Human Molecular Genetics Advance Access originally published online on March 6, 2007
Human Molecular Genetics 2007 16(8):972-981; doi:10.1093/hmg/ddm041
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Tissue-specific imprinting of the ZAC/PLAGL1 tumour suppressor gene results from variable utilization of monoallelic and biallelic promoters
Section of Genetics, Leeds Institute of Molecular Medicine, University of Leeds, St. James's University Hospital, Leeds LS9 7TF, UK
* To whom correspondence should be addressed at: Tel: +44 1133438649; Fax: +44 1133438702; Email: ed.t.bonthron{at}leeds.ac.uk
Received November 9, 2006; Revised January 3, 2007; Accepted February 23, 2007
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The tumour suppressor gene ZAC/PLAGL1 is widely expressed in many human tissues during fetal development and throughout life. It encodes a DNA-binding protein which shares with p53 the ability to regulate apoptosis and cell cycle arrest concurrently. Owing to its anti-proliferative properties, down-regulation or loss of ZAC is believed to deregulate cell growth, and loss of expression has been observed in a number of different cancers. In addition, overexpression of ZAC during fetal development is believed to underlie the rare disorder transient neonatal diabetes mellitus (TNDM). Imprinted expression of ZAC has been demonstrated in many human and mouse tissues, although biallelic transcription has been noted in human peripheral blood leucocytes (PBL). We report here the identification of a second ZAC promoter, which is responsible for the observed biallelic expression. The promoter lies within a previously uncharacterized CpG island ~55 kb upstream of the imprinted CpG island. In PBL, the imprinted CpG island (P1) is differentially methylated and produces monoallelic transcripts, as in other tissues. However, biallelic transcripts predominate and are derived from the alternative CpG island (P2), which is unmethylated. Biallelic P2 expression was also found in adult pancreas, and ZAC expression from this promoter was identified at a low level in all adult human tissues tested. These findings show that regulation of ZAC expression is more complex than previously realized. The existence of the apparently independently-regulated P2 promoter has important implications for the study of ZAC dysregulation in cancer and TNDM.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Genomic imprinting is an epigenetic phenomenon that results in preferential gene expression from one allele in a parent-of-origin-dependent manner. Loss of imprinting (LOI) at specific loci is seen in a number of pathological settings; inherited LOI results in a number of characteristic developmental abnormalities, whereas somatically acquired LOI is implicated in the cellular growth deregulation in some cancers. ZAC (also known as PLAGL1 or Lot-1) was first identified in rat ovarian surface epithelial cells which lost the gene during malignant transformation (1). The gene was shown to be expressed ubiquitously but to be lost from 38% of human ovarian cancers. The mouse orthologue Zac1 was identified independently in a functional screen for genes which induce the type I receptor (PACAP1-R) for the hormone pituitary adenylate cyclase activating polypeptide (PACAP) (2). This peptide is a potent insulin secretagogue and an important mediator of autocrine control of insulin secretion in the pancreatic islet. Subsequently, we identified ZAC during a screen for imprinted genes and identified it as the most likely candidate gene for transient neonatal diabetes mellitus (TNDM) (3).
ZAC encodes a zinc finger protein of the C2H2 type, thought to act as a transcription factor. It shares with p53 the ability to induce both apoptosis and cell cycle arrest. Two protein isoforms are produced, containing either seven or five zinc fingers (4). ZAC has been found to inhibit tumour cell growth and induce apoptosis and G1 arrest. Significant down-regulation is observed with EGF, TGF
and the tumour-promoting agent 12-O-tetradecanoylphorbol-13-acetate; this and other observations indicate that ZAC expression is probably regulated via the MAPK pathway (5). In addition, it is known to be a transcriptional activator, co-activator or repressor of androgen, oestrogen, glucocorticoid and thyroid nuclear receptors (6).
The ZAC gene is imprinted; it contains a differentially methylated region (DMR) that acts as a promoter directing synthesis of transcripts from the paternal (unmethylated) allele in most human (3) and mouse tissues (7). Such monoallelic expression has been observed in human placenta (3,8) in six human fetal tissues (heart, kidney, muscle, spinal cord, adrenal gland and lung) (3) and in skin fibroblasts (9). However, we and others have previously noted biallelic expression in peripheral blood leucocytes (PBL) (3,9). In the mouse, monoallelic Zac1 expression occurs in pituitary, ovary, lung, heart and brain, with biallelic expression in liver and to a lesser extent in kidney and skeletal muscle (10).
Overexpression of ZAC, either secondary to paternal uniparental disomy of 6q24 or to epigenetic alterations at the ZAC DMR, has been implicated in TNDM, a rare disease with an incidence of one in 400 000 live births (9). Affected children have severe intrauterine growth retardation and develop persistent hyperglycaemia and low or undetectable levels of insulin within the first 6 weeks of life. Transgenic mice which overexpress the gene mimic the features of TNDM and appear to have impaired development of the endocrine pancreas (11). Mice lacking Zac1 display intrauterine growth restriction and altered expression of a number of other imprinted genes believed to be important in controlling intrauterine growth (12).
Interpretation of epigenetic and genetic alterations at the ZAC locus, especially in neoplasia, requires an understanding of the normal pattern of expression of the gene. As the expression pattern of ZAC is tissue-specific, the mechanisms leading to loss of ZAC function may be different in different tumour types. As the 6q24 region is a frequent target for deletions in B cell lymphomas (13), we have been particularly interested in defining the basis for biallelic ZAC expression in haematopoietic tissue.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ZAC transcripts in PBL arise from an alternative CpG island
ZAC expression has been observed only from the paternal allele in most human tissues, although biallelic expression has been reported in PBL (3,9). For insight into this tissue-specific imprinting effect, we conducted a bioinformatic analysis of the chromosomal region close to the gene. Five spliced expressed sequence tags (ESTs) were found in the UCSC Genome Bioinformatics database, which start within a second CpG island ~55 kb upstream of the previously characterized, differentially methylated CpG island (designated P1). Three of the five sequences (AA831875 [GenBank] , AI436373 [GenBank] , BE646490 [GenBank] ) were derived from B cell libraries, indicating that the new CpG island (P2) might have particular significance in relation to ZAC expression in blood. Complete sequencing of the IMAGE clones showed that although they spliced onto ZAC non-coding exons, none of them included any part of the ZAC coding region (all of them have their 3' end within the large non-coding exon 3b) (see below). More recently, a further eight ESTs have been deposited in the database, which also start from the P2 CpG island. Of these, six sequences are derived from blood cell libraries, one is from a synovial membrane library and one is from a spleen library (14).
We conducted RT–PCR experiments to confirm that transcripts are produced from the P2 island in PBL. The P2 CpG island spans a region of ~994 bp (corresponding to nucleotides 82001–82994 in PAC AL031390 [GenBank] ) and contains at least 113 CpG sites. The exon located within the P2 island extends from at least nucleotide 82153 at the 5' end (as determined by the most 5' EST: DA675185 [GenBank] ) to the splice donor site at nucleotide 82284. Forward primers were designed to this exon, and reverse primers were designed to an exon within the ZAC 5'-untranslated region (5'-UTR) (exon 3a; Fig. 1A). Two different transcripts were amplified from leucocytes, and sequencing showed them to be identical to the ESTs in the database. The two sizes corresponded to the presence or absence of an alternatively spliced 40 bp exon (exon 2; Fig.1A).
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None of the published ESTs extends to the coding sequence, but all utilize exons known to be part of the ZAC 5'-UTR. In order to demonstrate that the P2 transcripts are likely to be protein-coding, we used RT–PCR to characterize alternatively spliced transcripts which start from the first exon of P2 and include one or both of the ZAC protein-coding exons. By combining the same forward primer in the first exon of P2 with a reverse primer in the last coding exon of ZAC, eight different partial transcripts were amplified and cloned. Sequencing confirmed in each case that the P2 transcripts extend to the coding region (Fig. 1A). Complex alternative splicing of the 5'-UTR was observed, and both the long and short ZAC protein isoforms were represented. These sequences have been deposited in the GenBank database (NCBI; see Materials and Methods). Our cloning strategy was not exhaustive, and the isolated transcripts may represent only a proportion of the alternatively spliced forms present in this tissue.
The same approach was used to clone P1 transcripts from leucocyte cDNA, using the same reverse primer combined with a forward primer designed to the P1 first exon. The P1 transcripts isolated again represented both long and short forms of the protein (Fig. 1B). Complex alternative splicing of the 5'-UTR was observed, and exon usage was very similar between the two promoters. Alternative splicing of the P1 transcripts was comparable to full-length P1 transcripts from other tissues which have been deposited in GenBank. These results demonstrated that in PBL, ZAC expression is derived from both promoters simultaneously.
For comparison, the same primer sets were used with adult human pancreas cDNA, and similar, alternatively spliced ZAC transcripts were amplified and sequenced. Unexpectedly, in addition to P1 transcripts, we were able to isolate and sequence P2 transcripts from this tissue. Subsequently, we have also isolated P2 transcripts from other tissues by RT–PCR, indicating that ZAC is not solely expressed from the imprinted promoter in tissues other than blood (data not shown, and real-time RT–PCR data, Fig. 4, given subsequently). Combining the data from leucocytes and pancreas, we observed that in both tissues, P1 transcripts more frequently encode the long protein isoform (containing seven zinc fingers) than the short form (containing five zinc fingers). The reverse is true for P2 transcripts. Given that the seven-finger transcripts yield an RT–PCR product 476 bp larger than the five-finger transcript, the predominance of the former among P1-derived transcripts is unlikely to be the result of an amplification bias. Only the novel PBL-derived transcripts have been deposited in GenBank; however, in total, 18 alternatively spliced P1-derived and 20 P2-derived cDNAs were sequenced from PBL and pancreas, of which 13 and seven, respectively, were of the seven-finger type. As the seven-finger and five-finger ZAC isoforms are reported to show functional differences (4), a difference in the splicing ratio between P1 and P2 transcripts could affect the biological function of the two promoters. This hypothesis will require further testing.
The P2 CpG island is unmethylated
To assess whether, like the known ZAC promoter, the novel promoter is imprinted, we analysed the methylation status of part of the P2 CpG island which includes the first exon. Methylation analysis of a similar portion of the P1 island was also carried out. Genomic DNA was extracted from PBL from two individuals and modified by standard methods using sodium bisulphite. Following bisulphite treatment, the two CpG islands were PCR-amplified from each individual and cloned. The primers were designed so that methylated and non-methylated sequences would be amplified non-selectively. Sequencing analysis was performed on a minimum of 20 clones for each CpG island.
Sequence data showed that the P1 CpG island is differentially-methylated in both individuals (Fig. 2A), consistent with previous reports of this CpG island in other tissues (3). It therefore seemed likely that, as in other tissues, P1-directed transcription would be monoallelic. In contrast, the P2 island was unmethylated (Fig. 2A), suggesting that transcripts derived from this island are more likely to be biallelic. Methylation analysis of the P2 island was repeated using DNA extracted from buccal cells and skin fibroblasts from different individuals. In each case, P2 was again unmethylated, indicating that this is probably the typical state of this CpG island and is not tissue-specific to PBL (Fig. 2B).
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Biallelic expression of ZAC from P2 occurs in leucocytes and pancreas
Given the unmethylated state of the P2 promoter, a biallelic origin for ZAC transcripts derived from P2 was anticipated. To test this, we used a single nucleotide polymorphism (SNP) within an exon in the 5'-UTR to define monoallelic or biallelic expression (SNP rs2092894; dbSNP, NCBI). The SNP lies within exon 3b (Fig. 3A), corresponding to nucleotide 13217 of PAC AL109755 [GenBank] . Partial-length transcripts amplified from pancreas and leucocyte cDNAs showed that exon 3b can be spliced to either of the first exons in both of these tissues. Exon 3b appears to be a very large exon, which was not represented in the transcripts we earlier sequenced (Fig. 1), due to the RT–PCR bias favouring smaller products. Some of the ESTs in the UCSC database, which are derived from the P2 promoter, extend beyond exon 3a into this region, but are not long enough to include the next splice junction (and thereby show the full 3' extent of exon 3b). An estimate can be made by comparing the sequence data from two ZAC ESTs from human fetal kidney (accession nos CR749329 [GenBank] and BX537397 [GenBank] ). These sequences extend from the 3' end into exon 3b, but the 5' end is missing. If these sequences align with the others, it would indicate that exon 3b potentially spans the sequence between exon 3a and exon 4d, resulting in a single spliced exon ~4.2 kb in size.
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Owing to the close proximity to the first exon, in a heterozygous individual, SNP 135217 is informative for both promoters. We identified two heterozygotes by amplifying and sequencing the SNP site using PBL genomic DNA from seven individuals. For these heterozygotes, we used primers to the P2 first exon and to exon 3b to amplify transcripts from corresponding leucocyte cDNA. The resulting amplicons were cloned and ~15 individual clones were sequenced for each individual. As predicted, P2 transcripts were derived from both alleles, confirming biallelic expression from this promoter (Fig. 3B and Table 1). The corresponding P1 transcript was amplified using a forward primer to the P1 first exon combined with the same exon 3b reverse primer. A similar number of clones were sequenced and only one allele was found, indicating monoallelic expression from the P1 promoter.
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The procedure was repeated using adult pancreas cDNA. P2 transcripts containing SNP 13217 were amplified and 10 individual clones were sequenced. (Although genomic DNA was not available for this sample, the source was tissue from a single individual who proved to be heterozygous for this SNP.) Sequencing of amplified cDNA clones showed that two alleles were present, indicating that biallelic ZAC expression from the P2 promoter also occurs in pancreas. As in leucocytes, the corresponding P1 transcripts were found to be monoallelic (Table 1).
Quantification of ZAC expression
To quantify expression of the various ZAC transcripts, real-time RT–PCR experiments were carried out using Taqman probes (Applied Biosystems). These assays were also used to test a panel of human tissues for the presence or absence of P2 transcripts (Fig. 4). Assays were designed to the 5' end of the transcripts in order to compare expression levels from each promoter and to capture as many transcripts as possible. The RNA used was derived from tissues pooled from several donors, except for the pancreas and leucocyte samples which were from single donors.
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Forward primers were designed to either the P1 or P2 first exons, and a common reverse primer was designed to exon 3a (Fig. 4A). This exon was chosen, as we have found that it is the only 5'-UTR exon to be incorporated into all ZAC transcripts (see Fig. 1). Consequently, a primer within this exon should amplify the 3' end of all of the ZAC transcripts produced. As alternative splicing of exon 2 could interfere with transcript quantification, separate assays were designed to the four possible splice variants (Fig. 4A). Previous RT–PCR experiments had not revealed any other 5'-UTR exons in this region, which were utilized in leucocytes or pancreas (Fig. 1 and unpublished data), although one of the P2 ESTs in the UCSC database contains an extended exon 2 (Accession no. AW275124 [GenBank] , derived from a pooled tissue library). To eliminate the possibility of interference caused by splice variants, reverse primers were designed to cross the splice junction between exons 2 and 3. In addition, for each assay, a unique Taqman probe was designed to bind to the first splice junction within the transcript to maximize specificity.
All four assays were used on a panel of human cDNA from different tissues. P2 transcripts were found in all the tissues that we tested, in addition to transcripts from the imprinted promoter (Fig. 4B and Table 2). Expression levels varied between tissues but generally P1 transcripts predominate. The exception was PBL, where P2 transcripts are very highly expressed and there is very low expression from P1. Repeating the assays with leucocyte cDNA from two more individuals (Fig. 4B; ‘leucocytes2’ and ‘leucocytes3’) showed some variation in absolute levels of the different transcripts, but a consistent overall profile. These data explain the reported biallelic expression of ZAC in peripheral blood. In most tissues, the level of P2 transcripts is swamped by P1 transcripts, and this is the likely reason why only imprinted expression has been observed previously. Interestingly, two tissues showed approximately equal levels of P1 and P2 transcripts—liver and spleen. If tested, we would predict that ZAC would appear to be biallelically expressed in these tissues, as observed in blood.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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We originally demonstrated that the differentially methylated CpG island referred to here as P1 represented a promoter of the ZAC gene, leading to the prediction and demonstration of monoallelic ZAC expression from the paternal allele. This has been found to be the case in human placenta (3,8), in several fetal tissues (3) and in skin fibroblasts (9). One exception has been PBL, where biallelic expression has been reported (3,9). In mouse, imprinting of the orthologue Zac1 is conserved, and the gene has been shown to be paternally expressed in adult tissues (pituitary, ovary, lung, heart and brain) (7,10). Biallelic expression was observed in mouse liver and to a lesser extent in skeletal muscle and kidney (10). These observations in both human and mouse could indicate either a relaxation of imprinting in certain tissues or the presence of one or more alternative promoters.
We have identified a novel, alternative promoter for human ZAC, which explains this apparent tissue-specific phenomenon. Analysis of the original (P1) CpG island in PBL shows that its differential methylation is maintained. By linking a single heterozygous SNP site to either promoter exon, we have further shown unequivocally that only monoallelic expression occurs from the P1 promoter in PBL. In contrast, the alternative promoter (P2) is located within a second, unmethylated CpG island. Using the same SNP linked to the alternative P2 promoter, we have demonstrated that biallelic transcription occurs from this promoter simultaneously with monoallelic transcription from P1. The biallelic ZAC expression previously reported in blood cells thus corresponds to a mix of a high expression level of biallelic P2 transcripts and a low level of monoallelic P1 transcripts. We observed that even though P2 was biallelically expressed, the A allele, which represented the imprinted allele for P1 (which we assume to be maternal), appeared to be expressed at a lower level (Table 1). At present, it is unclear whether the apparent over-representation of the G allele is significant. It might represent PCR bias (as all three heterozygotes have G as the paternal allele) or it may represent unequal expression from maternal and paternal alleles, suggesting that epigenetic or chromatin modifications of the P1 island on the maternal allele also have indirect effects on the transcription from the P2 island. Biallelic expression in some mouse tissues further suggests that there may be an alternative promoter equivalent to P2 in this species.
Unexpectedly, we found that use of the alternative P2 promoter was not restricted to leucocytes but was also seen in pancreas, with biallelic transcripts being produced at the same time as monoallelic transcripts are being produced from the imprinted promoter. We have therefore quantified the contributions of P1 and P2 in various tissues, using real-time RT–PCR assays with highly specific Taqman probes. We have shown that the P2 promoter is active in all the adult tissues that we have tested, but to varying degrees. In addition, the level of expression from P1 also varies, with the highest level in placenta, a tissue where key imprinted genes have been shown to regulate placental function (15). In PBL, biallelic P2 transcripts predominate, and this is strikingly different from the other tissues. This explains the previously observed biallelic expression, but the reason for the difference in promoter usage remains to be elucidated. In most tissues, P2 transcripts were expressed at a much lower level compared with P1 transcripts, explaining the apparently monoallelic expression in these tissues. Transcripts from both promoters were expressed at approximately equal levels in liver and spleen, and we would therefore predict that these tissues would show biallelic expression of ZAC if tested.
Although previous descriptions represent the ZAC gene as spanning ~70 kb and having several 5' non-coding exons (3,16), our present data show that it must span at least 125 kb. P2 transcripts are spliced with similar complexity to P1 transcripts, including to the coding exon of the gene, indicating that they encode functional protein. Further experiments will need to define the functional consequences of this and to unravel the mechanism by which the two promoters function in tandem. The reasons for the complex splicing of the 5'-UTR within ZAC transcripts are not yet known, but these exons may contribute to mRNA stability, translational efficiency or other regulatory roles (17). Of the transcript variants we sequenced, P1A was the smallest and consisted only of the first exon, exon 3a and two coding exons (Fig. 1). Several of the transcripts were found to contain upstream AUGs and open reading frames. In other genes, these (and other features of the 5'-UTR) are known to control the translation efficiency of the main ORF by affecting the mode of ribosome entry onto the RNA and subsequent protein synthesis (18). More promoters and 5' start sites for ZAC may exist, which are yet to be discovered. Three ESTs deposited by Kimura et al. (14) begin in a 5' untranslated exon, which may represent a third start site (Accession nos DA814732 [GenBank] ; DB104173 [GenBank] ; DB177852 [GenBank] ).
We also observed that the different promoters preferentially encode different protein isoforms of ZAC: the long form (containing seven zinc fingers) is more frequently encoded by P1 transcripts and the short form by P2 (containing five zinc fingers). The two protein variants have the same anti-proliferative activity, but the long protein isoform is more efficient at inducing apoptosis, whereas the short form has a greater ability to initiate cell cycle arrest (4). Our findings may therefore indicate a functional distinctness or bias for the different promoters, and further work will allow us to test this observation. The tissue-specific variation in the levels of both P1 and P2 transcripts implies dominant regulatory influences for cis- and trans-acting mechanisms other than methylation of the P1 CpG island. We have also found no evidence that P2 promoter activity is regulated by methylation of its CpG island (these data and unpublished results).
We have shown that P2 transcripts are expressed in pancreas, where they may have significant effects on the pathogenesis of TNDM, a disorder that is believed to result from ZAC overexpression. Most patients with TNDM are found to have a duplication of 6q24 or uniparental isodisomy of chromosome 6. In the former case, the P2 promoter is also likely be duplicated, with consequent overexpression of P2 transcripts in addition to P1 transcripts. Some patients without chromosomal abnormalities have been shown to have a relaxation of ZAC imprinting, and this has been measured by looking for biallelic expression in skin fibroblasts (9). As we have found P2 expression in skin fibroblasts, P2 transcripts could have a potentially confounding effect on these results (data not shown). Methylation of the P1 CpG island has been shown to be reduced in some patients, and this has been thought to be the cause of ZAC overexpression, resulting in TNDM (19). However, for a subset of patients, no defect in ZAC has yet been found (19), and in these patients, the possibility of aberrant expression from the P2 promoter may be borne in mind.
The discovery of a second promoter for ZAC also has important implications for understanding its role in tumorigenesis. Loss of the chromosomal region surrounding the gene or loss of its expression has been found in a number of tumours including ovarian and breast tumours, squamous cell carcinomas and pituitary adenomas (20–23). Hypermethylation of the P1 CpG island has been demonstrated in ovarian cancer, which would be predicted to result in reduced expression and consequential deregulation of cell growth (24). It is possible that tumorigenesis could be associated with differential usage of the two promoters such as that seen for other imprinted genes (25). As the regulation of this gene is likely to be more complex than previously anticipated, the activity of the P2 promoter must also be taken into account when considering the loss of this gene in tumours.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Methylation analysis of CpG islands
Unmethylated cytosines in genomic DNA were converted to uracil, using a sodium bisulphite modification protocol, as described previously (3). PCR of the P1 and P2 islands was carried out using primers which amplify both methylated and unmethylated sequences. These were, for P1, zac1: dGTGTTTAGGATAGTGTTTGGTT and zac9: dCCCAACCRTATCTAAATCAAAACT, and for P2, CpG2F: dGGAGGAGTAAGATTTGAATTGAGG and CpG2R: dCCCTACCTCACCTACCTTTCC. PCR conditions were as follows: denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 30 s, annealing at 56°C for 30 s (or 58°C for 30 s for P2) and extension at 72°C for 30 s, followed by a final extension step of 72°C for 3 min. PCR products were gel-extracted, purified using the Geneclean II kit (QBioGene) and ligated into pGEM T-Easy vector (Promega) according to the manufacturer's protocol. Ligations were transformed into E. coli DH10B and plasmid extracted from cultures of recombinant colonies using the Qiaprep Spin kit (Qiagen). Approximately 20 clones per sample were sequenced. The CpG islands as defined by UCSC Genome Browser (March 2006 Assembly) are as follows: P1, 931 bp (144371540–144370610, 5'–3') containing 118 CpGs and P2, 994 bp (144427581–144426588, 5'–3') containing 113 CpGs. The portions cloned for methylation analysis were as follows: P1, 539 bp (144371640–144371102) and P2, 371 bp (144427640–144427270) (Fig. 2).
Sequencing
Miniprep DNA was sequenced using the DYEnamic ET Terminator kit (Amersham), and the sequencing reactions were analysed on a MegaBace 500 sequencer (Amersham). The sequence data from the partial ZAC transcripts isolated from PBL (shown in Fig. 1) have been deposited in GenBank (NCBI). Accession nos are P2A: EF100438
[GenBank]
; P2B: EF100439
[GenBank]
; P2C: EF100440
[GenBank]
; P2D: EF100441
[GenBank]
; P2E: EF100442
[GenBank]
; P2H: EF100443
[GenBank]
; P2I: EF100444
[GenBank]
; P2J: EF100445
[GenBank]
; P1A: EF100446
[GenBank]
; P1B: EF100447
[GenBank]
; P1C: EF100448
[GenBank]
; P1D: EF100449
[GenBank]
; P1E: EF100450
[GenBank]
; P1F: EF100451
[GenBank]
; P1G: EF100452
[GenBank]
.
SNP analysis
For identification of biallelic or monoallelic expression of ZAC, SNP rs2092894 was used (dbSNP, NCBI), located at nucleotide 13217 of PAC accession no. AL109755
[GenBank]
. The allele frequencies of this SNP are 0.508:0.492 for the A:G alleles in the European population. Primers used to amplify genomic DNA surrounding the SNP were Snp1F (dTGGTGGACCCTACCTCAGTT) and Snp1R (dCAAGAGGACACGCTAAGAACG). For RT–PCR of ZAC transcripts, a forward primer specific for each alternative exon 1 was used, combined with a common reverse primer (P1 forward primer: dAGCCGTGCTCACAGCTCAG; P2 forward primer: dCGGACTCCAGAACTTTCCAA; reverse primer: Snp1F, discussed earlier). Leucocyte and pancreas RNAs (BD Biosciences) were from single donors and were reverse-transcribed using the Thermoscript II kit (Invitrogen), according to the manufacturer's instructions.
Real time RT–PCR
A panel of human tissue RNAs (obtained from Ambion, Stratagene, BD Biosciences) was used to prepare cDNA, using random hexamers (Thermoscript II kit, Invitrogen). All RNAs were derived from pooled tissues from several donors, except for the three leucocyte samples and the pancreas samples which came from single individuals. Taqman primers and probes were obtained from Applied Biosystems. Quantitative real-time PCR was carried out using Universal Taqman PCR Mastermix according to the manufacturer's instructions, and the reactions were performed and analysed on an ABI 7500 Real-Time PCR system. An assay for the endogenous control gene RPLP0/36B4 (Applied Biosystems) was carried out in tandem with each ZAC assay and used to normalize the quantity of ZAC transcript in each sample. All samples were run in triplicate.
Primer and probe sequences are as follows:
Assay 1a, probe 6FAM-dCCGCAGGATTGTTCC; forward dGGCCTCGCCTGAGCT; reverse dGCAATCAAAAGCCAATCACGATGTT;
Assay 1b, probe 6FAM-dCCGCAGGGATTGCTGT; forward dGGCCTCGCCTGAGCT; reverse dAGGCAGCAGCCACATTAGAC;
Assay 2a, probe 6FAM-dCAGGAACAATCCGCCGCCAA; forward dCCGGACTCCAGAACTTTCCAA; reverse dGCAATCAAAAGCCAATCACGATGTT;
Assay 2b, probe 6FAM-dCAGCAATCCCGCCGCCAA; forward dCCGGACTCCAGAACTTTCCAA; reverse dAGGCAGCAGCCACATTAGAC.
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ACKNOWLEDGEMENTS |
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This research was funded by Leukaemia Research Fund grant 00/56, by the West Riding Medical Research Trust and by a Yorkshire Cancer Research Pump-Priming grant to E.V. and D.T.B.
Conflict of Interest statement. None declared.
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REFERENCES |
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