CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

Priority Report

Phuongmai Nguyen,1 Hengmi Cui,2 Kheem S. Bisht,1 Lunching Sun,1 Krish Patel,1 Richard S. Lee,2 Hiroyuki Kugoh,3 Mitsuo Oshimura,3 Andrew P. Feinberg,2 and David Gius1

1Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, NIH, Bethesda, Maryland; 2Institute of Genetic Medicine and Departments of Medicine, Molecular Biology, and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland; and 3Department of Biomedical Science, Institute of Regenerative Medicine, Graduate School of Medical Science, Tottori University, Tottori, Japan

Abstract the imprinted maternal H19 allele (4). CTCF binds to the The CTCF paralog BORIS (brother of the regulator of unmethylated DNA of the differentially methylated region (DMR), imprinted sites) is an insulator DNA-binding protein thought limiting access to an enhancer shared between H19 and IGF2 and to play a role in chromatin organization and gene expression. causing silencing of the maternal IGF2 allele (5). Under normal physiologic conditions, BORIS is predominantly The CTCF paralog, CTCFL or BORIS (brother of the regulator of expressed during embryonic male germ cell development; imprinted sites), is thought to be predominantly expressed in testis; it has also been detected in >100 cancer cell lines representing all however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or major forms of human tumors (6). BORIS shares an 11 zinc finger cancer-testis gene. It has been suggested that BORIS may be a domain with CTCF (6); however, CTCF and BORIS differ pro-proliferative factor, whereas CTCF favors antiprolifera- significantly in their NH2 and COOH termini, suggesting that these tion. BORIS and CTCF share similar zinc finger DNA-binding regions may interact with different binding partners, altering gene domains and seem to bind to identical target sequences. Thus, expression by different mechanisms. Indeed, in contrast to CTCF, one critical question is the mechanism governing the DNA- BORIS seems to activate gene expression (7, 8). These results binding specificity of these two proteins when both are pre- suggest that CTCF and BORIS use a similar DNA sequence to elicit sent in tumor cells. Chromatin immunoprecipitation (ChIP) in very different changes in gene expression: one suppresses and the HCT116 cells and their hypermethylated variant showed that other induces gene expression at the same locus. It has recently been shown that the human genome contains BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using 13,804 CTCF DNA-binding sites in potential insulators of the human both whole-cell extracts and in vitro translated CTCF and genome, and although these sequences are significantly different BORIS protein, and methylation-specific ChIP PCR showed fromtranscriptional start sites, their distribution correlated with that BORIS is a methylation-independent DNA-binding pro- gene expression (9). Studies have also shown that CTCF is down- tein. Finally, experiments in murine hybrid cells containing regulated in lobular carcinoma in situ of the breast (10), suggesting a either the maternal or paternal human chromosome 11 potential role of the CTCF DNA-binding target sequences in showed that BORIS preferentially binds to the methylated carcinogenesis. Thus, a model could be proposed whereby the paternal H19 differentially methylated region, suggesting a aberrant reexpression of BORIS, which is observed in tumor cells (6), mechanism in which the affinity of CTCF for the unmethylated might in turn activate a large number of genes resulting in a cellular maternal allele directs the DNA binding of BORIS toward phenotype permissive for transformation. However, both CTCF and the paternal allele. [Cancer Res 2008;68(14):5546–51] BORIS seemto be expressed in tumorcells, suggesting a potential competition between these two proteins for CTCF DNA-binding sequences that may subsequently determine the expression of Introduction nearby target genes. Thus, one critical question about the activity of Insulator DNA sequences are thought to partition the genome these related insulator DNA-binding proteins is the mechanism by into functional chromosomal domains to regulate gene transcrip- which they specifically bind to CTCF target sequences. tion (1). The compartmentalization of chromatin into distinct regulatory domains seems to alter interactions between target Materials and Methods genes and nearby cis-acting enhancer elements (2). CTCF was the first human insulator DNA-binding protein identified that is a Cell lines, cell culture, and plasmids. HCT116 cells (human colon (À/À) ubiquitously expressed, highly conserved, zinc finger protein that carcinoma) and the methyltransferase somatic knockouts DNMT1 / (À/À) has multiple roles in gene regulation (3), including regulation of DNMT3B double knockout (DKO) cells were cultured in McCoy’s 5A medium containing 10% heat-inactivated fetal bovine serum (FBS). The murine hybrid A911P or A911M cells were grown in DMEM with 10% FBS. Media were supplemented with penicillin (100 units/mL) and Note: Supplementary data for this article are available at Cancer Research Online A (http://cancerres.aacrjournals.org/). streptomycin (100 g/mL). Primers were used to PCR the DMR region P. Nguyen and H. Cui contributed equally to this work. (Supplementary Data) and this fragment was cloned into ptk-Luc (Clontech, Requests for reprints: David Gius, Radiation Oncology Branch, National Cancer Inc.) and transient assay was done as previously described (11). Institute, NIH, 9000 Rockville Pike, Bethesda, MD 20892. Phone: 301-496-5457; Fax: 301- Chromatin immunoprecipitation. Chromatin immunoprecipitation 480-5439; E-mail: [email protected]. I2008 American Association for Cancer Research. (ChIP) assays were done using the Upstate Biotechnology, Inc. kit (12). doi:10.1158/0008-5472.CAN-08-1005 ChIP was performed using CTCF (C-20; Santa Cruz Biotechnology, Inc.) or

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BORIS (Supplementary Figs. S1 and S2) antibodies. Purified DNA samples mentary Fig. S2) antibody. Primers were used that overlap the pri- were analyzed by real-time quantitative PCR (QPCR) using SYBR Green PCR mary H19 CTCF DNA-binding site that has been shown to regulate Master Mix (Applied Biosystems) with the ABI Prism 7500 Detection System IGF2/H19 gene expression (2). ChIP of Tera-1 testicular tumor or (Applied Biosystems) with primers to the human H19 DMR (Supplementary HCT116 colon cancer cells followed by PCR or QPCR yielded a Materials and Methods). Data were collected and analyzed by comparative significant enrichment of the H19 DMR with both CTCF and BORIS C methods. T antibodies (Fig. 1B–D). As a negative control, an H19 genomic region Bisulfite pyrosequencing and methylation-specific ChIP PCR. DNA (10 Ag) was treated with sodiumbisulfite according to established methods without a CTCF consensus site was amplified (data not shown) (12). Treated DNA was resuspended in 40 mL of distilled water for PCR. All using primers that have been previously described (14). These results primers are listed in Supplementary Materials and Methods. PCR products show that, like CTCF, BORIS binds to the H19 DMR (15). were used directly for pyrosequencing according to the manufacturer’s BORIS preferentially binds to the methylated H19 DMR. instruction (Biotage). Methylation-specific ChIP PCR (MS-ChIP-PCR) was Although BORIS and CTCF both seemto bind to the H19 DMR, the carried out in HCT116 cells. Following ChIP with an anti-BORIS or anti- mechanism governing DNA binding is unknown, and it seemed CTCF antibody, the pulled-down chromatin was subjected to bisulfite reasonable to suggest that methylation might determine the conversion using the EZ DNA Methylation Gold kit (Zymo Research). specificity of binding. To address this idea, two cell lines were Primer sets were designed using Methyl Primer Express software (Applied used, (a) HCT116 cells and (b) HCT116 somatic DKO cells, which A Biosystems). Each reaction contained 10 L Fast Cycling Taq Master mix have both DNMT1 and DNMT3B deleted. These cells have been (Qiagen), 5 AL of converted DNA, and 1 AL each of 0.3 Amol/L forward and previously characterized; the methylation is decreased f95% in reverse primer. QPCR and PCR were done as described above. Preparation of nuclear extracts and electromobility shift assay. the DKO cells (13) compared with the parental HCT116 cells. Nuclear extracts were prepared as previously described method (11). In vitro The methylation status of the H19 DMR was determined by BORIS and CTCF protein was prepared using Expressway In Vitro Protein bisulfite pyrosequencing (12) and clearly showed hypomethylation Synthesis System(Invitrogen). For the electromobility shift assay (EMSA) of the DMR in the DKO compared with HCT116 cells (Fig. 2A). methylation analysis of BORIS and CTCF DNA binding, 1 Ag of PCR product ChIP analysis with an anti-BORIS antibody showed that BORIS was used. Each reaction consisted of 30 AL total volume containing 1 Ag binding was highest in the HCT116 cells and lowest in the DNA, 4 units of SssI methylase enzyme, and buffer, and reactions were unmethylated DKO cells (Fig. 2B, left). In contrast, CTCF DNA carried out at 37jC for 1 h. The same procedure was performed for binding is highest in the unmethylated DKO cells and lowest in the unmethylated DNA. The DNA reactions were gel purified, and 50 ng of DNA 32 HCT116 cells (Fig. 2B, right). Western blot analysis showed similar were used for labeling with [ P]ATP. For each EMSA, 10,000 cpmof labeled BORIS and CTCF protein levels in all HCT116 cell lines, ruling out DNA were used with in vitro transcribed CTCF or BORIS protein. that DNA binding is a function of total BORIS or CTCF levels (Supplementary Fig. S3). These experiments suggest that methyl- Results ation may be at least one mechanism governing the DNA binding BORIS binds to the H19 DMR in cultured cells. We initially of CTCF or BORIS. determined whether BORIS, like CTCF (3), binds to the DMR of Although these experiments suggest that methylation may the H19 locus (Fig. 1A). ChIP analysis was done using either an regulate CTCF and BORIS DNA binding, they do not a priori anti-CTCF (Santa Cruz Biotechnology) or an anti-BORIS (Supple- reveal the binding characteristics in a tissue culture model system.

Figure 1. BORIS is an H19 DMR DNA-binding protein. A, diagram of the H19 region and the location of the primers used for ChIP analysis. B, BORIS binds to the H19 DMR in testicular tumor cells. Tera-1 testicular tumor cells were fixed with 1% formaldehyde to cross-link protein-DNA interactions and then sonicated, and fixed cells were immunoprecipitated with either an anti-CTCF or anti-BORIS (SupplementaryFig. S2) antibody.DNA was eluted and purified before analysis byreal-time QPCR with primers that cover the CTCF-binding site in the H19 DMR region. BORIS binds to the H19 DMR in HCT116 colon tumor cells as measured byPCR ( C) and QPCR (D). HCT116 cells were harvested and CTCF and BORIS DNA binding was measured as described above. All ChIP experiments were done in triplicate. Columns, mean; bars, SD. www.aacrjournals.org 5547 Cancer Res 2008; 68: (14). July 15, 2008

Figure 2. BORIS preferentiallybinds to methylated H19 DMR. A, the H19 DMR is hypomethylated in somatic DNMT1/DNMT3B DKO cell lines. HCT116 (top) and the HCT116-derived DNMT1(À/À)/DNMT3B(À/À) somatic DKOs (bottom) cells were analyzed by bisulfite pyrosequencing at the H19 (CTCF-binding site 1) DMR. B, BORIS and CTCF bind to the H19 DMR depending on cellular DNMT status. HCT116 and DKO cells were examined byChIP analysiswith either an anti-BORIS (left) or anti-CTCF (right) antibodyto determine relative levels of CTCF and BORIS binding to the H19 DMR. C, BORIS preferentiallybinds to methylatedDNA. MS-ChIP-PCR was done using HCT116 cells. In these experiments, cells were harvested followed byChIP with either an anti-BORIS or anti-CTCF antibody . The immunoprecipitated DNA was bisulfite treated and subjected to PCR with either methylated or unmethylated primer sets complementary to the H19 DMR. All ChIP experiments were done in triplicate. D, H19 expression is decreased in DKO cells. HCT116 and DKO cells were harvested and total RNA was isolated and puritychecked, and levels of H19 RNA were determined using real-time PCR with Taqman probes. All results are the mean of at least three separate experiments. Results are presented as H19 relative RNA levels where 1.0 represents H19 RNA level in control HCT116 cells. All experiments were normalized to glyceraldehyde-3-phosphate dehydrogenase. E, transfection of a luciferase reporter plasmids contains the H19 DMR cloned upstream of the ptk-Luc (DMR-tk-Luc) into HCT116 or DKO cells. Cells were also transfected with cytomegalovirus-h-gal and results are presented as luciferase/h-galactosidase relative units. 1.0 represents luciferase activityin DKO cells transfected with DMR-tk-Luc. All results are the mean of at least three experiments, all done in duplicate. The resul ts of individual transfections varied by<25%. Columns, mean; bars, SD. Statistical significance was established byStudent’s t test. *, P < 0.05.

To confirmthe role of methylationin DNA binding, MS-ChIP-PCR DNA (Fig. 2C, left). In contrast, CTCF seems to preferentially bind was done in HCT116 cells. In these experiments, ChIP was to unmethylated chromatin (Fig. 2C, right). performed with either an anti-BORIS or an anti-CTCF antibody and H19 gene expression is decreased in HCT-derived DKO cells. the pulled-down chromatin was then subjected to bisulfite The results above show that BORIS preferentially binds to the H19 conversion. These samples were subsequently used for methyla- DMR in HCT116 cells, whereas CTCF preferentially binds to the tion-specific PCR with primers (Methyl Primer Express software) H19 DMR in DKO cells (Fig. 2B). Thus, it seemed logical to suggest designed to detect unmethylated versus methylated DNA. These that the expression of H19 would correlate with the levels of BORIS experiments showed that BORIS preferentially binds to methylated or CTCF binding to the DMR. As such, HCT116 and DKO cells

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. BORIS Is a Methylation-Independent Binding Protein were harvested and H19 RNA levels were determined by RT-PCR. lated oligonucleotide containing a CTCF DNA-binding consensus These experiments showed a roughly 70% decrease in H19 RNA sequence. EMSA analysis showed that the DNA binding of BORIS levels in DKO compared with HCT116 cells (Fig. 2D). The H19 DMR was unaltered by the methylation status of the oligo (Fig. 3B, left, was subsequently cloned upstreamof ptk-Luc and this plasmid lane 2 versus lane 4). In contrast, CTCF bound to the 32P-labeled (DMR-tk-Luc) was transfected into both HCT116 and DKO cells. A unmethylated oligo (Fig. 3B, right, lane 6) but not to the SssI- roughly 5-fold in increase in luciferase activity was observed in methylated oligo (lane 8). The results of these experiments imply HCT116 cells transfected with DMR-tk-Luc compared with DKO that, at least in vitro, BORIS seems to bind equally well to both cells (Fig. 2E). methylated and unmethylated DNA, suggesting that BORIS, in BORIS binding is methylation independent. EMSAs and contrast to CTCF, is a methylation-independent DNA-binding supershift experiments were done using HCT116 nuclear lysates protein. mixed with a 32P-labeled SssI-methylated oligonucleotide con- Small interfering RNA knockdown of CTCF increases BORIS taining the H19 DMR CTCF sequence. Supershift experiments binding to the H19 DMR. Because the results above suggested with either an anti-BORIS (Fig. 3A, lanes 3 and 4) or an anti-CTCF that BORIS can bind to unoccupied unmethylated CTCF DNA- (lanes 7 and 8) antibody showed that BORIS, but not CTCF, binds binding sites in a cell-free system, we hypothesized that BORIS may to methylated DNA. In contrast, supershift experiments using an be excluded frombinding to unmethylated CTCF DNA-binding sites unmethylated 32P-labeled oligonucleotide showed preferential simply due to the competitive occupancy of CTCF. To investigate this CTCF binding (Supplementary Fig. S4A) with some minimal idea, HCT116 cells were treated with either control or CTCF small binding of BORIS (Supplementary Fig. S4B, lane 2 versus lane 4). interfering RNA (siRNA; Supplementary Fig. S5) followed by ChIP This was our first indication that BORIS might also bind to with either an anti-CTCF or an anti-BORIS antibody. These results unmethylated DNA. clearly showed a decrease in CTCF (Fig. 3C, left) and an increase in Follow-up experiments were done using in vitro translated BORIS (right) binding to the H19 DMR in the absence of normal BORIS and CTCF with either an SssI-methylated or an unmethy- CTCF protein (Supplementary Fig. S5). MS-ChIP-PCR (described

Figure 3. BORIS, but not CTCF, DNA binding is methylation independent. A, BORIS, but not CTCF, binds to a methylated H19 DMR CTCF DNA-binding sequence. HCT116 cells were harvested and nuclear cell extracts were used for EMSA with a 32P-labeled SssI-methylated oligonucleotide containing the H19 CTCF-binding sequence. Lanes 1 and 5, probe alone; lanes 2 to 4 and 6 to 8, incubated with nuclear extract. Supershift assays were done by preincubating extracts with increasing concentrations (+ or ++) of either an anti-BORIS (lanes 3 and 4) or an anti-CTCF antibody( lanes 7 and 8). Arrows, position of the protein-DNA complex and free probe; small arrows, shifted bands. B, BORIS, but not CTCF, DNA binding in vitro is independent on methylation. EMSAs were done with in vitro translated BORIS (left) or CTCF (right) with either an unmethylated oligonucleotide containing a CTCF-binding site (lanes 1, 2, 5, and 6) or an identical SssI-methylated oligonucleotide (lanes 3, 4, 7, and 8). All gel and supershift experiments were done in triplicate. Sections of fluorograms from native gels using a Typhoon phosphorimager are shown. Arrows, the supershift as well as the protein-CTCF-DNA complex and free unbound oligonucleotide probe. C and D, siRNA knockdown of CTCF decreases CTCF and increases BORIS binding to the H19 DMR. HCT116 cells were transfected with either control (Cont)orCTCF siRNA (see SupplementaryFig. S5 for decreased CTCF levels). ChIP was done followed byeither ( C) QPCR with primers that cover the H19 DMR or (D) MS-ChIP-QPCR via bisulfite treatment and QPCR with either methylated or unmethylated primer sets to the H19 DMR. All ChIP experiments were done in triplicate. Columns, mean; bars, SD. Statistical significance was established byStudent’s t test. *, P < 0.05. www.aacrjournals.org 5549 Cancer Res 2008; 68: (14). July 15, 2008

Figure 4. BORIS preferentiallybinds to the paternal H19 DMR. A, BORIS binds preferentiallyto the paternal allele. A911M and A911P mouse hybridcells that contain human chromosome 11 of either the paternal (left) or maternal (right) origin, respectively, were analyzed via ChIP using either an anti-CTCF or an anti-BORIS antibodywith primers to the H19 DMR. DNA was eluted and analyzed by QPCR with primers to the human H19 DMR (Fig. 1A). All ChIP experiments were done in triplicate. Columns, mean; bars, SD. B and C, methylation-specific analysis of the paternal and maternal H19 DMR in the murine hybrid cell lines. A911M and A911P mouse hybrid cells were harvested, bisulfite treated, and subjected to QPCR with either methylated or unmethylated primer sets to the human H19 DMR (B)or analyzed by bisulfite pyrosequencing (C). above) confirmed that siRNA knockdown of CTCF significantly Discussion increased BORIS binding to unmethylated DNA (Fig. 3D, 3rd versus BORIS and CTCF share similar zinc finger DNA-binding domains 4th column) with little change to methylated DNA (1st versus 2nd and bind to the same cis-acting DNA-binding element; however, column). siRNA knockdown of CTCF proportionally decreased CTCF their NH -terminal and carboxy amino acid sequences are distinct, DNA binding (Supplementary Fig. S6). These results suggest that 2 suggesting that these two insulator DNA-binding proteins may CTCF may preferentially bind to unmethylated DNA, and if these have divergent cellular functions (6, 17). Whereas CTCF plays a sites become unoccupied, then BORIS can subsequently bind. central role in IGF2/H19 imprinting (4), the intracellular function BORIS binding is limited to the paternal H19 DMR. It is well of BORIS is unclear. However, it is well established that BORIS is established that the H19 DMR fromthe maternalallele is expressed in malignancies, and in this setting, both CTCF and hypomethylated, whereas the paternal allele is methylated and that this plays a key role in the regulation of IGF2 imprinting BORIS are present in tumor cells (6). Thus, the mechanism govern- (14, 15). To address whether BORIS binds to the H19 DMR of either ing either CTCF or BORIS DNA binding to CTCF DNA-binding the maternal or paternal origin, two murine hybrid cell lines that target sequences is very likely to provide insight into the cellular contain human chromosome 11 of either paternal (A911P) or role of these two insulator DNA-binding proteins and the sub- maternal (A911M) origin were used (16). ChIP analysis with an sequent expression of downstream genes, some of which may be anti-BORIS antibody showed preferential binding to the paternal prosurvival or pro-proliferative. allele (Fig. 4A, left) with minimal binding to the maternal allele In this work, we found that BORIS DNA-binding activity is largely (right). In contrast, ChIP analysis with an anti-CTCF antibody independent of the methylation status of its target sequence. showed preferential binding of CTCF to the maternal allele (right) In contrast, CTCF DNA binding is limited to unmethylated DNA. as has been previously shown (16) with very little binding to the We also showed that BORIS preferentially binds to the paternal paternal allele (left). Methylation-specific PCR (Fig. 4B) and bisul- H19 DMR using matched murine hybrid cells that contain human fite pyrosequencing (Fig. 4C) of these two murine hybrid cell lines chromosome 11 of either maternal or paternal origin. In con- clearly showed that the maternal H19 DMR is hypomethylated, trast, CTCF seems to preferentially bind to the maternal H19 whereas the paternal H19 DMR is methylated. DMR. These results are surprising because these two proteins

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. BORIS Is a Methylation-Independent Binding Protein contain a significant degree of homology in their zinc finger DNA- methylated promoter regions. However, the empirical fact that binding domains and both bind to identical cis-acting DNA CTCF only binds to the unmethylated DMR suggests that the regulator elements (6). siRNA knockdown of CTCF decreased DNA binding of BORIS and CTCF in tumor cells is quite com- CTCF binding to the DMR with a reciprocal increase in binding by plicated, and although the DNA-binding domains are highly sim- BORIS, an effect that seems to involve binding of BORIS to unmeth- ilar, there might be key difference that leads to different binding ylated DNA. properties. In addition, different NH2 and COOH termini of these Thus, these results suggest a regulatory mechanism whereby two proteins might interact with different protein complexes BORIS preferentially binds to methylated cis-acting CTCF DNA- leading to dissimilar DNA-binding behavior. The answers to these binding sites because it is prevented frombinding to unmethylated questions should help to better understand the binding dynamic CTCF DNA-binding sites by the physical occupancy of CTCF. under different physiologic conditions leading to better under- In isolation, BORIS preferentially binds to methylated DNA-binding standing of the role of BORIS and CTCF in normal and malig- sites but also binds readily to unmethylated DNA-binding sites. nant physiology. In contrast, when both CTCF and BORIS are present within the cell, which is the case in tumor (HCT116) and immortalized Disclosure of Potential Conflicts of Interest (A911P/A911M) cell lines, BORIS can bind to unmethylated CTCF No potential conflicts of interest were disclosed. DNA-binding sites only when CTCF is displaced. This explains why BORIS preferentially binds to the methylated paternal H19 DMR in the hybrid mouse cells. Acknowledgments In conclusion, these results strongly suggest that DNA methyl- Received 3/24/2008; revised 5/7/2008; accepted 5/12/2008. Grant support: CA65145 (A.P. Feinberg) and the Intramural Research Program of ation may be at least one mechanism governing the specificity the NIH, the National Cancer Institute, and the Center for Cancer Research. of insulator DNA-binding proteins in the regulation of target The costs of publication of this article were defrayed in part by the payment of page gene expression. Because CTCF seems to be ubiquitously expressed charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. in all cells, we would expect that the main targets of BORIS We thank Melissa Stauffer, Ph.D. (Scientific Editing Solutions) for editorial in tumor cells would be the induction of genes that contain assistance.

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Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2008 American Association for Cancer Research. CTCFL/BORIS Is a Methylation-Independent DNA-Binding Protein That Preferentially Binds to the Paternal H19 Differentially Methylated Region

Phuongmai Nguyen, Hengmi Cui, Kheem S. Bisht, et al.

Cancer Res 2008;68:5546-5551.

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