertile patients, especially those with oligospermia, may carry a higher risk of transmitting incorrect primary imprints to their offspring, highlighting the need for more research into ART.
Human Molecular Genetics 12/2007; 16(21):2542-51. · 7.64 Impact Factor
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ABSTRACT: It has been proposed that the human endometrium may contain a population of adult stem cells that are responsible for its remarkable regenerative capability. Recently, a subset of stem cells or progenitor cells in adult tissue has been identified as side-population cells (SP cells) displaying low staining with Hoechst 33342 by fluorescence-activated cell sorter (FACS) analysis. In this study, we isolated SP cells from the human endometrium and analysed their properties.
Endometrial cells were obtained using enzymatic digestion from uterine hysterectomy for the treatment of uterine myoma and stained with Hoechst 33342 dye either alone or in combination with verapamil. The cells were then analysed using FACS.
SP cells were present among normal human endometrial cells. Most SP cells were enriched in the CD9(-)CD13(-) fraction. These SP cells showed long-term repopulating properties and produced gland (CD9(+))- and stroma (CD13(+))-like cells. CD9(-)CD13(-) cells isolated from the endometrium also generated gland- or stroma-like cells.
SP cells in the human endometrium can function as progenitor cells. This is the first report of the phenotype of SP cells from normal human endometrial cells.
Human Reproduction 06/2007; 22(5):1214-23. · 4.47 Impact Factor
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ABSTRACT: We previously demonstrated that that the Ras/ER/MDM2 pathway was critical for NIH3T3 cell transformation. In this study, we examined the effect of blocking this pathway on cell growth in gynecologic cancer cells.
(1) The levels of MDM2, ER, p53 and p21 in endometrial or ovarian cancer cell lines were investigated and compared with that in normal cells by Western blots. (2) The effects of MEK-inhibitor and/or anti-estrogen, and siRNA of MDM2 on cell growth, tumorigenicity in nude mice were examined.
The MDM2 level was enhanced in cancer cells compared with normal cells. Treatment with MEK inhibitor(U0126) resulted in a reduced MDM2 level, enhanced p53 and p21 levels and inhibited cell growth by the induction of premature senescence. The effect of MEK inhibitor on cell growth was affected by ER levels and functions. Treatment with low-dose MEK inhibitor in combination with anti-estrogen (ICI182,780) had a more inhibitory effect on cell growth compared to treatment with MEK inhibitor or anti-estrogen alone in cancer cells. Down-regulation of the MDM2 level by siRNA resulted in the inhibition of growth in cancer cells.
The blockage of the MAPK/ER/MDM2 pathway suppress cell proliferation and it is supposed as a new molecular target therapy in estrogen-dependent gynecologic cancers, such as endometrial or ovarian cancer.
Gynecologic Oncology 06/2007; 105(2):341-50. · 3.89 Impact Factor
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ABSTRACT: Human papillomavirus type 18 (HPV18) is frequently detected in cervical cancer cells. The viral proteins E6 and E7 are expressed consistently and have oncogenic activities. The E7 protein binds to a tumor suppressor, the retinoblastoma gene product (pRB), however, leading to the stabilization of tumor suppressor, p53 protein. On the other hand, another viral product, E6, forms complexes with p53 and abrogates its function, resulting in tumor progression. These facts imply that the E6 oncogene is one of the ideal targets for directed gene therapy in HPV-positive cervical cancer. In this study, we tried photodynamic antisense regulation of the antiapoptotic E6 expression using a photocross-linking reagent, 4,5',8-trimethylpsoralen, conjugated oligo(nucleoside phosphorothioate) (Ps-S-Oligo). This photodynamic antisense strategy effectively elicited the apoptotic death of HPV18-positive cervical cancer cells through the selective repression of E6 mRNA and consequent stabilization of p53 protein. E7-mediated signals potentially activated the p53 function and mobilized the p53 pathway to deliver pro-apoptotic signals to the cancer cells, leading to the suppression of in vivo tumorigenesis. An extremely low concentration of cisplatin in addition to Ps-S-Oligos further up-regulated p53 activity, provoking massive apoptotic induction. These results suggest that the photodynamic antisense strategy has the great therapeutic potential in HPV-positive cervical cancers.
Oligonucleotides 02/2007; 17(1):66-79. · 2.80 Impact Factor
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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 genes, HYMAI and PLAGL1 (LOT1/ZAC), are located within human chromosome 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.
Genomics 12/2006; 88(5):650-8. · 3.02 Impact Factor
