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Tumor-associated Zinc Finger Mutations in the CTCF Transcription Factor Selectively Alter Its DNA-binding Specificity¹

Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 [G. N. F., J. E. U., J. M. M., M. D. W., Y. J. H., P. E. N., S. J. C.]; Laboratory of Immunopathology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, Maryland 20892 [C. F. Q., D. I. L., E. M. P., H. C. M., V. V. L.]; Department of Biological Sciences, University of Essex, Essex CO4 3SQ, United Kingdom [E. M. K.]; Cross Cancer Institute, Edmonton, Alberta, T6G 1Z2 Canada [P. E. G.]; Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 [A. P. F.]; Department of Pathology, University of Leiden, NL-2300 RA Leiden, the Netherlands [A-M. C-J., E. W. M., C. J. C.]; Department of Urology, School of Medicine, Chiba University, Chiba 260, Japan [H. S., A. K.]; Department of Clinical Genetics, Karolinska Hospital, 510401 Stockholm, Sweden [A. L.]; and Laboratoire de Génétique Moléculaire, Institut Bergonié, 33076 Bordeaux, France [F. D-B.]

	ABSTRACT

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
CTCF is a widely expressed 11-zinc finger (ZF) transcription factor that is involved in different aspects of gene regulation including promoter activation or repression, hormone-responsive gene silencing, methylation-dependent chromatin insulation, and genomic imprinting. Because CTCF targets include oncogenes and tumor suppressor genes, we screened over 100 human tumor samples for mutations that might disrupt CTCF activity. We did not observe any CTCF mutations leading to truncations/premature stops. Rather, in breast, prostate, and Wilms’ tumors, we observed four different CTCF somatic missense mutations involving amino acids within the ZF domain. Each ZF mutation abrogated CTCF binding to a subset of target sites within the promoters/insulators of certain genes involved in regulating cell proliferation but did not alter binding to the regulatory sequences of other genes. These observations suggest that CTCF may represent a novel tumor suppressor gene that displays tumor-specific "change of function" rather than complete "loss of function."

	Introduction

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
CTCF was originally identified as a ZF³ protein that binds and represses the chicken and mammalian MYC promoters (1, 2, 3) . CTCF encodes a highly conserved, widely expressed transcription factor harboring 11 ZFs, which mediate binding to specific DNA sequences. CTCF target sites have been characterized in the promoters, insulators, and other regulatory regions of several genes (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11) , are unusually long (50 bp), and differ greatly in sequence. Remarkably, CTCF uses different subsets of its individual ZFs to bind these diverse regulatory sequences (3 , 5 , 11) . This multivalent nature of CTCF most likely underlies its remarkable versatility in regulating gene expression. Indeed, CTCF has been implicated in both promoter activation (4) and repression (1, 2, 3) , in hormone-responsive gene silencing (5 , 11) , and in methylation-dependent chromatin insulation and genomic imprinting (6, 7, 8, 9, 10) .

Given the diverse roles that CTCF plays in normal gene expression and its likely involvement in regulating the expression of several genes directly implicated in cancer, i.e., MYC, ARF, PIM1, PLK, and Igf2, we hypothesized that CTCF might undergo genetic alterations in some human cancers. This assumption was supported by the observation that CTCF maps within the smallest region of overlap for LOH that has been observed at chromosome 16q22.1 in breast, prostate, and Wilms’ tumors (12 , 13) .

In this report, we describe the first examples of the tumor-specific missense mutations in the ZF domain of CTCF identified in breast, prostate, and Wilms’ tumors. These mutations did not completely abrogate DNA-binding by CTCF. Rather, they selectively altered the spectrum of CTCF binding such that CTCF interaction with the promoters/insulators of genes involved in regulating cell proliferation (MYC, ARF, PIM1, PLK, and Igf2) was reduced severely, whereas CTCF binding to other loci, including the ß-globin insulator, lysozyme silencer, or APP promoter, was unaffected. CTCF may represent a novel type of tumor suppressor gene, mutations of which display selective "change of function" rather than complete "loss of function" in contributing to the malignant phenotype.

	Materials and Methods

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
Cloning the Human CTCF Genomic Locus.
A human fibroblast genomic DNA library (Stratagene) was screened by standard procedures with the probes harboring either 5'-untranslated region sequence or the full-length human CTCF cDNA (3) . Fourteen positive lambda clones were isolated, mapped, and ordered, and most of the exon-containing fragments were subcloned into the Bluescript II SK+ vector and sequenced. Each exon-intron boundary was determined by comparing the cDNA and genomic sequences.

Paired Normal/Tumor DNA Samples.
Prostate cancer DNA samples were obtained at the Department of Urology, School of Medicine Chiba University (Chiba, Japan). Wilms’ tumor DNA samples were obtained from the Department of Pediatrics, Cross Cancer Institute, (Edmonton, Alberta, Canada) and the National Wilms’ Tumor Study Bank. Breast cancer samples were obtained from the Department of Pathology, Leiden University (Leiden, the Netherlands); from the Department of Clinical Genetics, Karolinska Hospital (Stockholm, Sweden); and from the Laboratoire de Génétique Moléculaire, Institut Bergonié (Bordeaux, France). Blood and tumor samples were obtained with the patient’s consent and the approval of the local ethics committee at each institution.

Protein Truncation Test.
The E1 exon that encodes the entire NH₂-terminal domain of CTCF (Fig. 1A) was PCR amplified from normal and tumor DNA samples with the 75-nucleotide-long forward primer 5'-gcgcaatTGTAATACGACTCACTATAgcgcAGGAGGgtttttaccATGgaaggtgaatgcagtcgaagccattgtg-3' containing the T7 promoter (shown in bold), a ribosome-binding site (italic), a spacer followed by the complementary sequence to the beginning of the human CTCF cDNA open reading frame (underlined), a reverse primer at the end of the E1 exon sequence followed by the stop codons in three frames, and a poly(A) tail. Purified PCR products were used as DNA templates for [³⁵S]Met-labeled protein synthesis with the TnT reticulocyte lysate coupled in vitro transcription-translation system (Promega) and SDS-PAGE analyses as described (3 , 11) .

View larger version (30K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Identification of tumor-specific missense mutations within the CTCF zinc finger domain. A, genomic organization of the human CTCF. The filled-in and open boxes show protein coding and noncoding exons, respectively. Arrow, transcription initiation, defined by the 5' end of the longest human CTCF cDNA clone (3) . Estimated size of introns is in kilobases and of exons is in bp. B, detection of tumor-specific missense mutations within the CTCF ZF DNA-binding domain by the PCR-mediated SSCP and DNA sequencing. These analyses documented mutations in one breast tumor (K344E), one prostate tumor (H345R), and two Wilms’ tumors (R339W and R448Q). Each of the four mutations consisted of a single nucleotide substitution resulting in a missense codon that was not present in normal tissue controls (upper sequencing panels). Numbering on DNA sequences corresponds to a given sequencing reaction rather than to CTCF cDNA nucleotide numbering. The sequences are shown only for one strand, whereas both strands were sequenced. C, schematic drawing of the wild-type CTCF protein encoded by the human CTCF cDNA shows the NH2-terminal and COOH-terminal domains of CTCF and the DNA binding domain of CTCF composed of 10 C2H2-class and 1 C2HC-class ZFs. The enlarged diagrams indicate the tumor-specific CTCF missense mutations that result in amino acid substitutions within ZF3 and ZF7 at positions critical for DNA base recognition or ZF formation.

LOH Analysis.
LOH of CTCF was determined by allele quantification using semiquantitative PCR analysis with 16q polymorphic markers localized telomeric and centromeric to the CTCF locus.⁴ The reduction of the signal intensity of >50% was considered evidence for LOH (13 , 15, 16, 17, 18) .

Gel Mobility Shift Assay.
The 11-ZF DNA binding domain and full-length human CTCF proteins were synthesized from the pCITE-11ZF and the pCITE-7.1 constructs, respectively (3 , 11) , with the TnT reticulocyte lysate coupled in vitro transcription-translation system (Promega). Site-specific mutagenesis in the pCITE-7.1 construct recreating each tumor-specific mutation in the full-length CTCF protein was carried out with the "Quick Change" site-directed mutagenesis kit (Stratagene). Both the presence of the correctly mutated nucleotide in the resulting plasmids and the absence of any inadvertently introduced mutations in the ZF domain were determined by DNA sequencing. Twelve 120–200-bp-long DNA fragments containing previously identified different CTCF target sites that are listed in Fig. 2A , were ³²P-labeled, gel purified, and used as DNA probes for gel mobility shift assays with equal amounts of the in vitro translated CTCF proteins as described (3 , 11) . Binding reactions were carried out in the buffer containing standard PBS with 5 mM MgCl₂, 0.1 mM ZnSO₄, 1 mM DTT, 0.1% NP40, and 10% glycerol in the presence of poly(deoxyinosinic-deoxycytidylic acid). Reaction mixtures of 20 µl of final volume were incubated for 30 min at room temperature and then analyzed on 5% nondenaturing PAGE run in 0.5x Tris-borate-EDTA buffer.

View larger version (87K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Tumor-specific CTCF ZF mutations selectively alter CTCF binding to different CTCF target sites. A, list of CTCF target sites analyzed by gel mobility shift assay for binding with the wild-type and mutant CTCF proteins. For each previously identified CTS (1 2 3 4 5 6 7 8 9 10 11) are shown the sequences bound by CTCF as determined by DNase I footprinting. CTCF contact guanine nucleotides determined by methylation interference assay are indicated in bold. Note that CTCF target sequences are quite divergent secondary to the ability of CTCF to use different combinations of its ZFs to specifically recognize different sequences (3 , 5 , 11) . B, gel mobility shift assays with the in vitro translated full-length wild-type (WT) and the tumor-specific mutant CTCF proteins. The DNA fragments harboring the CTCF target sites that are listed in A were 32P-labeled and used as probes. CTSs, CTCF target sites. The CTCF mutants are identified by abbreviations indicated at the top of each panel as KE (K344E), RW (R339W), HR (H345R), and RQ (R448Q). Reticulocyte TnT mixture (Promega) with no template for in vitro transcription-translation (Lanes Empty) and the 11 ZF domain protein (Lanes 11 ZF) were included in the DNA-binding reactions as negative and positive controls, respectively. The positions of the unbound (F), of the full-length CTCF-bound (B), and of the 11 ZF-bound (11ZF) DNA probes are indicated to the left of each panel. Summaries of the gel shift analyses are shown in a banner along the top of all panels. +, retention of the DNA binding activity by each mutant CTCF protein nearly equal to that of the wild-type protein; -, complete loss of DNA binding; +/-, retention of residual binding activity. These experiments showed that all mutations resulted in complete loss or severe reduction of CTCF binding to 9 of 12 targets analyzed while having no effect on binding to the other 3. The first group of targets included CTCF target sites in known growth-regulatory genes, MYC, PIM1, PLK, p19ARF, and Igf2/H19, and in the thyroid hormone receptor-responsive silencer element 144 of an unknown gene. The second group of sites, for which binding was unaffected by the mutations, comprised those found in ß-globin, APP, and lysozyme genes. Quantitation of CTCF binding affinity to several CTCF sites by surface plasmon resonance as described earlier (9) showed that the ZF mutations did not noticeably reduce CTCF binding affinity for the ß-globin site (1013-1012 M-1), whereas binding of the CTCF mutants to the Igf2/H19 sites was decreased severely from 1012 M-1 for the wild-type CTCF protein to 107-106 M-1 for K344E and R339W mutants and to undetectable levels for the R448Q and H345R mutants.

Database Accession Numbers.
The GenBank/EMBL database accession numbers for DNA sequences of CTCF exons E₁ through E₁₀, together with their flanking regions and indicated positions/sequences of the forward and reverse SSCP primers used for mutation detection, are AF145468 through AF145477, respectively.

	Results and Discussion

Top ABSTRACT Introduction Materials and Methods Results and Discussion REFERENCES

 
We isolated previously the human CTCF cDNA (3) and used it as a probe to clone the human CTCF genomic locus. Fig. 1A shows that the entire NH₂ terminus of the CTCF protein is encoded by the exon E1, whereas the COOH-terminal domain is split between exons E9 and E10, and the 11 ZFs are distributed in exons E2 to E8 with several ZFs being split across neighboring exons. In comparison to the avian CTCF gene that consists of eight exons (20) , the human CTCF has 12 exons with the introns harboring a high density of different classes of Alu-repeats (described in the corresponding GenBank entries). The additional human CTCF introns, however, do not change the CTCF cDNA open reading frame, which is overall 93% conserved between avian and humans with 100% identity in the ZF domain (3) .

As detailed in "Materials and Methods," we initially used the protein truncation assay to screen for tumor-specific mutations within the NH₂-terminus encoding first exon of CTCF (Fig. 1A) in paired normal-tumor DNA samples. These samples included 31 cases of sporadic breast cancer (15 , 16) , 40 prostate cancers (17) , and 59 Wilms’ tumors (18) . We did not identify any frameshift or truncating mutations at the CTCF NH₂ terminus using this approach. However, in an analysis of the CTCF ZF domain using SSCP screening and DNA sequencing, we documented missense codon mutations in one breast tumor, two Wilms’ tumors, and a prostate tumor (Fig. 1B) . These ZF mutations were tumor specific because corresponding normal peripheral blood lymphocyte DNA from each of these 4 individuals did not display such mutations. DNA samples from the tumors with ZF mutations were also examined for LOH with 16q polymorphic markers located centromeric and telomeric to CTCF (13 , 15, 16, 17, 18) . In the four tumors harboring CTCF mutations, each mutant allele was found associated with loss of the normal CTCF allele.

Mutations K344E, H345R, and R339W from breast, prostate and Wilms’ tumors, respectively, clustered in CTCF ZF3, whereas the other Wilms’ tumor mutation, R448Q, was in ZF7 (Fig. 1, B and C) . The DNA binding domain of CTCF is composed of 10 C₂H₂-class and one C₂HC-class ZFs (Fig. 1C) . These types of ZFs insert into the major groove of DNA to make specific contacts with nucleotides by amino acids at positions -1, 2, 3, and 6 (21) . Both Wilms’ tumor mutations (the ZF3 R339W and the ZF7 R448Q), which eliminate the guanine-contacting arginines at positions 6 and -1, respectively, therefore likely represent amino acid changes critical for specific interaction with DNA (Fig. 1C) . The H345R mutation in the prostate tumor likely disrupts the third ZF by eliminating the His residue critical for ZF formation (Fig. 1C) . The adjacent amino acid is altered by K344E mutation in the breast tumor; a mutation at the identical site in the third ZF of the TFIIIA has been shown to result in a loss of function (22) . Thus, each of the four identified tumor-specific point mutations in the CTCF ZF domain result in a missense codon at a position predicted to be critical for ZF formation or DNA base recognition.

Previously, in our experiments with ZF deletions, we observed that CTCF uses different combinations of its 11 ZFs to bind to different CTCF target sequences (3 , 5 , 11) . To test whether the observed tumor-specific CTCF ZF point mutations altered the specificity of CTCF binding to different targets, wild-type and mutant full-length CTCF proteins were synthesized and analyzed by gel mobility shift assay for binding to the 12 CTCF target sites presented in Fig. 2A . Fig. 2B shows that the tumor-specific CTCF mutations did not completely abrogate CTCF DNA-binding; rather, they selectively inhibited binding to some but not other CTCF target sites. Moreover, the four different tumor-specific missense mutations in the CTCF ZF domain all exhibited a remarkable functional similarity, displaying diminished binding (compared with the wild type CTCF) to target sites within the promoters/insulators of certain genes involved in regulating cell proliferation (MYC, PLK, PIM-1, p19ARF, and Igf2/H19) but exhibiting unaltered binding to the APPß, ß-globin, and lysozyme regulatory sequences (Fig. 2B) .

The clustering of these tumor-derived CTCF mutations within ZF 3 and 7, the striking similarity in the functional consequences of these distinct mutations, and the tumor-specific nature of these mutations together strongly suggest that these CTCF mutations are not random polymorphisms but rather have significant biological consequences that likely contribute to the malignant phenotype. Indeed, we would predict that growth-regulatory genes such as MYC, p19ARF, and Igf2, to which CTCF binding is disrupted by the observed CTCF ZF mutations, might exhibit dysregulated expression in the tumors harboring these mutations. Because of a deficiency of adequate RNA from the corresponding tumor samples, we could not directly test this hypothesis. However, we used the p19ARF promoter to determine whether the diminished binding to CTCF target sites displayed by the tumor-specific CTCF ZF mutations had functional consequences. In comparison with the wild-type CTCF, all four of the CTCF ZF mutations that inhibited CTCF binding to the target site within the p19ARF promoter (Fig. 2) resulted in a complete loss of CTCF-mediated activation of this promoter (Fig. 3) . In contrast, in similar experiments with the APPß promoter, to which the CTCF mutations showed unaltered binding (Fig. 2) , the mutations had no effect on the APPß promoter activation by CTCF (data not shown). Thus, the tumor-derived CTCF ZF mutations that result in the loss of binding of CTCF to its specific target sequence also result in a loss of its transcriptional activity at the corresponding gene.

View larger version (15K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. The tumor-specific CTCF mutations display a loss of transcriptional activation of the p19ARF promoter. CV-1 cells were cotransfected with the ARF promoter luciferase reporter (1 µg/plate), and 5 µg of either the wild-type (Wt) CTCF expression vector or an expression vector harboring the indicated ZF-mutant CTCF (see Fig. 2 legend for identification) or an expression vector harboring the COOH-terminal CTCF region (C) that was used as a negative control for CTCF binding. Compared with the wild-type CTCF, the ZF-mutant CTCF proteins showed only a baseline level of the promoter activation defined by the signal observed with the empty expression vector or the COOH-terminal region of CTCF. Western analysis indicated that the production of CTCF proteins of expected size was nearly equal for each expression vector (not shown). Results are plotted as the average values of three independent experiments. Bars, SD.

Tumor-specific mutations of the classic tumor suppressor genes often result in a complete loss of their functional activity. Although our LOH analysis indicates that all of the tumors harboring CTCF mutations have lost one normal CTCF allele, in our screen of these tumors we did not identify any mutations that might lead to complete loss of CTCF function in the retained allele. Rather the observed tumor-specific CTCF ZF mutations selectively alter CTCF DNA binding specificity without completely abrogating CTCF function. Therefore, it is not surprising that such point mutations conferring selective loss of CTCF function appear infrequent (only four mutations found of 134 tumors examined). We hypothesize that certain CTCF activities, such as maintaining transcriptional insulator activity at specific loci or preserving presently unknown but critical regulatory functions, are essential in maintaining cell viability, and that complete loss of CTCF function might lead to cell death in both normal and/or malignant cells. Consistent with this hypothesis we have invariably noted CTCF expression in all cell types examined including both normal and transformed cells and have also observed that CTCF nullizygous mice exhibit embryonic lethality that occurs at a very early, preimplantation stage.⁵ In contrast to complete loss of CTCF activity, the tumor-specific CTCF mutations that we have identified suggest that selective loss of CTCF function may be well tolerated and indeed might confer a selective growth advantage. Thus, the loss of binding of the tumor-specific CTCF mutants to the promoters/insulators of growth-regulatory genes such as MYC, p19ARF, and Igf2 (Fig. 2) may dysregulate their expression and contribute to the malignant phenotype. Similarly, epigenetic phenomenon such as the tumor-specific methylation of CTCF binding sites within the Igf2/H19 insulator domain observed earlier (25) might also disrupt CTCF function at selective sites, leading to dysregulated cell proliferation. We suggest that such genetic- or epigenetic-directed specific shifts in the CTCF binding spectrum to functionally distinct regulatory sites may represent a novel mechanism involving "change of function" rather than "loss of function" for a transcription factor to become oncogenic.

	ACKNOWLEDGMENTS

 
We thank D. Levens, R. Ohlsson, S. Tapscott, and C. Kemp for critical reading of the manuscript and T. Awad for the Fig. 1 art work. We thank M. Groudine for providing genomic MYC plasmid, D. K. Ferris for the human PLK genomic clone, M. Lilley and A. Berns for plasmids containing human and mouse Pim-1 promoters, A. Bell and G. Felsenfeld for the ß-globin HS4-containing plasmids, A. Vostrov and W. Quitschke for the APP promoter plasmid, and C. Sherr for the p19ARF promoter plasmid.

	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 Grants RO1 CA71732 and RO1 CA68360 (to V. V. L. and G. N. F.) and by the National Institute of Allergy and Infectious Diseases (to V. V. L.).

² To whom requests for reprints should addressed, at Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue N, C2-023, P. O. Box 19024, Seattle, WA 98109. Phone: (206) 667-4468; Fax: (206) 667-6523; E-mail: gfilippo{at}fhcrc.org. (for G. N. F.); or at Laboratory of Immunopathology, NIAID, NIH, 7 Center Drive, Room 7/303, MSC 0760, Bethesda, MD 20892. Phone: (301) 435-1690; Fax: (301) 402-0077; E-mail:vlobanenkov{at}niad.nih.gov (for V. V. L.).

³ The abbreviations used are: ZF, zinc finger; LOH, loss of heterozygosity; SSCP, single-strand conformation polymorphism.

⁴ The maps are available at www.ls.lanl.gov/images/sigma_images.

⁵ G. N. Filippova et al., unpublished data.

Received 9/24/01. Accepted 11/14/01.

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