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Loss of Imprinting of Insulin-like Growth Factor-II in Wilms’ Tumor Commonly Involves Altered Methylation but not Mutations of CTCF or Its Binding Site¹

Institute of Genetic Medicine [H. C., E. L. N., J. D. R., P. O., S. A. B., A. P. F.] and Departments of Medicine [H. C., A. P. F.], Oncology [A. P. F.], and Molecular Biology and Genetics [A. P. F.], Graduate Programs in Human Genetics [E. L. N.], and Biochemistry, Cell and Molecular Biology [J. D. R.], Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, and National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892 [V. V. L.]

	ABSTRACT

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Loss of imprinting (LOI) is the most common molecular abnormality in Wilms’ tumor (WT), other embryonal cancers, and most other tumor types. LOI in WT involves activation of the normally silent maternal allele of the insulin-like growth factor-II (IGF2) gene, silencing of the normally active maternal allele of the H19 gene, and aberrant methylation of a differentially methylated region (DMR) upstream of the maternal copy of H19. Recently, the transcription factor CTCF, which binds to the H19 DMR, has been implicated in the maintenance of H19 and IGF2 imprinting. Here, we show that mutations in the CTCF gene or in the H19 DMR do not occur at significant frequency in WT, nor is there transcriptional silencing of CTCF. We also confirm that methylation of the H19 DMR in WT with LOI includes the CTCF core consensus site. However, some WTs with normal imprinting of IGF2 also show aberrant methylation of CTCF binding sites, indicating that methylation of these sites is necessary but not sufficient for LOI in WT.

	Introduction

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Genomic imprinting is an epigenetic modification of a specific parental chromosome in the gamete or zygote that leads to preferential expression of genes on that chromosome in somatic cells of the offspring. Several genes important in cancer are imprinted, including IGF2,³ p57^KIP2, and ARH1 (1, 2, 3, 4, 5) . We and others have shown previously that LOI occurs commonly in cancer and can lead to activation of the silent copy of growth promoting genes such as IGF2 (2 , 3) or silencing of the active copy of growth inhibitory genes such as p57^KIP2 (4) . We and others have also shown that LOI of IGF2 in WT is linked to aberrant methylation of a DMR upstream of the H19 gene (6 , 7) . One of the effects of methylation of this DMR that has been shown in normal cells is the abrogation of binding of the transcription factor CTCF (8, 9, 10, 11) , and CTCF can discriminate differentially methylated DMRs on the paternal versus maternal alleles in vivo (8) .

The CTCF gene product was originally identified as a transcription factor for myc and other genes (12) and later also was found to be an insulator protein that isolates enhancers from promoters, leading to transcriptional repression (13) . Recently, four groups simultaneously reported that CTCF is also involved in the regulation of the Igf2/H19 imprinting cluster (8, 9, 10, 11) . Binding of CTCF to the H19 DMR prevents the access of one or more enhancers telomeric to the H19 gene, preventing their interaction with the Igf2 promoter (8, 9, 10, 11) . Insulator activity is abolished by methylation of the H19 DMR in mouse, leading to activation of Igf2 in reporter constructs (8, 9, 10, 11) . Interestingly, both CTCF and its binding sequences in the H19 DMR are conserved between human and mouse, suggesting that a similar mechanism may apply in humans.

Because of the association of CTCF with the regulation of normal imprinting, we examined this gene in WT with LOI, comparing to normal fetal kidney the tissue from which WT are derived. The complete coding sequence of CTCF and flanking intronic sequence was examined for mutations in 25 samples. In addition, CTCF binding sites were examined for mutations, and levels of CTCF mRNA was assayed by RTQ-PCR. Finally, genomic bisulfite sequencing was performed to examine DNA methylation. Here we show that CTCF disruption does not commonly involve genetic alterations in the sequence of the CTCF gene or its binding site within the H19 DMR. We also confirm by bisulfite sequencing that the methylation we previously observed in the H19 DMR (6) includes site-specific methylation of the CpGs within the CTCF binding sites that is known to abrogate CTCF binding. Thus, functional disruption of CTCF in WT arises most commonly by an epigenetic rather than a genetic mechanism.

	Materials and Methods

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DNA and RNA Preparation.
DNA and RNA were isolated from tissues snap-frozen in liquid nitrogen. DNA was extracted as described (14) . RNA was prepared using the RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer’s protocol. All specimens were obtained from Johns Hopkins Hospital, the Cooperative Human Tissue Network, or the University of Washington Fetal Tissue Bank.

Detection of Mutation.
To detect mutation of both the CTCF gene and CTCF binding sites upstream of H19, direct PCR sequencing of genomic DNA was carried out. For the CTCF gene, the entirety of all coding exons as well as flanking intronic sequence were screened. About 200 ng of genomic DNA were amplified using the primers listed in Table 1 under the following conditions: 94°C for 1 min; 36 PCR cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min; and 72°C for 10 min. Rather than analyze only CTCF binding sites within the H19 DMR, we performed sequence analysis of the entire DMR, corresponding to GenBank nucleotides 2057 to 8070 (accession no. AF087017). The primers used are provided in Table 1 and the same conditions described above were used. All of the PCR products were purified using the QIAEX II gel extraction kit (Qiagen) and directly sequenced with an ABI Prism 377 DNA sequencer using the BigDye Terminator Cycle Sequencing kit and following the manufacturer’s protocol (PE Applied Biosystems, Foster City, CA).

Identification of LOH.
LOH on chromosome 16 was identified using microsatellite marker D16S3095. PCR was carried out using 1 µl of genomic DNA (0.1 µg) in a final volume of 10 µl containing 0.1 µM of each primer, 0.15 mM dNTP, 1.5 mM MgCl₂, 1x PCR buffer, and 0.06 units Taq polymerase (LTI, Bethesda, MD). In each reaction, one primer was 5'-end-labeled. The PCR products were analyzed on 6% denatured polyacrylamide gels.⁴

RTQ-PCR.
RTQ-PCR was performed on an ABI Prism 7700 Sequence Detection System (Applied Biosystems) in a 25-µl reaction containing 12.5 µl of 2x Taqman Master Mix, 900 nm of forward and reverse primers, and 200 nm of Taqman probe, according to the manufacturer’s recommendation. Primers to detect CTCF mRNA were designed to span an intron-exon boundary (exons 9–10; GenBank accession nos. AF145476 and AF145477): 5'-CAGAACCAACCAGCCCAAA-3' and 5'-AACTATAATGTTCTCAATTGCACCTGTATT-3'. The TaqMan probe VIC-AACCAGCCAACAGCTATCATTCAGGTTGAA-TAMRA also spanned the exon-intron boundary. The input amount of cDNA was normalized using a Taqman primer-probe set for ß-actin (Applied Biosystems).

Analysis of DNA Methylation.
To confirm that the previously reported methylation of the H19 DMR (6 , 7) included the CTCF binding region, we performed bisulfite genomic sequencing. Bisulfite treatment was carried out using the CpG Genome DNA Modification kit (Intergen, Purchase, NY) with the following modifications of the manufacturer’s protocol: denatured genomic DNA (4 µg) was incubated at 55°C in the dark overnight in 1100 µl of freshly prepared Reagent I, with subsequent column purification with the QIAquick PCR purification kit (Qiagen). Purified DNA was treated at 37°C for 15 min with freshly prepared 3 M NaOH to a final concentration of 0.3 M NaOH. Then the DNA was precipitated with ethanol and dissolved in 40 µl of 10 mM Tris (pH 8)-1 mM EDTA for nested PCR. PCR products were purified on 2% agarose gels for direct sequencing as described above. The annealing temperature was 55°C. The first round of PCR primers were: 5'-GTATAGGTATTTTTGGAGGTTTTTTA-3' and 5'-CCTAAAATAAATCAAACACATAACCC-3'. The second PCR primers were: 5'-GAGGTTTTTTATTTTAGTTTTGG-3' and 5'-ACTATAATATATAAACCTACAC-3'. For sequencing individual clones, the PCR products were subcloned into a TA Cloning vector (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions, and 10–15 clones were selected for sequencing.

	Results

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No Mutations of the CTCF Gene in WT.
To test the hypothesis that the CTCF gene might be mutated in WT, we first screened all of the coding sequence (10 exons) and flanking intronic sequence of CTCF in 15 WT patients with LOI. Although one polymorphism in the 3'-UTR was identified, none of these patients’ tumors exhibited any change in the coding sequence (Table 3) . Thus, mutation of CTCF is not a common mechanism of LOI in WT.

DNA samples from all ten of these tumors were sequenced over all 10 coding exons of CTCF, including the flanking intronic sequence. As with the nonselected set of WTs, none of these tumors showed mutations in CTCF. Therefore, CTCF is not mutated at appreciable frequency in WT, and CTCF does not appear to be the WT tumor suppressor gene on 16q.

CTCF mRNA Levels Are Comparable in WT with and without LOI.
Because no CTCF mutations were observed in WT, we compared levels of CTCF mRNA quantitatively between WTs with normal imprinting and those with LOI of IGF2. There was no significant difference in the expression level of CTCF between these two groups. The average normalized expression level for WTs with normal imprinting (n = 24) was 2.62 ± 2.66 (relative units normalized to ß-actin), compared with 2.72 ± 2.26 for WTs with LOI (n = 24; not statistically significant; two-tailed t test). However, tumors overall showed a 2.2-fold higher level of expression compared with fetal kidney (2.68 ± 2.44, n = 48 compared with 1.22 ± 0.35, n = 26; P = 0.0035), although no significant difference was seen comparing tumors overall to matched normal kidney from the same patients (n = 48; paired t test).

No Mutations of the H19 DMR in WT.
As frequent mutations were not found in CTCF, we examined the DMR upstream of the H19 gene to which CTCF binds (8 , 11) . This region in humans is located from 5.2 to 0.3 kb upstream of the start site of transcription. We used direct PCR sequencing to the entire DMR, i.e., from 5.2 to 0.3 kb upstream of the start site, in 15 WTs with LOI of IGF2. No somatic mutations were found anywhere within the region in any tumors (Table 3) , although 10 distinct SNPs were identified that were present in both tumor and normal DNA (Table 2) . Thus, neither CTCF nor the DMR with which it associates are mutated at appreciable frequencies in WT.

View larger version (21K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Complete methylation of a CTCF binding site in H19 DMR in WT. Top, wild type genomic DNA sequence. Bisulfite treated DNA, derived from a WT with LOI and from a normal fetal kidney (FK), was PCR amplified and analyzed by DNA sequencing. Note that the unmethylated cytosine is chemically converted to uracil by bisulfite treatment, leading to a thymine in the chromatogram, whereas methylated cytosine remains unchanged after bisulfite treatment. In WTs with LOI of IGF2, all of cytosines in CpG dinucleotides were methylated, whereas in fetal kidney with normal imprinting, the cytosine in CpG dinucleotides was half methylated. The chromatogram is shown at high amplitude for ease of peak discrimination, although they are truncated on the display. The same results were obtained from individual sequencing of cloned PCR products.

View larger version (29K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Allele-specific methylation status in fetal tissue and WT. Bisulfite treatment and PCR of genomic DNA was the same as in Fig. 1 , but the PCR products were subcloned prior to sequencing, to link a single-nucleotide polymorphism to the CTCF binding site. Ten to 15 clones were sequenced for each sample. Each line represents a separate clone. •, methylated CpG sites; , unmethylated CpG sites. Boxed area, the core CTCF-binding site. Monoallelic methylation was observed in fetal tissues, whereas biallelic methylation was seen in WTs with LOI of IGF2.

	Discussion

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There are two major results of this report. First, we show that CTCF mutations were not found in any of 15 WTs with LOI. In support of this result, we also examined 10 additional tumors with LOH involving chromosome 16, the chromosome on which CTCF is localized. In none of these cases were mutations found, indicating that these mutations may not occur at significant frequency.

However, we and our collaborators have found two rare missense mutations in CTCF zinc fingers 3 and 7 among 59 WTs selected for 16q22 LOH. These mutations were clearly functional, because they resulted in a selective loss of CTCF binding to the H19 DMR but not to the ß-globin gene insulator.⁵ Nevertheless, the low frequency of these events suggests that genetic disruption of CTCF itself is rare in WT, and there must be another tumor suppressor gene on 16q.

Similarly, there was no significant difference in levels of CTCF mRNA in WT with LOI, compared with tumors with normal imprinting, although there was an 2-fold increased level of expression of CTCF in WT overall. We also report here that cis-acting CTCF target sequences within the H19 DMR also did not show mutations in any of 15 tumors analyzed. However, 10 polymorphisms were identified within the H19 DMR at frequencies of 12–39% (Table 2) .

The second major result of this report is that methylation of the H19 DMR includes CTCF sequences in WTs with LOI. Although this is not a surprising result, inasmuch as altered DNA methylation of this region has been shown at a gross level by us and others (6 , 7) , we confirmed this observation by bisulfite sequencing. It has been shown previously that methylation can disrupt the action of the CTCF insulator (8, 9, 10, 11) , and therefore methylation of these sequences is a potential mechanism for LOI in tumors. However, whether this is the initial change in tumors with LOI, or other epigenetic changes are important, remains to be determined. For example, there are two DMRs within the IGF2 gene that may serve independent roles in the regulation of IGF2 imprinting in cancer. Recently, altered methylation of a CTCF binding site in the H19 DMR was described by Nakagawa et al. (18) in colorectal cancers with LOI of IGF2. However, we had previously shown that LOI of IGF2 affects both tumor and matched normal mucosa of such patients (14) . However, in the study of Nakagawa et al. (18) , normal methylation was generally observed in the normal mucosa with LOI, again consistent with the idea that CTCF is only one of several factors involved in the disruption of genomic imprinting in cancer. In addition, that study did not examine all potential CTCF binding sites, nor did this; and it will be important to couple such analyses with detailed functional studies of CTCF binding as well as with analysis of DMRs within IGF2 itself. The results obtained here may also direct future studies to the role of aberrant methylation of CTCF target sequences in the deregulation of other potential target sites, such as the INK4a and myc genes.

Unexpectedly, we found that about half of WTs with normal imprinting of IGF2 also showed aberrant methylation of the H19 DMR. This is consistent with the idea that aberrant methylation is necessary but not sufficient, and that CTCF is only one of several factors involved in the disruption of genomic imprinting in cancer. Our finding of the general absence of conventional genetic alterations suggests that, unlike the analysis of conventional tumor suppressor genes in cancer, future studies of imprinting of IGF2 in cancer must be focused upon epigenetic alterations of the sequences that regulate genomic imprinting.

	ACKNOWLEDGMENTS

 
We thank Hiroshi Uejima for thoughtful comments and Melinda Graber for preparing the manuscript.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grant CA65145.

² To whom requests for reprints should be addressed, at 1064 Ross, Johns Hopkins Medical School, 720 Rutland Avenue, Baltimore, MD 21205. Phone: (410) 614-3489; Fax: (410) 614-9819; E-mail: afeinberg{at}jhu.edu

³ The abbreviations used are: IGF2, insulin-like growth factor-II; LOI, loss of imprinting; WT, Wilms’ tumor; DMR, differentially methylated region; LOH, loss of heterozygosity; SNP, single nucleotide polymorphism; RTQ-PCR, real-time quantitative polymerase chain reaction.

⁴ These primer sequences were obtained at Internet address: http://www.ncbi.nlm.nih.gov.

⁵ G. N. Filippova, H. Cui, C. F. Qi, D. I. Loukinov, E. M. Pugacheva, J. E. Ulmer, J. M. Moore, Y. J. Hu, H. Moon, J. Breen, P. E. Grundy, et al. Cancer-associated selective disruption of complexes formed by the multifunctional CTCF protein and varying DNA targets involves mutations in the multivalent Zinc finger domain of the protein or binding abrogation by aberrant CpG-methylation of the targets, submitted for publication.

Received 2/19/01. Accepted 5/16/01.

	REFERENCES

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