Human Molecular Genetics, 2001, Vol. 10, No. 14 1475-1483
© 2001 Oxford University Press
A conserved imprinting control region at the HYMAI/ZAC domain is implicated in transient neonatal diabetes mellitus
Wellcome/CRC Institute of Cancer and Developmental Biology, and Physiological Laboratory, University of Cambridge, Tennis Court Road, Cambridge, CB2 1QR, UK, 1Department of Molecular and Cell Genetics, School of Life Sciences, Faculty of Medicine, Tottori University, Nishimachi 86, Yonago, Tottori 683-8503, Japan, 2Department of Public Health, Asahikawa Medical College, Midorigaoka-Higashi 2111, Asahikawa, Hokkaido, 078-8510, Japan and 3Department of Reproductive Physiology and Endocrinology, Medical Institute of Bioregulation, Kyusyu University, 4546 Tsurumihara, Beppu, Oita 874-0838, Japan
Received March 23, 2001; Revised and Accepted May 2, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Transient neonatal diabetes mellitus (TNDM) is associated with intra-uterine growth retardation, dehydration and a lack of insulin. Some TNDM patients exhibit paternal uniparental disomy (UPD) of chromosome 6q24, where at least two imprinted genes, HYMAI and ZAC, have so far been characterized. Here we show that the differentially methylated CpG island that partially overlaps mZac1 and mHymai at the syntenic mouse locus is a likely imprinting control region (ICR) for the
120–200 kb domain. The region is unmethylated in sperm but probably methylated in oocytes, a difference that persists between parental alleles throughout pre- and post-implantation development. We also show that within this ICR, there is a region that exhibits a high degree of homology between mouse and human. Using a reporter expression assay, we demonstrate that this conserved region acts as a strong transcriptional repressor when methylated. Finally, we provide in vivo evidence that in the majority of TNDM patients with a normal karyotype, there is a loss of methylation within the highly homologous region. We propose that this ICR regulates expression of imprinted genes within the domain; epigenetic or genetic mutations of this region probably result in TNDM, possibly by affecting expression of ZAC in the pancreas and/or the pituitary. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting is a gamete-specific modification which causes differential expression of the two parental alleles in somatic cells (1,2). Differentially methylated regions (DMRs) are commonly associated with imprinted genes. A loss of the differential methylation at DMRs can affect transcription at imprinted domains, resulting in either biallelic expression or repression of imprinted genes (3). Differences in the methylation of parental alleles are initiated in the embryonic germ-line, are heritable after fertilization and persist during development and into adulthood (4). Mutations which affect the epigenetic states of imprinted loci underlie a number of diseases, including developmental abnormalities, congenital diseases and malignant tumours (5). Such an epigenetic mechanism is also implicated in the pathogenesis of transient neonatal diabetes mellitus (TNDM), a disease associated with intra-uterine growth failure, dehydration, hypergylcaemia and failure to thrive, usually resolving within the first 6 months of life (6,7). In this context, some cases of TNDM are associated with paternal uniparental disomy (UPD) or paternal duplication of chromosome 6 (8,9). Gardner et al. (10) have suggested that imprinted genes which map to human chromosome 6q24 probably play a role in TNDM.
Two paternally expressed human genes, HYMAI and ZAC, are located in this region (11–13). HYMAI generates an untranslated mRNA of unknown function. Gardner et al. (14) reported TNDM patients with a methylation defect at the differentially methylated CpG island that partially overlaps HYMAI, which strongly implicates the importance of this region in TNDM. ZAC/LOT1, a previously characterized putative tumour supressor gene (15–18), is located 50 kb downstream of HYMAI and acts as a transcriptional regulator of a receptor for pituitary adenylate cyclase activating peptide (PACAP) (19). PACAP is produced by pancreatic neural cells and is an intra-islet regulator of insulin release (20). ZAC can also induce G1 arrest and apoptosis (15,17), suggesting that it may be a mediator of pancreatic ß cell proliferation and death.
In this study, we first examined the size of the human imprinted region at chromosome 6q24, which spans 200 kb between the FK506 isoform and KIAA0680 genes. To facilitate the molecular analysis of TNDM in an experimentally tractable organism, we have analysed the genomic organization of the murine mHymai/mZac1 locus and their embryonic expression patterns. We find that mHymai and mZac1 overlap at a differentially methylated CpG island, which is homologous to the DMR at the syntenic human locus. The mouse DMR apparently functions as a methylation imprinting mark and we demonstrate that this conserved region acts as a strong transcriptional repressor when methylated. Finally, we provide in vivo evidence that in the majority of TNDM patients with a normal karyotype, there is a loss of methylation within the highly homologous region. We propose that this imprinting control region (ICR) plays a role in regulating expression of imprinted genes within the domain.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Characterization of the HYMAI/ZAC imprinted domain in human and mouse
To identify control element(s) at the human 6q24 imprinting region (syntenic to mouse chromosome 10A), we first defined the boundaries of this domain, within which the imprinted genes, HYMAI (AF241534) (13) and ZAC/LOT1, are located (11–13). A database search revealed four overlapping P1 artificial chromosomes (PACs) containing five known genes, four CpG islands (Fig. 1A) and several expressed sequence tag (EST) clusters. Both KIAA0680 (accession no. AB014580) and FK506 isoform (accession no. AF100751) are biallelically expressed transcripts which presumably define the borders of this imprinted domain. In the mouse, we also identified a conserved EST, antisense to and downstream of mZac, which shows predominant expression of the maternal allele in all organs except for the testis and liver (T. Arima, manuscript in preparation). Recently, Huang and Stallcup (21) reported a new variant of mZac1 (mZac1b; accession no. AF147785), which interacts with nuclear receptors and their coactivators and has both positive and negative roles in regulating nuclear receptor function, depending on cell type. We established that mZac1b is imprinted just as mZac1a (data not shown; T. Arima, manuscript in preparation).
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Expression profile of mouse Hymai and Zac1
Next, we examined the expression patterns of the previously identified imprinted genes in the region, mHymai and mZac1, in 13.5 days post-coitum (d.p.c.) (E13.5) mouse embryos. Despite their genomic proximity, these genes showed dissimilar expression patterns; mHymai was expressed in liver, sclerotome, telencephalon and placenta (Fig. 1B and C), whereas mZac1 was expressed in lung, tongue and sclerotome, but not in placenta (Fig. 1D and E). Since expression of mZac is of particular interest in the context of TNDM (see below), we examined its expression during pancreogenesis. Expression of mZac1 is indeed detected in the pancreas (Fig. 1F). We compared the expression pattern of mZac1 with those of several genes required for pancreatic development (see Materials and Methods), in particular the endocrine marker Insulin (Fig. 1G). The expression of mZac1 at E13.5 was localized to the periphery of the pancreas (Fig. 1F) and did not appear to overlap with the expression of Insulin, suggesting that mZac1 is not expressed in pancreatic ß cells (Fig. 1G).
Identification of a mouse CpG island homologous to the human DMR of HYMAI
Another objective of this work was to identify cis-regulatory elements responsible for controlling imprinting in this region. During the identification of the mouse Hymai gene, we screened a mouse PAC library using a 1.0 kb probe from the human HYMAI gene. SpeI–SalI (6 kb) and KpnI–KpnI (5 kb) fragments were subcloned from the PAC clone, sequenced (GenBank accession no. AF314094) and analysed using the NIX programme (HGMP). Figure 2A shows the density and the percentage of CpG dinucleotides in the 2.2 kb genomic sequence. A region of 594 bp was identified as a CpG island (CpG score 0.96, GC score 63.81). Two simple A-rich repeats were identified upstream of the CpG island in the mouse sequence (Fig. 2A). A tandem direct repeat element of >500 bp (CA repeats) was also found 8 kb upstream of the CpG island. Figure 2B shows a dot plot comparing 1.2 kb of mouse genomic sequence, including the CpG island and 935 bp of human CpG-rich genomic sequence which we reported previously (13). These two sequences exhibited a high level of conservation (72%). Furthermore, these sequences were also found to be conserved in monkey, rat, dog, cow, rabbit and chicken (data not shown). As expected, high homology was detected between the human HYMAI gene and the mouse sequence. However, the primary sequence of this region also matched with exon 1 of mZac1, suggesting that Hymai and Zac are both within the CpG island in the mouse (Fig. 2C).
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Allele-specific methylation of the mouse CpG island
The mHymai/mZac1 CpG island was examined for parent-of-origin specific methylation differences. We used a 2.2 kb EcoRI fragment, including the CpG island, as a probe and a polymorphic SspI site at position 1252 (Fig. 2D) that was present in C57BL/6 mice (hereafter BL/6) but absent in Mus spretus mice. Genomic DNA from matings between BL/6, M.spretus and their F1 progeny (BL/6 x M.spretus, M.spretus x BL/6) was analysed using EcoRI and SspI, together with a methylation-sensitive restriction enzyme, NotI or SmaI (see Materials and Methods). Cumulative evidence from embryonic and adult tissues from different organs revealed the presence of a 2106 bp fragment that was diagnostic for the methylated maternal allele, and a 1548 bp fragment that was diagnostic for the unmethylated paternal allele (Fig. 2D). These results are consistent with our previous data showing that the homologous region is unmethylated in human sperm (13) as it is in the mouse (see below).
DMR is a methylation imprint mark
We performed bisulphite-modified genomic sequencing of the conserved region in the mouse DMR, which allows analysis of the methylation status of individual sites (22). After bisulphite treatment of different samples, PCR products were subcloned, sequenced and the methylation status of the sense strand at 23 CpG sites located between position 1603 and 1893 was determined (GenBank accession no. AF314094) (Fig. 3A). As shown, all 23 CpG sites were predominantly unmethylated in sperm DNA, consistent with the unmethylated state in human sperm (Fig. 3B). In contrast, in E3.5 blastocysts and E13.5 embryos, the percentage of methylation of the individually sequenced PCR products showed that nearly 50% of the residues were methylated (Fig. 3B). Importantly, the individual clones analysed were mostly either heavily methylated (representing the maternal allele) or unmethylated (representing the paternal allele). Therefore, the maternal-specific methylation of the DMR that is probably established in female germ cells, appears to be maintained during pre-implantation development when the genome is undergoing widespread demethylation (23), and persists thereafter in advanced embryos and adults. These results suggest that the conserved region represents a methylation imprint mark.
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Cell transfection assay
To determine whether the differential methylation region has the capacity to function as a regulatory element, we analysed this putative ICR from the human HYMAI/ZAC locus. We employed a transient transfection assay in HeLa cells in which a reporter gene (firefly luciferase) was expressed from an SV40 promoter. Fragments from the human ICR were assayed for their effect on transcription of the reporter when either unmethylated or methylated (Fig. 4). Amongst the fragments we tested, the 480 bp PX fragment (NheI–SmaI), which contains 47 CpG sites, was shown to act as a strong transcriptional silencer, but only when methylated (Fig. 4). Significantly, this is the highly homologous region within the ICR in human and mouse (Fig. 2B). In contrast, methylation of the 766 bp PY fragment (SacI–SmaI), which contains 53 CpG sites, caused negligible repression of the reporter gene (Fig. 4). Interestingly, the PX fragment is contained within a larger fragment (PZ) which has a lower repression activity. This may be due to the inclusion of the HYMAI promoter within the larger PZ fragment. This promoter may harbour both transcriptional activation and repression properties in this assay, as the PW fragment (containing the promoter region) does not show a significant increase in repressive activity when methylated, compared with the unmethylated fragment.
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This indicates that the conserved region within the ICR (PX) can function as a cis-regulatory element and is capable of strong transcriptional silencing in this assay. It is therefore possible that the ICR may serve a similar role at the endogenous locus in humans and mice: methylation of this region on the maternal chromosome may result in transcriptional regulation of the imprinted genes in this domain.
Methylation defects at the DMR in TNDM patients
TNDM patients were examined for aberrant methylation patterns at the conserved region of the DMR (Fig. 5A), using a bisulphite PCR assay of genomic DNA from 17 patients with this disease. This sample included seven patients with UPD for chromosome 6 (UPD6), four with partial duplications of this region and six with a normal chromosome complement (J. Inoue, manuscript in preparation). After PCR amplification of sodium bisulphite-treated DNA, restriction enzyme digests were performed. The enzymes were chosen for their recognition sequences which contain a CpG dinucleotide. In this assay, a methylated cytosine will maintain the enzyme recognition site and consequently will be cut, whereas an unmethylated cytosine is converted to thymidine, which will destroy the recognition site and remain undigested (Fig. 5B). Five out of six TNDM patients with a normal karyotype were found to have a loss of methylation on the maternal chromosome at eight CpG sites tested in this region (Fig. 5B, data shown for BsaAI). With only a single exception, all patients with UPD of chromosome 6 showed only an unmethylated pattern. In the UPD6 case where monoallelic methylation is maintained, it may be of interest to further characterize the methylated chromosome, as this presumably harbours a mutation preventing demethylation of this allele. The other patients exhibited a normal methylation profile. These results demonstrate that TNDM is associated with a loss of methylation at the conserved region in the DMR at the HYMAI/ZAC locus.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ZAC is the key candidate for TNDM since it is known to induce the PACAP-type 1 receptor (PAC1-R) (19). The PACAP itself is known to be localized in nerve terminals within pancreatic islets, where it contributes to glucose-stimulated insulin secretion (20). The predicted gain of function from biallelic expression of ZAC in TNDM patients (since UPD of the region also results in TNDM) may affect PAC1-R and, perhaps indirectly, PACAP. Loss of methylation in the conserved ICR may result in biallelic ZAC expression and possibly cause TNDM. We have shown that mZac is not expressed in the insulin-secreting ß cells of the pancreas, which suggests that any effect of ZAC on insulin secretion is likely to be indirect. ZAC expression in the pituitary and liver may also play a significant role in the production and release of insulin (15). In addition, loss of function of ZAC is associated with tumours which raises the possibility that epigenetic mutations resulting in the methylation of the ICR may contribute to cancer (16).
Since the conserved DMR proximal to HYMAI/mHymai is epigenetically modified and can induce transcriptional repression, it is likely to function as an ICR. From our detailed analysis (and T. Arima, manuscript in preparation), this is the most prominent region in this imprinted domain which is modified in the germ line. Such ICRs can have long-range effects on transcription of multiple genes in imprinted domains (24). It is likely that the DMR proximal to HYMAI/mHymai plays such a role in regulating expression of ZAC/mZac and possibly other as yet uncharacterized genes within this domain, such as the predominantly maternally expressed antisense transcript we have detected (T. Arima, manuscript in preparation). Loss of methylation at the DMR may result in biallelic expression of ZAC/mZac or other paternally expressed transcripts in the domain (or loss of expression of maternally expressed transcripts), resulting in the TNDM phenotype. Murine transgenic experiments will be able to address the effect of overexpression of imprinted genes at this locus. In addition, precise genetic deletions at the endogenous mouse locus will enable us to define the critical region of the DMR required for transcriptional regulation and assess its role in genomic imprinting. A functional role for the DMR region as an ICR capable of regulating gene expression within the imprinted domain is supported by our identification of a highly conserved region within the ICR that can induce transcriptional repression when methylated. Loss of methylation at the conserved region in vivo is a likely contributory factor responsible for TNDM in human patients.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Mouse PAC clone screening
The mouse PAC library from the UK HGMP Resource Centre was screened using a probe from human HYMAI (AI540783). One positive clone was identified, and SpeI–SalI (6 kb) and KpnI–KpnI (5 kb) fragments were subcloned into pBluescript II SK+ (Stratagene) for sequencing. Subsequently, BlastN search and motif analysis were performed by the computer programme NIX, available from the HGMP (www://hgmp.mrc.ac.uk).
Mouse strains and isolation of DNA and RNA
C57BL/6 (BL/6) females were naturally mated with M.spretus males to generate F1 mice. The F1 females were backcrossed to BL/6 males to generate F2 animals. Genomic DNA and total RNA were obtained from whole 13.5 d.p.c. embryos and various adult organs from F1 mice. Total RNA was prepared using RNAzol B (AMS Biotechnology, Witney, UK).
Methylation analysis
Genomic DNAs from mouse whole embryos, BL/6, M.spretus, F1 (BL/6 x M.spretus) and F2 (M.spretus x BL/6) were prepared as described previously (13) and digested with EcoRI and SspI, which recognize a polymorphic site, and a methylation sensitive endonuclease. Filters were probed with a [32P]dCTP-labelled 2.2 Kb EcoRI–EcoRI fragment spanning the murine CpG island.
Bisulphite PCR methylation assay
Mature spermatozoa were isolated from the vas deferens of adult male mice. Blastocysts were flushed from the uterus at E3.5. Genomic DNA was prepared as described previously (13). Sperm, blastocyst and E13.5 total embryo DNA (0.1 µg) were digested with EcoRI. Bisulphite treatment was carried out essentially as described previously (22). In each experiment, bisulphite-treated DNA was amplified by two rounds of nested PCR. Primer sequences were: BS1F, 5'-GAATTTTGATTTAGTTGGGGTGGGG-3' and BS1R, 5'-TCTACACTCAACACAACAACCCATCC-3' for the first round; and BS2F, 5'-AGGTGGGAGAATGTTTGGGGAGTTGTGG-3' and BS2R, 5'-TCACTACTTACCTACCCTAACC-3' for the second round. The amplified fragments were cloned into the pGEM-T vector (Promega). Individual clones were sequenced using a T7 primer.
The CpG methylation status within the HYMAI/ZAC DMR of 17 Japanese TNDM patients was analysed by treating genomic DNA as described above. PCR was carried out using the following primers which amplified the conserved region from within the DMR. HBSF, 5'-GTGTGGGTGTTGTTTAGTTTTTTT-3' and HBSR, 5'-AACTAAATAACAAATAACAAATACC-3'. PCR products were digested with appropriate restriction enzymes (BsiEI, TaqI, BsaAI, BssHII, HhaI) and electrophoresed on 2.5% agarose gels.
Transient transfection assay
To test the effect of DNA methylation on the human HYMAI/ZAC DMR we used a transient transfection assay in HeLa cells. Regions from the human DMR were subcloned; SacI–NheI fragment (–350–89 bp relative to the start site of the CpG island; PW), NheI–SmaI fragment (89–566 bp; PX), SacI–SmaI fragment (619–1385 bp; PY), SacI–SacI fragment (–350–619 bp; PZ), into pGL3-Promoter Firefly Luciferase reporter vector (Promega). Plasmids were prepared using a midiprep kit (Qiagen). In vitro DNA methylation was perfomed by incubation with CpG methylase (SssI methylase, New England Biolabs). DNA constructs (2 µg) were transfected into HeLa cells, cultured for 22 h, lysed and luciferase readings assayed. Firefly luciferase values were normalized against a co-transfected Renilla luciferase reporter, as described in the DLR assay protocol (Promega). Each construct was tested in triplicate in each experiment and the experiment was repeated three times.
In situ hybridization analysis
Mouse cDNA clones for Hymai, Zac1, Insulin, pdx-1, ngn3, isl1 and Glucagon were used to prepare sense and antisense riboprobes by in vitro transcription using the DIG RNA labelling kit (Boehringer Mannheim). Sagittal and transverse sections of 8 µm from mouse embryos and placentas at E13.5 were used for in situ hybridization, essentially as described previously (25,26). Sections were counterstained with eosin.
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ACKNOWLEDGEMENTS |
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We would like to thank Sheila Barton and Kathy Hilton for technical assistance and all members of the laboratory for their support and valuable suggestions, in particular J. Ainscough, R. John, M. Saitou and S. Khosla for their comments on the manuscript. This work was supported by a grant from the Wellcome Trust (M.A.S.) and the Newton Trust (T.A.). R.A.D. is a Wellcome Trust Prize Research Fellow, K.L.A. is supported by an Elmore Research Studentship from Gonville and Caius College, Cambridge.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +44 1223 334136; Fax: +44 1223 334089; Email: as10021@mole.bio.cam.ac.ukPresent address:Robert A. Drewell, 401 Barker Hall #3204, Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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 T. Barz, A. Hoffmann, M. Panhuysen, and D. Spengler Peroxisome Proliferator-Activated Receptor {gamma} Is a Zac Target Gene Mediating Zac Antiproliferation Cancer Res., December 15, 2006; 66(24): 11975 - 11982. [Abstract] [Full Text] [PDF]
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T. Arima, T. Kamikihara, T. Hayashida, K. Kato, T. Inoue, Y. Shirayoshi, M. Oshimura, H. Soejima, T. Mukai, and N. Wake ZAC, LIT1 (KCNQ1OT1) and p57KIP2 (CDKN1C) are in an imprinted gene network that may play a role in Beckwith-Wiedemann syndrome Nucleic Acids Res., May 11, 2005; 33(8): 2650 - 2660. [Abstract] [Full Text] [PDF]
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 G. Valerio, A. Franzese, M. Salerno, G. Muzzi, G. Cecere, K. I. Temple, and J. P. Shield {beta}-Cell Dysfunction in Classic Transient Neonatal Diabetes Is Characterized by Impaired Insulin Response to Glucose but Normal Response to Glucagon Diabetes Care, October 1, 2004; 27(10): 2405 - 2408. [Abstract] [Full Text] [PDF]
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 M. I. McCarthy Progress in defining the molecular basis of type 2 diabetes mellitus through susceptibility-gene identification Hum. Mol. Genet., April 1, 2004; 13(suppl_1): R33 - R41. [Abstract] [Full Text] [PDF]
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 A. Abdollahi, D. Pisarcik, D. Roberts, J. Weinstein, P. Cairns, and T. C. Hamilton LOT1 (PLAGL1/ZAC1), the Candidate Tumor Suppressor Gene at Chromosome 6q24-25, Is Epigenetically Regulated in Cancer J. Biol. Chem., February 14, 2003; 278(8): 6041 - 6049. [Abstract] [Full Text] [PDF]
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 A. Hoffmann, E. Ciani, J. Boeckardt, F. Holsboer, L. Journot, and D. Spengler Transcriptional Activities of the Zinc Finger Protein Zac Are Differentially Controlled by DNA Binding Mol. Cell. Biol., February 1, 2003; 23(3): 988 - 1003. [Abstract] [Full Text]
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I K Temple and J P H Shield Transient neonatal diabetes, a disorder of imprinting J. Med. Genet., December 1, 2002; 39(12): 872 - 875. [Abstract] [Full Text] [PDF]
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E Marquis, J J Robert, C Bouvattier, C Bellanne-Chantelot, C Junien, and C Diatloff-Zito Major difference in aetiology and phenotypic abnormalities between transient and permanent neonatal diabetes J. Med. Genet., May 1, 2002; 39(5): 370 - 374. [Full Text] [PDF]
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 R. A. Drewell, K. L. Arney, T. Arima, S. C. Barton, J. D. Brenton, and M. A. Surani Novel conserved elements upstream of the H19 gene are transcribed and act as mesodermal enhancers Development, January 3, 2002; 129(5): 1205 - 1213. [Abstract] [Full Text] [PDF]
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 L. Z. Strichman-Almashanu, R. S. Lee, P. O. Onyango, E. Perlman, F. Flam, M. B. Frieman, and A. P. Feinberg A Genome-Wide Screen for Normally Methylated Human CpG Islands That Can Identify Novel Imprinted Genes Genome Res., April 1, 2002; 12(4): 543 - 554. [Abstract] [Full Text] [PDF]
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