Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (39)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lee, M. P.
Right arrow Articles by Feinberg, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M. P.
Right arrow Articles by Feinberg, A. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Human Molecular Genetics Pages 683-690  

Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting
Introduction
Results
   Molecular cloning of TSSC4
   Molecular cloning of TSSC6
   Tissue-specific expression of TSSC4 and TSSC6
   TSSC4 and TSSC6 were not imprinted in any tissues examined
Discussion
Materials And Methods
   cDNA cloning and mapping
   Isolation of DNA and RNA from tissues
   Northern blot hybridization
   Identification of transcribed polymorphisms in TSSC4 and TSSC6
   Analysis of allele-specific expression in fetal tissues
Acknowledgements
References

Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting

Two novel genes in the center of the 11p15 imprinted domain escape genomic imprinting

Maxwell P. Lee1, Sheri Brandenburg1, Gregory M. Landes3, Mark Adams4, Glenn Miller3 and Andrew P. Feinberg1,2,*

Departments of 1Medicine, 2Molecular Biology and Genetics, and Oncology, Johns Hopkins University School of Medicine, 1064 Ross, 720 Rutland Avenue, Baltimore, MD 21205, USA, 3Genzyme Research and Development, One Mountain Road, Framingham, MA 01701, USA and 4The Institute for Genome Research, 9712 Medical Center Drive, Rockville, MD 20856, USA

Received November 27, 1998; Revised and Accepted January 17, 1999

DDBJ/EMBL/GenBank accession nos AF125568 and AF125569

We previously reported the isolation of a 2.5 Mb tumor-suppressing subchromosomal transferable fragment (STF) from human chromosome 11p15 and the identification of nine known genes and four novel genes within this STF. We now report the isolation of two novel cDNAs, designated here as TSSC4 and TSSC6 (tumor-suppressing STF cDNA 4 and 6), located within the STF. TSSC4 and TSSC6 encode predicted proteins of 329 and 290 amino acids, respectively, with no close similarity to previously reported proteins. TSSC4 and TSSC6 are both located in the center of a 1 Mb imprinted domain, which contains the imprinted genes TSSC3, TSSC5, p57KIP2, KVLQT1, ASCL2, IGF2 and H19. However, we found that neither TSSC4 nor TSSC6 was significantly imprinted in any of the fetal or extra-embryonic tissues examined. Based on this result, the imprinted gene domain of 11p15 appears to contain at least two imprinted subdomains, between which TSSC4 and TSSC6 substantially escape imprinting, due either to lack of initial silencing or to an early developmental relaxation of imprinting.

INTRODUCTION

Human chromosome 11p15.5 is one of the most heavily studied genomic regions, since it harbors multiple imprinted genes and, thus, may constitute a relatively large imprinted chromosomal domain. In addition, 11p15 harbors several genes, mutations or altered imprinting of which are associated with human disease. These include Beckwith-Wiedemann syndrome (BWS), an autosomal dominant disorder, characterized by prenatal overgrowth and predisposition to cancer; and Wilms’ tumor 2 (WT2), defined by frequent loss of heterozygosity (LOH) in embryonal tumors such as Wilms’ and other embryonal cancers, as well as lung, breast and ovarian cancer. These disorders appear to involve imprinted genes, because BWS is transmitted predominantly through the mother, and balanced germline chromosomal rearrangements in BWS, as well as LOH in embryonal cancers, exclusively involve the maternally inherited allele (reviewed in ref. 1).

We and others have identified seven imprinted genes on 11p15: (i) IGF2, which encodes an important autocrine growth factor in cancer (2-5); (ii) H19, an untranslated RNA whose imprinting regulates IGF2 (6); (iii) ASCL2, a homolog of Drosophila achaete-scute expressed in the trophoblast (7); (iv) KVLQT1, coding for a voltage-gated potassium channel (8); (v) p57KIP2, encoding a cyclin-dependent kinase inhibitor (9); (vi) TSSC5 (also known as IMPT1/BWR1A/ORCTL2), which codes for a predicted transmembrane transporter (10-12); and (vii) TSSC3 (also known as IPL) (13,14), a homolog of a mouse apoptosis-inducing gene (15). With the exception of IGF2, all of these genes are expressed from the maternal allele.

We and others have also found direct evidence of a role for five of these genes in disease: (i) IGF2 undergoes loss of imprinting (LOI) in 70% of Wilms’ tumors and about half of all adult cancers, with activation of the normally silent maternal allele (2,5); (ii) H19 shows epigenetic silencing in some tumors with LOI of IGF2 (16); (iii) KVLQT1 is rearranged in most BWS patients with balanced germline chromosomal rearrangements (8); (iv) p57KIP2 is mutated in 5% of BWS patients and shows epigenetic silencing and/or LOI in all Wilms’ tumors (17-21); and (v) TSSC5 undergoes germline mutations in Wilms’ tumor and somatic mutation in lung cancer (11). Because of the large number of imprinted genes on 11p15, spanning ~1 Mb, it is generally believed that 11p15 represents one of two known large (>1 Mb) imprinted domains in the human genome, the other being the Prader-Willi/Angelman syndrome domain of 15q11-13 (22,23). We previously isolated a subchromosomal transferable fragment (STF) which suppresses in vitro growth of the rhabdomyosarcoma cell line RD, confirming the existence of one or more tumor suppressor genes within this region (24). The STF spans ~2.5 Mb, with D11S12 at its proximal end and D11S1318 at its distal end (25). Here we report two novel genes, within a cluster of imprinted genes in this STF, that are not imprinted in any of the fetal or extra-embryonic tissues examined.

RESULTS

Molecular cloning of TSSC4

We previously reported the mapping and isolation of 13 genes within an STF from human chromosome 11p15.5 (11,25) of which seven were imprinted (2-14). To continue our effort of systematic isolation and characterization of genes from this region, we isolated 12 exons from BAC clones 161F14 and 318P18, which are located between KvLQT1 and IGF2 (Fig. 1A), and are part of a genomic contig that we had isolated earlier (25). This region has been relatively less well characterized than regions located more centromeric or telomeric to this interval (25). As described in Materials and Methods, we identified two expressed sequence tag (EST) contigs within this interval, THC176530 and THC156825. Sequencing of these contigs revealed the presence of a single open reading frame (ORF) encoding a protein of 329 amino acids, within a cDNA of 1463 bp. This gene is designated here as TSSC4 (tumor-suppressing STF cDNA 4; GenBank accession no. AF125568). The first ATG of the ORF is located at nucleotide 182, and the stop codon is at nucleotide 1171 (Fig. 2A). A BLAST search indicated the absence of any protein homologous to TSSC4. Motif analysis using the GCG software package identified one predicted protein kinase A (PKA) site at Ser311. TSSC4 also contained five predicted protein kinase C (PKC) phosphorylation sites, at Ser90, Ser129, Ser146, Ser248 and Ser307 (Fig. 2A). Comparison of TSSC4 and genomic sequence (GenBank accession no. AC000376) indicated that TSSC4 contains two exons, and the entire coding region is located within the second exon (Figs 1B and 2A).


Figure 1. Map of TSSC4 and TSSC6 in 11p15.5 and polymorphisms within them. (A) Transcript map. The arrows indicate transcriptional orientation. 161F14 and 318P18 are BACs containingTSSC4 and TSSC6. (B) Intron-exon structure of TSSC4. The polymorphic sites at nucleotides 1290 and 1353 are indicated by a single asterisk because of their proximity. The sequences of primers TSSC4-114, TSSC4-112 and TSSC4-212 are given in Materials and Methods. (C) Partial intron-exon structure of TSSC6. The polymorphic site at nucleotide 89 is indicated by an asterisk. The sequences of primers TSSC6-1A, TSSC6-1B, TSSC6-105 and TSSC6-202 are given in Materials and Methods.

   A
   B

Figure 2. DNA and predicted protein sequence of (A) TSSC4. The first methionine of the ORF is at nucleotide 182 and the stop codon is at nucleotide 1171. (B) TSSC6. The first methionine of the ORF is at nucleotide 146 and the stop codon is at nucleotide 1018. The arrows indicate the positions of intron-exon boundaries.

Molecular cloning of TSSC6

In a similar manner, we identified an additional EST contig, THC133845, within this interval. Sequencing of EST clone 235072 within the contig did not reveal a long ORF. However, RT-PCR using primers designed from the 5[prime] and 3[prime] end sequences of 235072, followed by direct sequencing of the RT-PCR product, revealed an ORF of 290 amino acids, within a cDNA of 1241 bp (Fig. 2B). This gene was termed TSSC6 (tumor-suppressing STF cDNA 6; GenBank accession no. AF125569). Sequence comparison of TSSC6 and genomic sequence (GenBank accession no. AC002536) indicated that TSSC6 contains nine exons (Fig. 1C). EST clone 235072 lacks exon 4, causing a shift in the reading frame, most likely because of aberrant RNA splicing (26). The first ATG is located at nucleotide 146, and the stop codon TGA is located at nucleotide 1018 (Fig. 2B). A Psi-BLAST search revealed that TSSC6 shows weak similarity to the rat TAPA1 gene (E = 0.019), which belongs to the tetramembrane-spanning protein family. Interestingly, the human TAPA1 gene is located 60 kb centromeric to TSSC6, suggesting that one of the genes may have arisen in part by duplication followed by considerable sequence divergence (Fig. 1A). Motif analysis indicates the presence of one potential PKA site at Ser18, three potential casein kinase sites at Thr53, Thr87 and Thr138, three potential PKC sites at Thr53, Thr208 and Ser248, and one potential tyrosine kinase phosphorylation site at Tyr24 (Fig.2B).

Mapping of TSSC4 and TSSC6 by Southern hybridization to our previously established genomic contig (25) indicated the follow-ing gene order: cen-p57KIP2-KvLQT1-TSSC4-TAPA1-TSSC6-ASCL2-TH-IGF2-tel. The transcriptional orientation for TSSC4, TAPA1 and TSSC6 is from telomere to centromere (Fig. 1A).

Tissue-specific expression of TSSC4 and TSSC6

To assess the pattern of tissue-specific expression of TSSC4, we performed northern blot hybridization using human multiple tissue northern blots. We detected a 1.6 kb transcript of TSSC4 in fetal brain, lung, liver and kidney (Fig. 3A), suggesting that the isolated cDNA is full length. TSSC4 was also expressed to varying degrees in all adult tissues (Fig. 3B-D).


Figure 3. Northern blot analysis of TSSC4. (A) Fetal tissues; (B-D) adult tissues. The filters were hybridized using TSSC4 as a probe, and the film was exposed for 24 h. SM, skeletal muscle; SI, small intestine; PBL, peripheral blood leukocytes.

We were not able to detect a signal using TSSC6 on the same MTN blots, suggesting that TSSC6 is expressed at low levels. However, we were able to amplify TSSC6 by RT-PCR from all tissues tested, suggesting ubiquitous low level expression.

TSSC4 and TSSC6 were not imprinted in any tissues examined

Since both TSSC4 and TSSC6 are located within the 11p15 imprinted gene domain, we next examined allele-specific expression of TSSC4 and TSSC6. The individual exons of each gene were sequenced from 10 separate fetal samples, using four primer sets to examine the two exons of TSSC4 (one set for exon 1 and three sets for exon 2), and nine primer sets to examine the nine exons of TSSC6. Two transcribed polymorphisms were identified within exon 2 of TSSC4, at nucleotides 1290 and 1353. A transition at nucleotide 1290 from G to A removes a restriction endonuclease site for BanI, while a transition at nucleotide 1353 from C to T adds a HindIII site. We thus used HindIII and BanI to type 35 fetal DNA samples. Four of these were heterozygous (data not shown), which was also confirmed directly by DNA sequencing (Fig. 4).


Figure 4. Biallelic expression of TSSC4 in tissues of four separate fetuses (A-D). DNA indicates genomic DNA. The sources of RNA for RT-PCR are indicated. The C1353T polymorphism was used for analysis of allele-specific expression in (A) and (B), and the G1290A polymorphism was used in (C) and (D).

In order to assess the imprinting status of TSSC4, we carried out RT-PCR using primer pair TSSC4-112/TSSC4-212, generating a 235 bp cDNA product from exon 2 (Fig. 1B), which includes both the G1290A and C1353T polymorphisms. We then directly sequenced the RT-PCR products derived from 15 tissues of four fetuses, to analyze allele-specific gene expression. No RT-PCR products were detected when RT reactions were carried out in the absence of reverse transcriptase, indicating that there was no genomic DNA contamination.

All 15 fetal tissues showed biallelic expression of TSSC4 (Fig. 4). For example, the genomic DNA of fetus 1 clearly showed equal representation of both C and T alleles at nucleotide 1353, the same as for genomic cDNA. Analysis of RNA by RT-PCR also showed equal representation of both alleles in all fetal tissues, including liver, limb, lung and heart (Fig. 4A). Similarly, fetus 2 was heterozygous for the C1353T polymorphism (Fig. 4B). Both alleles were expressed from fetal lung, kidney and intestine (Fig. 4B), again indicating biallelic expression of TSSC4 in these tissues. Furthermore, four tissues from fetus 3, heterozygous for the G1290A polymorphism, also showed biallelic expression (Fig. 4C). The heart and lung of fetus 4 showed equal representation of both alleles (Fig. 4D). However, in two of 15 tissues tested, the intestine and limb of fetus 4, the two alleles were not expressed precisely equally. In these tissues, the paternal A allele was expressed ~2-fold more abundantly than the maternal G allele. The ratio of expression of the two alleles was much less than that of other imprinted genes in this region (from 4- to 10-fold for TSSC3, to >20-fold for IGF2, H19 and p57KIP2).

To rule out any possibility of DNA contamination, we also carried out RT-PCR using primer TSSC4-114, located in exon 1, and TSSC4-212, located in exon 2 (Fig. 1B). As in the previous experiments, biallelic expression of TSSC4 was observed in all cases examined (data not shown). The absence of significant imprinting of TSSC4 was a surprising result, given the fact that it lies in the middle of the 11p15 imprinted gene domain (Fig. 1A).

These results were also confirmed using a polymorphic HindIII site at nucleotide 1353. Fetuses 1 and 2 were heterozygous (Fig. 6). RT-PCR followed by HindIII digestion revealed biallelic expression of TSSC4 in all eight fetal tissues analyzed. Quantitation using a Phosphorimager indicated that the ratios of a to b alleles, normalizing for fragment size, using genomic DNA as a standard, were 1.22, 0.90, 1.05, 1.04, 1.07, 0.90, 1.07 and 1.37 for adrenal gland, kidney, intestine, lung, liver, heart, lung and limb, respectively (Fig. 6). The absence of a PCR product in the -RT lanes indicated the absence of DNA contamination. These results are consistent with those obtained by DNA sequencing (Fig. 4).

We next examined the imprinting status of TSSC6. Using primer pair TSSC6-1A/TSSC6-1B to amplify exon 1 from genomic DNA (Fig. 1C), we identified a polymorphism changing G at nucleotide 89 to A. We typed 27 fetuses and found seven heterozygotes. We then used primer TSSC6-105 located in exon 1 as a forward primer and TSSC6-202 located in exon 4 as the reverse primer for RT-PCR analysis (Fig. 1C), analyzing 35 tissues from five separate fetuses. All 35 tissues displayed biallelic expression. For example, DNA from fetus 5 showed equal representation of G and A alleles at nucleotide 89 (Fig. 5A, DNA), indicating that it was heterozygous. RT-PCR analysis revealed equal representation of both G and A alleles in fetal pancreas, liver, spleen, kidney and testis (Fig. 5A, and data not shown). Similar biallelic expression was seen in fetuses 6 and 7, including the kidney, lung, pancreas, adrenal, spleen, eye, thymus, skin, gut, heart, limb, liver and brain (Fig. 5B and C, and data not shown). Fetus 8 also showed nearly equal biallelic expression in most tissues (Fig. 5D, and data not shown), although there was an ~1.3-fold ratio of expression paternal allele over maternal allele in the lung and brain. This also is in marked contrast to the ratio of expression of other imprinted genes in this region. Finally, examination of two placentas and one amnion showed approximately equal expression of the two alleles, equal to genomic DNA, indicating that TSSC4 is not significantly imprinted in extra-embryonic tissue.


Figure 5. Biallelic expression of TSSC6 in tissues of four separate fetuses (A-D). Labeling is as in Figure 4. The G89A polymorphism was used for analysis of allele-specific expression.


Figure 6. Biallelic expression of TSSC4 analyzed by restriction enzyme digestion. The uncut and cut alleles correspond to C1353 (labeled a) and T1353 (labeled b), respectively. The following tissues in fetuses 1 (cDNA lanes 10-17, genomic DNA lane 18) and 2 (cDNA lanes 1-8, genomic DNA lane 9) were examined, in sets of four (-RT followed in the figure by +RT of the same tissues): liver, heart, lung and limb (fetus 1); adrenal, kidney, intestine and limb (fetus 2). Phosphorimager analysis showed an allele ratio (normalized for DNA content) of 0.90 to 1.37.

DISCUSSION

We have molecularly cloned two novel genes, TSSC4 and TSSC6, within a cluster of imprinted genes on 11p15. We have established the gene order as follows: cen-hNAP2-TSSC3-TSSC5 -p57KIP2-KvLQT1-TSSC4-TAPA1-TSSC6-ASCL2-TH-IGF2- H19-L23-tel. TSSC4 and TSSC6 both lie within a tumor-suppressing STF from 11p15 (24) and thus are potential tumor suppressor genes.

These results have important implications for the understanding of genomic imprinting. TSSC3, TSSC5, p57KIP2, KvLQT1, ASCL2, IGF2 and H19 previously were shown to be imprinted (2-14). However, we demonstrate here that despite their location in the middle of these genes, both TSSC4 and TSSC6 were not significantly imprinted in any of the fetal or extra-embryonic tissues examined. The ratio of allele-specific expression measured quantitatively was 0.90- to 1.37-fold. While there may be a slight allelic preference in some tissues, TSSC4 and TSSC6 are not substantially imprinted when compared with other genes in this region (4- to >20-fold allelic ratio). This relaxation could arise either by escape from imprinting or by a developmental relaxation of imprinting specific to this region and occurring before day 53 of gestation, when the earliest fetuses could be examined. Consistent with the latter idea, the mouse homolog of TAPA1, which lies between TSSC4 and TSSC6 in humans, has been shown to be imprinted until e8.5, after which imprinting is relaxed (27). Thus, TSSC4 and TSSC6 might also show early developmental relaxation of imprinting, which might have already occurred in the human fetuses. However, mouse and human imprinting are not necessarily the same, as mouse Kvlqt1 also loses imprinting after e9.5 (27,28), but the human gene remains imprinted throughout prenatal development except in the heart (8).

Regardless of whether an imprint fails to include these genes initially or is erased early in development, these data imply the existence of separately regulated subdomains within the 11p15 imprinted domain. This may explain why germline mutations of mouse H19 disrupt Igf2 but have no effect on more distant imprinted genes in cis, such as Kvlqt1 or Mash2 (the mouse homolog of ASCL2) (27).

The results described here predict that the two imprinted subdomains, separated by TSSC4-TSSC6, may define subtypes of human disease. Preliminary data from our laboratory suggest that this is so, as we have found LOI of a novel paternally expressed transcript, termed LIT1, within 11p15 in most patients with BWS (M.P. Lee, M.R. DeBaun, K. Mitsuya, H.L. Galonek, S. Brandenburg, M. Oshimura and A.P. Feinberg, submitted for publication) and LOI of LIT1 in this region is independent of LOI in the more telomeric region including IGF2 (M.P. Lee et al., submitted for publication). Conversely, we and others previously showed frequent LOI of IGF2 in Wilms’ tumor (2,5), and tumors with LOI of IGF2 show normal imprinting of LIT1 in the centromeric subdomain (K. Mitsuya, M. Meguro, M.P. Lee, A. Kashiwagi, M. Kohda, M. Katoh, T.C. Schultz, H. Kugoh, M.A. Yoshida, A.P. Feinberg and M. Oshimura, submitted for publication). Thus, the concept of two imprinted subdomains is consistent with observations from human disease. We hypothesize that alterations of the IGF2-H19 domain may be more specific for malignancy, and alterations of the p57KIP2- LIT1-KVLQT1 domain may be more specific for BWS (M.P. Lee et al., submitted for publication).

MATERIALS AND METHODS

cDNA cloning and mapping

We previously isolated 30 PAC, P1 and BAC clones spanning STF 74-1-6, from which 200 non-redundant trapped exons were isolated (25). The region within the STF spanned by BAC clones 161F14 and 318P18 was relatively unexplored by any laboratory. Twelve non-redundant exons initially were isolated from these BAC clones. Four of these exons were found within an unordered unmapped phase 1 genomic sequence of PAC pDJ5K6 in GenBank (accession no. AC000376). This sequence was then used to search the THC database at the Institute for Genome Research, identifying two EST contigs, THC176530 and THC156825. These two contigs ultimately were found to correspond to a single cDNA, TSSC4, as described in Results. Similarly, eight exons were found to correspond to the phase 1 sequence of PAC pDJ1075F20 in GenBank (accession no. AC002536). This sequence was then used to identify THC133845, from which TSSC6 ultimately was cloned, as described in Results. cDNA clones were sequenced and analyzed using BLAST (29), Sequencher (Genecode) and GCG (Genetics Computer Group). cDNA clones were mapped back to our previously published genomic contig (25) by Southern blot hybridization for determination of gene order, and to the GenBank genomic sequence for transcriptional orientation.

Isolation of DNA and RNA from tissues

Normal fetal tissues (53-81 days gestation) were obtained from the University of Washington Fetal Tissue Bank and were stored at -135°C until use. DNA was isolated by digestion with proteinase K (0.2 mg/ml) in the presence of 1% SDS in buffer TE9 (0.5 M Tris-HCl pH 9.0, 20 mM EDTA, 10 mM NaCl), at 50°C overnight, followed by phenol-chloroform extraction and ethanol precipitation. RNA was isolated using RNAzol B (Tel-Test), according to the manufacturer’s protocol.

Northern blot hybridization

Northern blot experiments were performed as described (16) on human multiple tissue northern blots (fetal MTN II, adult MTN, adult MTN III and adult IV; Clontech, Palo Alto, CA). DNA probes were synthesized by the random priming method (30). Autoradiograms were exposed for 24 h.

Identification of transcribed polymorphisms in TSSC4 and TSSC6

In order to identify transcribed polymorphisms in both genes, we sequenced both exons of TSSC4, using a total of four sets of primers, on each 10 fetal DNA samples as templates. Similarly, all nine exons of TSSC6 were sequenced using nine sets of primers. As described in Results, two polymorphisms were identified in TSSC4 and one in TSSC6. Primers used for typing the G1290A and C1353T polymorphisms in exon 2 of TSSC4 are as follows: TSSC4-112, 5[prime]-GAGATGGCCCAGCCTGACCCCACTGGC-3[prime]; and TSSC4-212, 5[prime]-AACCTTTATTGTCCCTACCAGGGAGCC-3[prime]. Primers used for typing the G89A polymorphism in exon 1 of TSSC6 are as follows: TSSC6-1A, 5[prime]-GTCTGCAGTGAAGGCCTCCTGAGACCA-3[prime]; and TSSC6-1B, 5[prime]-CACCATTCTGGCTGTGAGGCGTCCAGC-3[prime]. PCR reactions contained 0.5 µM primers, 0.2 mM dNTP, 50 ng of DNA, 1× PCR buffer (LTI, Gaithersburg, MD), 1.5 mM MgCl2 and 1 U of Taq DNA polymerase (LTI), in 25 µl, and they were performed with a Robocycler (Stratagene, La Jolla, CA) as follows: 40 cycles of 95°C for 45 s, 60°C for 30 s and 72°C for 1 min 30 s; followed by extension at 72°C for 10 min. PCR products were purified using Qiaex II (Qiagen, Valencia, CA) and directly sequenced, or treated with HindIII or BanI and analyzed by electrophoresis on 5% polyacrylamide gels.

Analysis of allele-specific expression in fetal tissues

RT-PCR and sequencing were used to analyze allele-specific gene expression. RNA was treated with RNase-free DNase (Boehringer Mannheim, Indianapolis, IN) and extracted with phenol-chloroform before the RT reaction. RT was carried out using AMV reverse transcriptase (Boehringer Mannheim). Primers used for RT-PCR are: TSSC4-112 and TSSC4-212 as given in the previous section; TSSC4-114, 5[prime]-AACCACTGGCACCCAGCAGCCAAGAGA-3[prime]; TSSC6-105, CTGAGCCTGTCTCTCACGGGACCGGGA-3[prime]; and TSSC6-202, AGGTGTCCAGCATGGCGTCCTCCACCT. RT-PCR products were purified from 2% agarose gels using Qiaex II (Qiagen). Restriction enzyme digestion or DNA sequencing of the RT-PCR products was used to assay allele-specific gene expression.

ACKNOWLEDGEMENTS

We thank Paul Grundy and the National Wilms Tumor Study for tumor specimens, and Pat Rusche for preparing the manuscript. We thank Patrick Onyango for discussions. This work was supported by NIH grants CA65145 and CA54358 (A.P.F.).

REFERENCES

1. Feinberg, A.P. (1998) Genomic imprinting and cancer. In Vogelstein, B. and Kinzler, K.W. (eds), The Genetic Basis of Human Cancer. Part 1. McGraw-Hill, New York, pp. 95-107.

2. Rainier, S., Johnson, L.A., Dobry, C.J., Ping, A.J., Grundy, P.E. and Feinberg, A.P. (1993) Relaxation of imprinted genes in human cancer. Nature, 362, 747-749. MEDLINE Abstract

3. Giannoukakis, N., Deal, C., Paquette, J., Goodyear, C.G. and Polychronakos, C. (1993) Parental genomic imprinting of the human IGF2 gene. Nature Genet., 4, 98-101. MEDLINE Abstract

4. Ohlsson, R., Nystrom, A., Pfeifer-Ohlsson, S., Tohonen, V., Hedborg, F., Schofield, P., Flam, F. and Ekstrom, T.J. (1993) IGF2 is parentally imprinted during human embryogenesis and in the Beckwith-Wiedemann syndrome. Nature Genet., 4, 94-97. MEDLINE Abstract

5. Ogawa, O., Eccles, M.R., Szeto, J., McNoe, L.A., Yun, K., Maw, M.A., Smith, P.J. and Reeve, A.E. (1993) Relaxation of insulin-like growth factor II gene imprinting implicated in Wilms' tumour. Nature, 362, 749-751. MEDLINE Abstract

6. Zhang, Y. and Tycko, B. (1992) Monoallelic expression of the human H19 gene. Nature Genet., 1, 40-44. MEDLINE Abstract

7. Alders, M., Hodges, M., Hadjantonakis, A.K., Postmus, J., van Wijk, I., Bliek, J., de Meulemeester, M., Westerveld, A., Guillemot, F., Oudejans, C., Little, P. and Mannens, M. (1997) The human Achaete-Scute homologue 2 (ASCL2,HASH2) maps to chromosome 11p15.5, close to IGF2 and is expressed in extravillus trophoblasts. Hum. Mol. Genet., 6, 859-867. MEDLINE Abstract

8. Lee, M.P., Hu, R.-J., Johnson, L.A. and Feinberg, A.P. (1997) Human KvLQT1 gene shows tissue-specific imprinting and encompasses Beckwith-Wiedemann syndrome chromosomal rearrangements. Nature Genet., 15, 181-185. MEDLINE Abstract

9. Matsuoka, S., Thompson, J.S., Edwards, M.C., Barletta, J.M., Grundy, P., Kalikin, L.M., Harper, J.W., Elledge, S.J. and Feinberg, A.P. (1996) Imprinting of the gene encoding a human cyclin-dependent kinase inhibitor, p57KIP2, on chromosome 11p15. Proc. Natl Acad. Sci. USA, 93, 3026-3030. MEDLINE Abstract

10. Cooper, P.R., Smilinich, N.J., Day, C.D., Nowak, N.J., Reid, L.H., Pearsall, R.S., Reece, M., Prawitt, D., Landers, J., Housman, D.E., Winterpacht, A., Zabel, B.U., Pelletier, J., Weissman, B.E., Shows, T.B. and Higgins, M.J. (1998) Divergently transcribed overlapping genes expressed in liver and kidney and located in the 11p15.5 imprinted domain. Genomics, 49, 38-51. MEDLINE Abstract

11. Lee, M.P., Reeves, C., Schmitt, A., Su, K., Connors, T.D., Hu, R.-J., Brandenburg, S., Lee, M.J., Miller, G. and Feinberg, A.P. (1998) Somatic mutation of TSSC5, a novel imprinted gene from human chromosome 11p15.5. Cancer Res., 58, 4155-4159. MEDLINE Abstract

12. Dao, D., Frank, D., Qian, N., O'Keefe, D., Vosatka, R.J., Walsh, C.P. and Tycko, B. (1998) IMPT1, an imprinted gene similar to polyspecific transporter and multi-drug resistance genes. Hum. Mol. Genet., 7, 597-608. MEDLINE Abstract

13. Lee, M.P. and Feinberg, A.P. (1998) Genomic imprinting of a human apoptosis gene homologue, TSSC3. Cancer Res., 58, 1052-1056. MEDLINE Abstract

14. Qian, N., Frank, D., O'Keefe, D., Dao, D., Zhao, L., Yuan, L., Wang, Q., Keating, M., Walsh, C. and Tycko, B. (1997) The IPL gene on chromosome 11p15.5 is imprinted in human and mice and is similar to TDAG51, implicated in Fas expression and apoptosis. Hum. Mol. Genet., 6, 2021-2029. MEDLINE Abstract

15. Park, C.G., Lee, S.Y., Kandala, G., Lee, S.Y. and Choi, Y. (1996) A novel gene product that couples TCR signaling to Fas(CD95) expression in activation-induced cell death. Immunity, 4, 583-591. MEDLINE Abstract

16. Steenman, M.J.C., Rainier, S., Dobry, C.J., Grundy, P., Horon, I.L. and Feinberg, A.P. (1994) Loss of imprinting of IGF2 is linked to reduced expression and abnormal methylation of H19 in Wilms' tumor. Nature Genet., 7, 433-439. MEDLINE Abstract

17. Hatada, I., Ohashi, H., Fukushima, Y., Kaneko, Y., Inoue, M., Komoto, Y., Okada, A., Ohishi, S., Nabetani, A., Morisaki, H., Nakayama, M., Niikawa, N. and Mukai, T. (1996) An imprinted gene p57KIP2 is mutated in Beckwith-Wiedemann syndrome. Nature Genet., 14, 171-173. MEDLINE Abstract

18. Hatada, I., Nabetani, A., Morisaki, H., Ohishi, S., Inoue, M., Niikawa, N., Komoto, K., Okada, A., Steichen, E., Ohashi, H., Fukushima, Y., Nakayama, M. and Mukai, T. (1997) New p57KIP2 mutations in Beckwith-Wiedemann syndrome. Hum. Genet., 100, 681-683. MEDLINE Abstract

19. Lee, M.P., DeBaun, M., Randhawa, G.S., Reichard, B.A. and Feinberg, A.P. (1997) Low frequency of p57KIP2 mutation in Beckwith-Wiedemann syndrome. Am. J. Hum. Genet., 61, 304-309. MEDLINE Abstract

20. O'Keefe, D., Dao, D., Zhao, L., Sanderson, R., Warburton, D., Weiss, L., Anyane-Yeboa, K. and Tycko, B. (1998) Coding mutations in p57KIP2 are present in some cases of Beckwith-Wiedemann syndrome but are rare or absent in Wilms' tumors. Am. J. Hum. Genet., 61, 295-303.

21. Thompson, J.S., Reese, K.J., DeBaun, M.R., Perlman, E.J. and Feinberg, A.P. (1996) Reduced expression of the cyclin-dependent kinase inhibitor p57KIP2 in Wilms tumor. Cancer Res., 56, 5723-5727. MEDLINE Abstract

22. Sutcliffe, J.S., Nakao, M., Christian, S., Orstavik, K.H., Tommerup, N., Ledbetter, D.H. and Beaudet, A.L. (1994) Deletions of a differentially methylated CpG island at the SNRPN gene define a putative imprinting control region. Nature Genet., 8, 52-58. MEDLINE Abstract

23. Buiting, K., Saitoh, S., Gross, S., Dittrich, B., Schwartz, S., Nicholls, R.D. and Horsthemke, B. (1995) Inherited microdeletions in the Angelman and Prader-Willi syndromes define an imprinting centre on human chromosome 15. Nature Genet., 9, 395-400. MEDLINE Abstract

24. Koi, M., Johnson, L.A., Kalikin, L.M., Little, P.F.R., Nakamura, Y. and Feinberg, A.P. (1993) Tumor cell growth arrest caused by subchromosomal transferable DNA fragments from human chromosome 11. Science, 260, 361-364. MEDLINE Abstract

25. Hu, R.-J., Lee, M.P., Connors, T.D., Johnson, L.A., Burn, T.C., Su, K., Landes, G.M. and Feinberg, A.P. (1997) A 2.5-Mb transcript map of a tumor-suppressing subchromosomal transferable fragment from 11p15.5, and isolation and sequence analysis of three novel genes. Genomics, 46, 9-17. MEDLINE Abstract

26. Lee, M.P. and Feinberg, A.P. (1997) Aberrant splicing but not mutations of TSG101 in human breast cancer. Cancer Res., 57, 3131-3134. MEDLINE Abstract

27. Caspary, T., Cleary, M.A., Baker, C.C., Guan, X.J. and Tilghman, S.M. (1998) Multiple mechanisms regulate imprinting of the mouse distal chromosome 7 gene cluster. Mol. Cell. Biol., 18, 3466-3474. MEDLINE Abstract

28. Gould, T.D. and Pfeifer, K. (1998) Imprinting of mouse Kvlqt1 is developmentally regulated. Hum. Mol. Genet., 7, 483-487. MEDLINE Abstract

29. Altshul, S.F., Gish, W., Miller, W., Myers, E.W. and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol., 215, 403-410. MEDLINE Abstract

30. Feinberg, A.P. and Vogelstein, B. (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem., 132, 6-13. MEDLINE Abstract

*To whom correspondence should be addressed. Tel: +1 410 614 3489; Fax: +1 410 614 9819; Email: afeinberg@jhu.edu

This page is run by Oxford University Press, Great Clarendon Street, Oxford OX2 6DP, as part of the OUP Journals
Comments and feedback: www-admin{at}oup.co.uk
Last modification: 10 Mar 1999
Copyright©Oxford University Press, 1999.
Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 J ANIM SCIHome page
I. Zaitoun and H. Khatib
Comparative genomic imprinting and expression analysis of six cattle genes
J Anim Sci, January 1, 2008; 86(1): 25 - 32.
[Abstract] [Full Text] [PDF]

Home page
 Cold Spring Harb Symp Quant BiolHome page
A.P. FEINBERG
A Genetic Approach to Cancer Epigenetics
Cold Spring Harb Symp Quant Biol, January 1, 2005; 70(0): 335 - 341.
[Abstract] [PDF]

Home page
 Genome ResHome page
M. Paulsen, T. Khare, C. Burgard, S. Tierling, and J. Walter
Evolution of the Beckwith-Wiedemann syndrome region in vertebrates
Genome Res., January 1, 2005; 15(1): 146 - 153.
[Abstract] [Full Text] [PDF]

Home page
 J. Med. Genet.Home page
B Kerr, M L Mucchielli, S Sigaudy, M Fabre, P Saunier, M A Voelckel, E Howard, R Elles, T O B Eden, G C Black, et al.
Is the locus for Costello syndrome on 11p?
J. Med. Genet., June 1, 2003; 40(6): 469 - 471.
[Full Text] [PDF]

Home page
 JCBHome page
N. L. Mahy, P. E. Perry, and W. A. Bickmore
Gene density and transcription influence the localization of chromatin outside of chromosome territories detectable by FISH
J. Cell Biol., December 9, 2002; 159(5): 753 - 763.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
J. M. Tarrant, J. Groom, D. Metcalf, R. Li, B. Borobokas, M. D. Wright, D. Tarlinton, and L. Robb
The Absence of Tssc6, a Member of the Tetraspanin Superfamily, Does Not Affect Lymphoid Development but Enhances In Vitro T-Cell Proliferative Responses
Mol. Cell. Biol., July 15, 2002; 22(14): 5006 - 5018.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
R. Weksberg, J. Nishikawa, O. Caluseriu, Y.-L. Fei, C. Shuman, C. Wei, L. Steele, J. Cameron, A. Smith, I. Ambus, et al.
Tumor development in the Beckwith-Wiedemann syndrome is associated with a variety of constitutional molecular 11p15 alterations including imprinting defects of KCNQ1OT1
Hum. Mol. Genet., December 1, 2001; 10(26): 2989 - 3000.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
R. M. John, J. F. -X. Ainscough, S. C. Barton, and M. A. Surani
Distant cis-elements regulate imprinted expression of the mouse p57 Kip2 (Cdkn1c) gene: implications for the human disorder, Beckwith-Wiedemann syndrome
Hum. Mol. Genet., July 1, 2001; 10(15): 1601 - 1609.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S. Engemann, M. Strodicke, M. Paulsen, O. Franck, R. Reinhardt, N. Lane, W. Reik, and J. Walter
Sequence and functional comparison in the Beckwith-Wiedemann region: implications for a novel imprinting centre and extended imprinting
Hum. Mol. Genet., November 1, 2000; 9(18): 2691 - 2706.
[Abstract] [Full Text] [PDF]

Home page
 Genome ResHome page
P. Onyango, W. Miller, J. Lehoczky, C. T. Leung, B. Birren, S. Wheelan, K. Dewar, and A. P. Feinberg
Sequence and Comparative Analysis of the Mouse 1-Megabase Region Orthologous to the Human 11p15 Imprinted Domain
Genome Res., November 1, 2000; 10(11): 1697 - 1710.
[Abstract] [Full Text]

Home page
 Am. J. Pathol.Home page
W. Hartmann, A. Waha, A. Koch, C. G. Goodyer, S. Albrecht, D. von Schweinitz, and T. Pietsch
p57KIP2 Is Not Mutated in Hepatoblastoma but Shows Increased Transcriptional Activity in a Comparative Analysis of the Three Imprinted Genes p57KIP2, IGF2, and H19
Am. J. Pathol., October 1, 2000; 157(4): 1393 - 1403.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
S.-i. Horike, K. Mitsuya, M. Meguro, N. Kotobuki, A. Kashiwagi, T. Notsu, T. C. Schulz, Y. Shirayoshi, and M. Oshimura
Targeted disruption of the human LIT1 locus defines a putative imprinting control element playing an essential role in Beckwith-Wiedemann syndrome
Hum. Mol. Genet., September 1, 2000; 9(14): 2075 - 2083.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
M. Paulsen, O. El-Maarri, S. Engemann, M. Strodicke, O. Franck, K. Davies, R. Reinhardt, W. Reik, and J. Walter
Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse
Hum. Mol. Genet., July 22, 2000; 9(12): 1829 - 1841.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
D. Prawitt, T. Enklaar, G. Klemm, B. Gartner, C. Spangenberg, A. Winterpacht, M. Higgins, J. Pelletier, and B. Zabel
Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression
Hum. Mol. Genet., January 22, 2000; 9(2): 203 - 216.
[Abstract] [Full Text] [PDF]

Home page
 Hum Mol GenetHome page
K. Mitsuya, M. Meguro, M. P. Lee, M. Katoh, T. C. Schulz, H. Kugoh, M. A. Yoshida, N. Niikawa, A. P. Feinberg, and M. Oshimura
LIT1, an imprinted antisense RNA in the human KvLQT1 locus identified by screening for differentially expressed transcripts using monochromosomal hybrids
Hum. Mol. Genet., July 1, 1999; 8(7): 1209 - 1217.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (39)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Lee, M. P.
Right arrow Articles by Feinberg, A. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, M. P.
Right arrow Articles by Feinberg, A. P.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?