Human Molecular Genetics, 2001, Vol. 10, No. 9 903-910
© 2001 Oxford University Press
The endothelin receptor B (EDNRB) promoter displays heterogeneous, site specific methylation patterns in normal and tumor cells
1Department of Biochemistry and Molecular Biology, University of Southern California/Norris Comprehensive Cancer Center, Los Angeles, CA 90089-9176, USA, 2Department of Urology, Hitachi General Hospital, 2-1-1 Jonancho, Hitachi-shi, Ibaraki, 317-0077, Japan and 3Department of Pathology and Microbiology, University of Nebraska Medical Center, Omaha, NE 68198, USA
Received 12 December 2000; Revised and Accepted 19 February 2001.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The 5' region for the endothelin receptor B (EDNRB) gene is a complex CpG island giving rise to four individual transcripts initiating within the island. Here, for the first time, we analyze the relationship between methylation and gene expression in a CpG island located in the 5' region of a gene with multiple transcription start sites. The CpG island was unmethylated in normal prostate and bladder tissue, whereas it became methylated in apparently normal colonic epithelium. Tumors derived from these tissues were frequently hypermethylated relative to the respective normal tissues. Analysis of 11 individual CpG sites located throughout the CpG island showed that specific sites with high methylation levels in several tumors were also methylated in normal tissues, suggesting that they might serve as foci for further de novo methylation. This region also had high levels of methylation in several cancer cell lines, and we found that a low methylation level in a small region within the 5' region correlated with expression of the 5'-most transcript. Interestingly, almost complete methylation 200–1000 bp downstream of the transcriptional start site did not block expression of this transcript. Finally, we show that treatment with 5-aza-2'-deoxycytidine can induce transcriptional activation of the four EDNRB transcripts. Our results show the existence of differential, tissue-dependent methylation at the EDNRB 5' region, suggest the existence of a spreading mechanism for de novo methylation, starting from methylation hotspots, and show that hypermethylation immediately 3' to a transcriptional start site does not prevent initiation.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
DNA methylation has been implicated in transcriptional repression (1) and the formation of condensed inactive chromatin (2,3). Methylation is required for normal mammalian development (4) and generally occurs at CpG dinucleotides (5), which are found in lower than expected frequency in the genome except in CpG islands. These are regions of
1 kb in length which are often associated with promoters or transcribed regions of genes and are generally not methylated (6). It has become increasingly clear that autosomal genes can be silenced in cancer by abnormal de novo methylation of CpG islands, leading to transcriptional downregulation of gene expression (7,8). Examples of genes that are frequent targets for de novo methylation include CDKN2A (p16/INK4A) (9,10), RB1 (11,12) and CDKN2B (p15/INK4B) (13), the mismatch repair gene MLH1 (14–17) and the estrogen receptor gene (ESR1) (18). The possible number of genes that may be targeted for de novo methylation in cancer is in all probability much higher than currently known, and scanning techniques, such as arbitrarily primed PCR (AP-PCR) (19), provide a rapid way to determine general patterns of methylation changes in cancer cells and allow us to find new genes that have undergone abnormal methylation changes during oncogenesis. Using the AP-PCR technique we found previously that the 5' region of the endothelin receptor B (EDNRB) gene is hypermethylated in cancer compared with white blood cell (WBC) DNA (20). Other investigators had previously shown that the EDNRB gene is abnormally methylated in prostate cancer (21), but the relationship of this methylation with expression was not examined. The role of EDNRB in mediating vaso-constriction has been well established. Mutations in the EDNRB gene also give rise to Hirschsprung’s disease (22), a condition characterized by megacolon and abnormal skin pigmentation (23). Furthermore, recent findings show that EDNRB signaling is necessary during development for proper migration of cells derived from the neural crest, including melanoblasts and enteric neuroblasts (24). A role for EDNRB in carcinogenesis has not yet been established; however, EDNRB joins a growing number of genes that are important for normal development and may become disregulated in cancer (25).
The EDNRB gene has a complex 5' region which we have shown recently to give rise to four different transcripts (20). Three of these transcripts encode for the EDNRB protein, but have unique splicing of the 5'-untranslated region (5'-UTR), whereas a more upstream transcript has the potential of generating a new EDNRB protein with a unique N-terminus. The roles and regulation of these novel transcripts are not known; however, the known 5' region of the EDNRB gene is a CpG island spanning 1 kb, and the transcriptional start sites for all four EDNRB transcripts are located within this CpG island. DNA methylation in promoter regions is known to decrease transcriptional activity, but its effect on multiple transcripts within the same 5' region has not been examined previously.
In the present study we studied the relationship between expression and DNA methylation in a CpG island with multiple transcriptional start sites, as this would extend our understanding of the connection between methylation and transcriptional silencing. Specifically, analysis of the 5' region of the EDNRB gene in a series of tumor and adjacent normal tissues of prostate, bladder and colon origin showed that normal prostate and bladder tissues are unmethylated, as expected, whereas normal colonic tissue revealed a moderate level of methylation. Furthermore, the tumor tissue was generally hypermethylated relative to the normal tissue. Our analysis included methylation data on 11 individual CpG sites spanning the whole island and, interestingly, several non-adjacent CpG sites showed high levels of methylation in tumor tissues and some of the normal samples. This pattern is suggestive of these sites serving as a seeding point for methylation. Methylation could potentially spread from these sites through the remainder of the CpG island, providing a potential mechanism to explain de novo methylation of CpG islands found in cancer cells. To elucidate the relationship between the 5' region methylation and expression of the four EDNRB transcripts, we analyzed expression and methylation in a panel of cancer cell lines. Low methylation levels in a small region within the CpG island correlated with expression of the EDNRB 
3 transcript, the 5'-most transcript in this cluster. Surprisingly, high levels of methylation immediately downstream (200–1000 bp) from EDNRB 3 did not block its transcriptional activity, which is in disagreement with other studies (26,27) performed in plasmid and episomal vectors, where methylation along the vector backbone does inhibit transcriptional activity. Lastly, using 5-aza-2'-deoxycytidine (5-Aza-CdR) we showed that expression of the four EDNRB transcripts could be induced by DNA demethylation, further supporting the role of methylation in the control of this complex promoter. Our findings suggest the existence of a spreading mechanism for de novo methylation starting from distinct methylation hotspots and show that hypermethylation in the 5' region of a gene does not necessarily equate with transcriptional silencing.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The EDNRB 5' region is a CpG island.
Our earlier AP-PCR analysis showed a fragment that was hypermethylated in tumor tissue compared with WBC DNA and which corresponded to the 5' region of the EDNRB gene. Sequencing upstream of the known sequence (GenBank accession no. D13162) showed that the 2000 bp encompassing the EDNRB 5' region and exon 1 is a CpG island, as defined by Antequera et al. (28) (Fig. 1). The CpG island has a GC content of 55% and a CpG observed/expected ratio of 0.74. Figure 1 also shows the transcriptional start sites for the four transcripts of the EDNRB gene located within this CpG island (20).
|
EDNRB 5' region methylation in normal and tumor tissue
A panel of tumor and adjacent normal tissues was screened to determine whether there were any changes in methylation levels at the EDNRB locus (Fig. 2). The methylation levels of 11 individual CpG sites in the EDNRB 5' region (Fig. 1) were first measured in tissues derived from the prostate, bladder and colon using the quantitative methylation sensitive-single nucleotide primer extension (Ms-SNuPE) technique. In normal prostate tissue the levels of methylation were generally low, as is expected for normal tissues (Fig. 2A) with increased methylation at CpG -130, the 5'-most CpG dinucleotide analyzed, which is located at the fringe of the CpG island. Prostate tumor tissue had mostly low methylation levels, nevertheless the levels were higher than in the normal adjacent tissue. Higher methylation levels measured in the 3' end of the CpG island were consistent with conclusions from a previous study (21). Normal bladder tissue was generally unmethylated, with the exception of CpG -130, where methylation was high (Fig. 2B) and two distinct patterns of methylation could be discerned in bladder tumors. Some bladder tumors showed low levels of methylation in the EDNRB 5' region, but substantial hypermethylation was observed in a subset of bladder tumors. Some sites were consistently heavily methylated (CpG 377, CpG 618, CpG 654, CpG 1115) in these tumors and might therefore represent sites targeted for preferential de novo methylation. Conversely, CpG 336 remained resistant to hypermethylation, even when adjacent CpGs were highly methylated.
|
In contrast to prostate and bladder normal tissues, apparently normal colonic epithelium obtained from patients with colorectal cancer was extensively methylated (Fig. 2C). Hypermethylation of other CpG islands in normal colonic mucosa has been documented previously and has been associated with aging (29). The EDNRB 5' region was also a target for increased methylation in colon tumors and CpG 377, CpG 618, CpG 654 and CpG 1115 had the highest levels of methylation, whereas CpG 336 again appeared to be protected from increased methylation, in a pattern analogous to the one observed in bladder tumors. These findings showed that in the EDNRB 5' regulatory region, prostate, bladder and colon normal tissue have methylation patterns that are particular to each tissue type. Our findings also showed that hypermethylation at the EDNRB 5' region is common in tumors, regardless of the tissue type. Furthermore, some sites within the CpG island appeared to be preferential targets for de novo methylation, whereas others seemed to be protected from hypermethylation changes.
EDNRB expression and methylation profile in cancer cell lines
Next, we asked if the hypermethylation in the EDNRB 5' region correlated with expression of any of the EDNRB transcripts. The methylation profiles of the 5' region in a panel of cancer cell lines were measured and compared with the expression of each of the four EDNRB transcripts in the same cell lines (Fig. 3). Figure 3A shows that of nine cell lines analyzed only the SK-Mel-28 melanoma cell line strongly expressed all four transcripts as was reported previously (20). EDNRB expression was detected in two additional cell lines. J82 expressed EDNRB 3 strongly and expressed EDNRB 1 and the original EDNRB transcript weakly, but did not express EDNRB 2. Weak expression of EDNRB 3 was detected in SW48 and the remaining cell lines did not express any EDNRB transcripts.
|
Methylation was not detected throughout the EDNRB 5' region in the SK-Mel-28 cell line (Fig. 3B), which correlated well with the high levels of expression of all EDNRB transcripts in this cell line. The methylation profile for the J82 cell line showed a bipartite pattern with the upstream region comprising five CpG sites (CpG -130 to CpG 336) having a lower level of methylation (26%) compared with the more downstream region comprising the latter six CpG sites, where the average methylation was considerably higher (75%). Therefore, the low level of methylation in the upstream region correlated with strong expression of EDNRB
3, but not of the other transcripts and high methylation levels downstream of the transcriptional start site did not block transcription of EDNRB 3.
EDNRB expression can be induced by 5-Aza-CdR treatment
We reasoned that if EDNRB expression was inhibited by DNA methylation, then treatment of cells with the demethylating agent 5-Aza-CdR would induce expression of EDNRB transcripts. The bladder cancer cell line T24 which had high methylation levels in the 5' region of EDNRB (Fig. 3B) and did not express any EDNRB transcript (Fig. 3A), was therefore treated with the drug and expression of EDNRB transcripts monitored as a function of time after treatment (Fig. 4A). Figure 4A shows a representative analysis of the kinetics of reactivation of EDNRB transcripts following 5-Aza-CdR treatment. The original, 1 and 3 transcripts were coordinately induced with peak expression seen at day 21 after treatment, suggesting a common regulatory mechanism. The EDNRB 2 transcript was induced more weakly and expression was detected on days 21 and 35 after treatment. The kinetics of expression were confirmed in a separate experiment (data not shown) which showed minor variations in maximal expression levels and timing. An additional five cell lines with low or no expression of EDNRB transcripts (SW48, 5637, SW837, HCT-15, HCT116) were treated with 5-Aza-CdR. In all cases reactivation of EDNRB transcripts was detected after treatment with the drug (data not shown) showing that 5-Aza-CdR-mediated transcript reactivation of EDNRB transcripts was not limited to the T24 cell line.
|
The methylation profile of the CpG island in 5-Aza-CdR-treated T24 cells was measured over a period of 35 days (Fig. 4B) to determine the possible relationship between methylation and expression. The initial methylation at this locus in the T24 cells was high, as was determined previously (Fig. 3B) and maximal demethylation was reached between days 3 and 6 after drug treatment. At day 21 post treatment remethylation was detectable and increased over time. This increasing methylation was followed by decreasing expression levels of the EDNRB transcripts. It is of note that the CpG sites within this island did not remethylate with equal kinetics, but that site specificity was detected. CpGs 165, 377 and 654 showed the most rapid rate of remethylation, whereas CpG 336 seemed protected. This pattern was similar to the one observed in tumor samples. Therefore, measurement of the kinetics of remethylation at the EDNRB 5' region showed that methylation was correlated with transcriptional activity and that there was preferential targeting of some CpG dinucleotides for remethylation.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
The purpose of this study was to investigate the role of DNA methylation in the regulation of the complex EDNRB 5' region, a CpG island containing three transcription start sites giving rise to four distinct mRNA transcripts. We have found increased levels of methylation in tumor compared with normal samples derived from colon, bladder and prostate tissues. A few individual CpG dinucleotides exhibited higher methylation levels in normal tissue and might represent seeding points from which methylation spreads in tumor samples. Transcription of EDNRB mRNA was detected even in the presence of high levels of methylation downstream of the transcriptional start site.
Hypermethylation in tumors relative to normal tissue has been described before in many instances (7,8) and it has been suggested that these changes can be useful as markers for the early detection of cancer cells. Hypermethylation in the EDNRB 5' region could potentially be used as a marker for malignancy and our extensive study of the EDNRB 5' region shows that selected CpGs within the island may be more reliable markers for malignancy. However, background noise particular to a tissue type must be taken into account. For example, a previous study in the EDNRB promoter found frequent hypermethylation in prostate tumors at the more 3' end of the EDNRB promoter (21), yet, we show that this hypermethylation seldom spreads to the more upstream sites in prostate tumors. Conversely, bladder and colon tumors are often highly methylated in the 5' region, with the highest methylation levels found around a few ‘hotspot’ sites. These hotspots could possibly represent the most appropriate CpGs to use as markers for the detection of early methylation changes. The intrinsic basal levels of methylation varied in each tissue so that prostate and bladder normal tissue were mostly unmethylated as expected. However, colon normal tissue had levels of methylation which sometimes exceeded the levels in prostate or bladder tumors. This situation is analogous to the one in the estrogen receptor (ESR1) promoter, where hypermethylation in normal colon tissue has been correlated to ageing (18). Therefore, differences in basal methylation levels vary between tissue types and must be taken into account when measuring hypermethylation events. Although the EDNRB 5' region displays abnormal methylation patterns in tumors of the colon, bladder and prostate, additional experiments will be needed to confirm its value as a prognostic marker for the detection of neoplastic changes.
Little is known about the mechanisms that result in CpG island hypermethylation in cancer. Proposed mechanisms include the binding of proteins, such as SpI, that prevent methylation of the island (30,31) or the presence of boundary sequences that demarcate methylated and unmethylated regions (32,33). We detected sites within the EDNRB 5' region that were methylated in normal tissue and showed further hypermethylation in tumor tissue. This result is suggestive of the presence of ‘hotspots’ for methylation, rather than a defined boundary between methylated and unmethylated sequences, and supports a model for a gradual increase of methylation originating from the core of the EDNRB island.
Previous studies using plasmid and episomal vectors (26,27) have used patch methylation techniques to show that methylation downstream of a promoter can decrease transcription levels. In the J82 cell line EDNRB 3 was expressed even in the presence of almost complete methylation in the region 200–1000 bp downstream of the transcriptional start site. Therefore, a promoter in its native chromosomal context may be subject to levels of transcriptional control not present in plasmids or episomes. We do not know whether this extensive methylation attenuated the transcription level, but the data clearly show that extensive methylation closely downstream of the initiation site does not completely silence a gene.
Demethylation using 5-Aza-CdR in T24 cells was of interest since it was revealed that activated transcripts could be coordinately expressed and there was a considerable lag time between the demethylation event and maximal expression levels. All four EDNRB transcripts were activated by treatment with 5-Aza-CdR and in three of the four transcripts the expression patterns observed over 35 days were surprising; expression was clearly coordinated between the individual transcripts. We expected to see preferential expression of one transcript, following the model of transcriptional interference (34–36). However, it should be noted that all EDNRB transcription start sites are located within one CpG island, and this is the first time a promoter of its type has been analyzed. It is possible that the close proximity of the transcription start sites opens up the chromatin of the whole region when one transcript is activated, thus resulting in coordinate expression of all the mRNAs. Another interesting observation was the fact that maximal expression levels were not obtained until several days after the treatment. Previous reports showed that p16/INK4A reactivation closely followed treatment with 5-Aza-CdR, and maximal expression was obtained 3–6 days after drug treatment (37). The different kinetics of expression and methylation at this 5' region together with the unique promoter architecture suggests that in addition to DNA methylation, other layers of control are at work in the regulation of expression of this complex 5' region.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Tissue samples
Human bladder, colon and prostate tumor tissue and adjacent normal tissue were obtained from patients from the Los Angeles County/University of Southern California Medical Center and the University of Southern California/Norris Comprehensive Cancer Center (Los Angeles, CA), following institutional guidelines. To ensure purity of sample, mucosal tissue was dissected from surrounding muscle and adipose tissue, as previously described (10,38,39). DNA and RNA was isolated as described previously (40).
Cell culture
Colorectal cancer cell lines (HCT116, LoVo, HCT-15, SW837, SW48) and bladder cancer cell lines (T24, J82, 5637) were obtained from the American Type Culture Collection (Rockville, MD). The melanoma cell line SK-Mel-28 was obtained from Memorial Sloan Kettering (New York, NY). All colorectal cancer cell lines and T24 and 5637 were cultured in McCoy’s 5A medium (Life Technologies). J82 was cultured in MEM (Life Technologies) supplemented with 0.1 mM non-essential amino acids and 1mM sodium pyruvate. SK-Mel-28 was cultured in DMEM (Life Technologies). All media were supplemented with 10% heat-inactivated FCS, 100 U/ml penicillin and 10 µg/ml streptomycin. Cell lines were maintained at 37°C, 5% CO2-95% air in standard tissue culture incubators under sterile conditions.
5-Aza-CdR treatment
Cells (3 x 105) were seeded in a 100 mm cell culture dish 24 h prior to treatment with 5-Aza-CdR (Sigma). Cells were incubated with 10–6 M 5-Aza-CdR for 24 h, medium was changed and cells were incubated for another 24 h with freshly prepared 10–6 M 5-Aza-CdR. At the conclusion of the treatment, medium was changed, and cells kept non-confluent and growing with medium changes every 3 days. The 5-Aza-CdR treatments were performed multiple times to ensure reproducibility of results. DNA and RNA were harvested at various timepoints as described (40).
RT–PCR
Reverse transcription was performed using total RNA (2.5 µg), MMLV-reverse transcriptase (Life Technologies) and random hexamers (Amersham Pharmacia Biotech) in a volume of 25 µl, as described previously (40). The sequences of the four EDNRB transcript variants have been described (20,41,42). We generated primers that would specifically amplify EDNRB 1, EDNRB 2 and EDNRB 3. Due to sequence constraints it is not possible to design primers that amplify only the original EDNRB transcript, so we designed primers that amplify both the original EDNRB transcript and EDNRB 1 and called the product EDNRB original + 1. PCR amplifications were performed using 100 ng cDNA, 1 U Expand High Fidelity PCR system enzyme mix (Roche Molecular Biochemicals), 0.2 mM each dNTP, 0.5 µM each sense and antisense primer and 1x Expand HF buffer in a total volume of 25 µl. Additionally, amplification of EDNRB original + 1 and EDNRB 2 required 10% DMSO.
All PCR amplifications started with an initial denaturation step at 94°C for 4 min and ended with a final extension step at 72°C for 10 min. PCR cycling conditions for EDNRB original + 1 were 36 cycles of 94°C for 45 s, 65°C for 60 s and 72°C for 60 s. The primers were EDNRB orig. + 1, 5'-GGC GGT ATT AGC GTT TGC AGC GAC TT-3' (sense), and EDNRB orig. + 1, 5'-GCC AGT CCT CTG CCA GCA GC-3' (antisense). PCR cycling conditions for EDNRB 1 were 38 cycles of 94°C for 45 s, 67°C for 60 s and 72°C for 60 s. Primers were EDNRB 1, 5'- AGC TTT GCC TGG GAC CCC CAT C-3' (sense), and EDNRB 1, 5'-GCC AGT CCT CTG CCA GCA GC-3' (antisense). PCR cycling conditions for EDNRB 2 were 35 cycles of 94°C for 45 s, 63°C for 60 s and 72°C for 60 s. Primers were EDNRB 2, 5'-GGA GCT GTA GCT CAG CCA GC-3' (sense), and EDNRB 2, 5'-GAG ATG GTG CGT GGC GGA GA-3' (antisense). PCR cycling conditions for EDNRB 3 were 34 cycles of 94°C for 45 s, 62°C for 30 s and 72°C for 45 s. Primers used were EDNRB 3, 5'-CGA GCA AAC GGT GGA GGC TAC A-3' (sense) and EDNRB 3, 5'-CGG CTG CAT GCT GCT ACC TG-3' (antisense). PCR products were resolved on a 2% agarose gel and transferred onto a Zeta-Probe membrane (Bio-Rad Laboratories) via alkaline transfer. Membranes were probed using a 5' end labeled oligonucleotide with sequence 5'-CTC TGA AAC TGC GGA GCG GCC AC-3' and exposed to autoradiographic BioMax MR film (Eastman Kodak).
Methylation analysis
Methylation status in the 5' region of the EDNRB gene was determined by Ms-SNuPE (43). This technique depends on chemical modification of DNA using sodium bisulfite, where unmethylated cytosine residues are converted to uracil residues but methylated cytosines remain unchanged. DNA samples were bisulfite-converted as described previously (44).
Ms-SNuPE was performed as described previously (43). In order to analyze a representative number of CpG dinucleotides spanning the 5' region of the EDNRB gene, four separate bisulfite-PCR products were generated. EDNRB segment 0 allowed for analysis of CpG dinucleotides at positions –130 and –8. EDNRB segment 1 allowed for analysis of CpG dinucleotides at positions 165, 291 and 336. EDNRB segment 2 allowed for analysis of CpG sites at positions 336, 377, 431, 618 and 654. EDNRB segment 3 allowed for analysis of CpG 994 and 1115. Numbering is relative to GenBank accession no. D13162. PCR and Ms-SNuPE conditions have been described previously (40).
Primers used for PCR were as follows:
EDNRB segment 0: 5'-GGG TAA AAT GAA GTA GAG TAA AGA GTA G-3' (sense); EDNRB segment 0: 5'-CTC TTC AAA TAA ACC CAA ATC AAA AAC AAA TTA TCA C-3' (antisense); EDNRB segment 1: 5'-TAA TTA TTA TTG ATG TTG TTT AGG T-3' (sense); EDNRB segment 1: 5'-TTC CAA CCT ACT CTA AAA AAA A-3' (antisense); EDNRB segment 2: 5'-TTT TAG AGT AGG TTG GAA TTT A-3' (sense); EDNRB segment 2: 5'-ACT CCC TAA CTA ACT AAA CT-3' (antisense); EDNRB segment 3: 5'-GGA GTT TTG TTT GGG ATT TTT ATT-3' (sense); EDNRB segment 3: 5'-ACA AAA CAC TTA AAT CAA CTA CC-3'(antisense).
Ms-SNuPE primers were as follows:
CpG-130: 5'-GAA GTA GAG TAA AGA GTA G-3';
CpG-8: 5'-GTT TTG TTT TAG TTT GGA GTT GT-3';
CpG 165: 5'-AGG GGA AAG GTT GTA G-3';
CpG 291: 5'-AGG TTA TAT TGT TTG GTA TTT T-3';
CpG 336: 5'-TTT GTA GTT TAA GGG AGG-3';
CpG 377: 5'-TGG AAT TTA GTT GGG TTT-3';
CpG 431: 5'-TTG TAT TTG GTT TGT TAG ATT-3';
CpG 618: 5'-GTT TGG AGG GAA TAG-3';
CpG 654: 5'-GTT GAT TTG AGA AGT TTT TG-3';
CpG 994: 5'-TGT ATA TTA TTT ATT TTT TTT GGT TA-3';
CpG 1115: 5'-TAA ATT TGA GTT ATT TTT GAG-3'.
|
ACKNOWLEDGEMENTS |
|---|
We thank Daniel Weisenberger for helpful discussions. This work was supported by grant USPHS R35 CA 49758 from the National Cancer Institute.
|
FOOTNOTES |
|---|
+ To whom correspondence should be addressed. Tel: +1 323 865 0816; Fax: +1 323 865 0102; E-mail: jones_p@ccnt.hsc.usc.edu
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
1 Bird, A.P. (1986) CpG-rich islands and the function of DNA methylation. Nature, 321, 209–213.[Medline]
2 Bird, A.P. and Wolffe, A.P. (1999) Methylation-induced repression—belts, braces, and chromatin. Cell, 99, 451–454.[Web of Science][Medline]
3 Keshet, I., Lieman-Hurwitz, J. and Cedar, H. (1986) DNA methylation affects the formation of active chromatin. Cell, 44, 535–543.[Web of Science][Medline]
4 Li, E., Bestor, T.H. and Jaenisch, R. (1992) Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell, 69, 915–926.[Web of Science][Medline]
5 Bird, A. (1992) The essentials of DNA methylation. Cell, 70, 5–8.[Web of Science][Medline]
6 Cooper, D.N. and Krawczak, M. (1989) Cytosine methylation and the fate of CpG dinucleotides in vertebrate genomes. Hum. Genet., 83, 181–188.[Web of Science][Medline]
7 Baylin, S.B. and Herman, J.G. (2000) DNA hypermethylation in tumorigenesis: epigenetics joins genetics. Trends Genet., 16, 168–174.[Web of Science][Medline]
8 Jones, P.A. and Laird, P.W. (1999) Cancer epigenetics comes of age. Nature Genet., 21, 163–167.[Web of Science][Medline]
9 Merlo, A., Herman, J.G., Mao, L., Lee, D.J., Gabrielson, E., Burger, P.C., Baylin, S.B. and Sidransky, D. (1995) 5' CpG island methylation is associated with transcriptional silencing of the tumour suppressor p16/CDKN2/MTS1 in human cancers. Nat. Med., 1, 686–692.[Web of Science][Medline]
10 Gonzalez-Zulueta, M., Bender, C.M., Yang, A.S., Nguyen, T., Beart, R.W., Van Tornout, J.M. and Jones, P.A. (1995) Methylation of the 5' CpG island of the p16/CDKN2 tumor suppressor gene in normal and transformed human tissues correlates with gene silencing. Cancer Res., 55, 4531–4535.
11 Ohtani-Fujita, N., Fujita, T., Aoike, A., Osifchin, N.E., Robbins, P.D. and Sakai, T. (1993) CpG methylation inactivates the promoter activity of the human retinoblastoma tumor-suppressor gene. Oncogene, 8, 1063–1067.[Web of Science][Medline]
12 Stirzaker, C., Millar, D.S., Paul, C.L., Warnecke, P.M., Harrison, J., Vincent, P.C., Frommer, M. and Clark, S.J. (1997) Extensive DNA methylation spanning the Rb promoter in retinoblastoma tumors. Cancer Res., 57, 2229–2237.
13 Herman, J.G., Jen, J., Merlo, A. and Baylin, S.B. (1996) Hypermethylation-associated inactivation indicates a tumor suppressor role for p15INK4B. Cancer Res., 56, 722–727.
14 Veigl, M.L., Kasturi, L., Olechnowicz, J., Ma, A.H., Lutterbaugh, J.D., Periyasamy, S., Li, G.M., Drummond, J., Modrich, P.L., Sedwick, W.D. et al. (1998) Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc. Natl Acad. Sci. USA, 95, 8698–8702.
15 Deng, G., Chen, A., Hong, J., Chae, H.S. and Kim, Y.S. (1999) Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res., 59, 2029–2033.
16 Herman, J.G., Umar, A., Polyak, K., Graff, J.R., Ahuja, N., Issa, J.P., Markowitz, S., Willson, J.K., Hamilton, S.R., Kinzler, K.W. et al. (1998) Incidence and functional consequences of hMLH1 promoter hypermethylation in colorectal carcinoma. Proc. Natl Acad. Sci. USA, 95, 6870–6875.
17 Kane, M.F., Loda, M., Gaida, G.M., Lipman, J., Mishra, R., Goldman, H., Jessup, J.M. and Kolodner, R. (1997) Methylation of the hMLH1 promoter correlates with lack of expression of hMLH1 in sporadic colon tumors and mismatch repair-defective human tumor cell lines. Cancer Res., 57, 808–811.
18 Issa, J.P., Ottaviano, Y.L., Celano, P., Hamilton, S.R., Davidson, N.E. and Baylin, S.B. (1994) Methylation of the oestrogen receptor CpG island links ageing and neoplasia in human colon. Nature Genet., 7, 536–540.[Web of Science][Medline]
19 Liang, G., Salem, C.E., Yu, M.C., Nguyen, H.D., Gonzales, F.A., Nguyen, T.T., Nichols, P.W. and Jones, P.A. (1998) DNA methylation differences associated with tumor tissues identified by genome scanning analysis. Genomics, 53, 260–268.[Web of Science][Medline]
20 Tsutsumi, M., Liang, G. and Jones, P.A. (1999) Novel endothelin B receptor transcripts with the potential of generating a new receptor. Gene, 228, 43–49.[Web of Science][Medline]
21 Nelson, J.B., Lee, W.H., Nguyen, S.H., Jarrard, D.F., Brooks, J.D., Magnuson, S.R., Opgenorth, T.J., Nelson, W.G. and Bova, G.S. (1997) Methylation of the 5' CpG island of the endothelin B receptor gene is common in human prostate cancer. Cancer Res., 57, 35–37.
22 Puffenberger, E.G., Hosoda, K., Washington, S.S., Nakao, K., deWit, D., Yanagisawa, M. and Chakravart, A. (1994) A missense mutation of the endothelin-B receptor gene in multigenic Hirschsprung’s disease. Cell, 79, 1257–1266.[Web of Science][Medline]
23 Jackson, I.J. (1997) Homologous pigmentation mutations in human, mouse and other model organisms. Hum. Mol. Genet., 6, 1613–1624.
24 Shin, M.K., Levorse, J.M., Ingram, R.S. and Tilghman, S.M. (1999) The temporal requirement for endothelin receptor-B signalling during neural crest development. Nature, 402, 496–501.[Medline]
25 Ford, H.L. (1998) Homeobox genes: a link between development, cell cycle, and cancer? Cell Biol. Int., 22, 397–400.[Web of Science][Medline]
26 Kass, S.U., Goddard, J.P. and Adams, R.L. (1993) Inactive chromatin spreads from a focus of methylation. Mol. Cell. Biol., 13, 7372–7379.
27 Hsieh, C.L. (1997) Stability of patch methylation and its impact in regions of transcriptional initiation and elongation. Mol. Cell Biol., 17, 5897–5904.[Abstract]
28 Antequera, F., Boyes, J. and Bird, A. (1990) High levels of de novo methylation and altered chromatin structure at CpG islands in cell lines. Cell, 62, 503–514.[Web of Science][Medline]
29 Ahuja, N., Li, Q., Mohan, A.L., Baylin, S.B. and Issa, J.P. (1998) Aging and DNA methylation in colorectal mucosa and cancer. Cancer Res., 58, 5489–5494.
30 Macleod, D., Charlton, J., Mullins, J. and Bird, A.P. (1994) Sp1 sites in the mouse aprt gene promoter are required to prevent methylation of the CpG island. Genes Dev., 8, 2282–2292.
31 Brandeis, M., Frank, D., Keshet, I., Siegfried, Z., Mendelsohn, M., Nemes, A., Temper, V., Razin, A. and Cedar, H. (1994) Sp1 elements protect a CpG island from de novo methylation. Nature, 371, 435–438.[Medline]
32 Melki, J.R., Vincent, P.C. and Clark, S.J. (1999) Cancer-specific region of hypermethylation identified within the HIC1 putative tumour suppressor gene in acute myeloid leukaemia. Leukemia, 13, 877–883.[Web of Science][Medline]
33 Millar, D.S., Paul, C.L., Molloy, P.L. and Clark, S.J. (2000) A distinct sequence (ATAAA)n separates methylated and unmethylated domains at the 5'-end of the GSTP1 CpG island. J. Biol. Chem., 275, 24893–24899.
34 Bateman, E. and Paule, M.R. (1988) Promoter occlusion during ribosomal RNA transcription. Cell, 54, 985–992.[Web of Science][Medline]
35 Wu, J., Grindlay, G.J., Bushel, P., Mendelsohn, L. and Allan, M. (1990) Negative regulation of the human epsilon-globin gene by transcriptional interference: role of an Alu repetitive element. Mol. Cell. Biol., 10, 1209–1216.
36 Proudfoot, N.J. (1986) Transcriptional interference and termination between duplicated alpha-globin gene constructs suggests a novel mechanism for gene regulation. Nature, 322, 562–565.[Medline]
37 Bender, C.M., Gonzalgo, M.L., Gonzales, F.A., Nguyen, C.T., Robertson, K.D. and Jones, P.A. (1999) Roles of cell division and gene transcription in the methylation of CpG islands. Mol. Cell. Biol., 19, 6690–6698.
38 Salem, C.E., Markl, I.D., Bender, C.M., Gonzales, F.A., Jones, P.A. and Liang, G. (2000) PAX6 methylation and ectopic expression in human tumor cells. Int. J. Cancer., 87, 179–185.[Web of Science][Medline]
39 Nguyen, T.T., Nguyen, C.T., Gonzales, F.A., Nichols, P.W., Yu, M.C. and Jones, P.A. (2000) Analysis of cyclin-dependent kinase inhibitor expression and methylation patterns in human prostate cancers. Prostate, 43, 233–242.[Web of Science][Medline]
40 Pao, M.M., Liang, G., Tsai, Y.C., Xiong, Z., Laird, P.W. and Jones, P.A. (2000) DNA methylator and mismatch repair phenotypes are not mutually exclusive in colorectal cancer cell lines. Oncogene, 19, 943–952.[Web of Science][Medline]
41 Ogawa, Y., Nakao, K., Arai, H., Nakagawa, O., Hosoda, K., Suga, S., Nakanishi, S. and Imura, H. (1991) Molecular cloning of a non-isopeptide-selective human endothelin receptor. Biochem. Biophys. Res. Commun., 178, 248–255.[Web of Science][Medline]
42 Arai, H., Nakao, K., Takaya, K., Hosoda, K., Ogawa, Y., Nakanishi, S. and Imura, H. (1993) The human endothelin-B receptor gene. Structural organization and chromosomal assignment. J. Biol. Chem., 268, 3463–3470.
43 Gonzalgo, M.L. and Jones, P.A. (1997) Rapid quantitation of methylation differences at specific sites using methylation-sensitive single nucleotide primer extension (Ms-SNuPE). Nucleic Acids Res., 25, 2529–2531.
44 Frommer, M., McDonald, L.E., Millar, D.S., Collis, C.M., Watt, F., Grigg, G.W., Molloy, P.L. and Paul, C.L. (1992) A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands. Proc. Natl Acad. Sci. USA, 89, 1827–1831.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  M. Kawaguchi, T. Toyama, R. Kaneko, T. Hirayama, Y. Kawamura, and T. Yagi Relationship between DNA Methylation States and Transcription of Individual Isoforms Encoded by the Protocadherin-{alpha} Gene Cluster J. Biol. Chem., May 2, 2008; 283(18): 12064 - 12075. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H.-R. Wang, C.-Y. Hsieh, Y.-C. Twu, and L.-C. Yu Expression of the Human Sda -1,4-N-Acetylgalactosaminyltransferase II Gene is Dependent on the Promoter Methylation Status Glycobiology, January 1, 2008; 18(1): 104 - 113. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Suzuki, S. Sato, Y. Arai, T. Shinohara, S. Tanaka, J. M. Greally, N. Hattori, and K. Shiota A new class of tissue-specifically methylated regions involving entire CpG islands in the mouse. Genes Cells, December 1, 2007; 12(12): 1305 - 1314. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Sakamoto, M. Suzuki, T. Abe, T. Hosoyama, E. Himeno, S. Tanaka, J. M Greally, N. Hattori, S. Yagi, and K. Shiota Cell type-specific methylation profiles occurring disproportionately in CpG-less regions that delineate developmental similarity Genes Cells, October 1, 2007; 12(10): 1123 - 1132. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Berger, C. C. Bernasconi, and L. Juillerat-Jeanneret Targeting the endothelin axis in human melanoma: combination of endothelin receptor antagonism and alkylating agents. Experimental Biology and Medicine, June 1, 2006; 231(6): 1111 - 1119. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Mercado, N. Vazquez, L. Song, R. Cortes, A. H. Enck, R. Welch, E. Delpire, G. Gamba, and D. B. Mount NH2-terminal heterogeneity in the KCC3 K+-Cl- cotransporter Am J Physiol Renal Physiol, December 1, 2005; 289(6): F1246 - F1261. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Fatemi, M. M. Pao, S. Jeong, E. N. Gal-Yam, G. Egger, D. J. Weisenberger, and P. A. Jones Footprinting of mammalian promoters: use of a CpG DNA methyltransferase revealing nucleosome positions at a single molecule level Nucleic Acids Res., November 27, 2005; 33(20): e176 - e176. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Auriol, L.-M. Billard, F. Magdinier, and R. Dante Specific binding of the methyl binding domain protein 2 at the BRCA1-NBR2 locus Nucleic Acids Res., July 28, 2005; 33(13): 4243 - 4254. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. G. Friedrich, D. J. Weisenberger, J. C. Cheng, S. Chandrasoma, K. D. Siegmund, M. L. Gonzalgo, M. I. Toma, H. Huland, C. Yoo, Y. C. Tsai, et al. Detection of Methylated Apoptosis-Associated Genes in Urine Sediments of Bladder Cancer Patients Clin. Cancer Res., November 15, 2004; 10(22): 7457 - 7465. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hattori, T. Abe, N. Hattori, M. Suzuki, T. Matsuyama, S. Yoshida, E. Li, and K. Shiota Preference of DNA Methyltransferases for CpG Islands in Mouse Embryonic Stem Cells Genome Res., September 1, 2004; 14(9): 1733 - 1740. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Gilbert, S. D. Gore, J. G. Herman, and M. A. Carducci The Clinical Application of Targeting Cancer through Histone Acetylation and Hypomethylation Clin. Cancer Res., July 15, 2004; 10(14): 4589 - 4596. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Lindsey, M. E. Lusher, J. A. Anderton, S. Bailey, R. J. Gilbertson, A. D.J. Pearson, D. W. Ellison, and S. C. Clifford Identification of tumour-specific epigenetic events in medulloblastoma development by hypermethylation profiling Carcinogenesis, May 1, 2004; 25(5): 661 - 668. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. Strathdee, B.R. Davies, J.K. Vass, N. Siddiqui, and R. Brown Cell type-specific methylation of an intronic CpG island controls expression of the MCJ gene Carcinogenesis, May 1, 2004; 25(5): 693 - 701. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yegnasubramanian, J. Kowalski, M. L. Gonzalgo, M. Zahurak, S. Piantadosi, P. C. Walsh, G. S. Bova, A. M. De Marzo, W. B. Isaacs, and W. G. Nelson Hypermethylation of CpG Islands in Primary and Metastatic Human Prostate Cancer Cancer Res., March 15, 2004; 64(6): 1975 - 1986. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C Jeronimo, R Henrique, P F Campos, J Oliveira, O L Caballero, C Lopes, and D Sidransky Endothelin B receptor gene hypermethylation in prostate adenocarcinoma J. Clin. Pathol., January 1, 2003; 56(1): 52 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Koizume, K. Tachibana, T. Sekiya, S. Hirohashi, and M. Shiraishi Heterogeneity in the modification and involvement of chromatin components of the CpG island of the silenced human CDH1 gene in cancer cells Nucleic Acids Res., November 1, 2002; 30(21): 4770 - 4780. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Nakagawa, G. J. Nuovo, E. E. Zervos, E. W. Martin Jr., R. Salovaara, L. A. Aaltonen, and A. de la Chapelle Age-related Hypermethylation of the 5' Region of MLH1 in Normal Colonic Mucosa Is Associated with Microsatellite-unstable Colorectal Cancer Development Cancer Res., October 1, 2001; 61(19): 6991 - 6995. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||