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. 2008 Jul 15;68(14):5546-51.
doi: 10.1158/0008-5472.CAN-08-1005.

CTCFL/BORIS is a methylation-independent DNA-binding protein that preferentially binds to the paternal H19 differentially methylated region

Phuongmai Nguyen  1 , Hengmi Cui, Kheem S Bisht, Lunching Sun, Krish Patel, Richard S Lee, Hiroyuki Kugoh, Mitsuo Oshimura, Andrew P Feinberg, David Gius
Affiliations

CTCFL/BORIS is a methylation-independent DNA-binding protein that preferentially binds to the paternal H19 differentially methylated region

Phuongmai Nguyen et al. Cancer Res. .

Abstract

The CTCF paralog BORIS (brother of the regulator of imprinted sites) is an insulator DNA-binding protein thought to play a role in chromatin organization and gene expression. Under normal physiologic conditions, BORIS is predominantly expressed during embryonic male germ cell development; however, it is also expressed in tumors and tumor cell lines and, as such, has been classified as a cancer-germline or cancer-testis gene. It has been suggested that BORIS may be a pro-proliferative factor, whereas CTCF favors antiproliferation. BORIS and CTCF share similar zinc finger DNA-binding domains and seem to bind to identical target sequences. Thus, one critical question is the mechanism governing the DNA-binding specificity of these two proteins when both are present in tumor cells. Chromatin immunoprecipitation (ChIP) in HCT116 cells and their hypermethylated variant showed that BORIS binds to methylated DNA sequences, whereas CTCF binds to unmethylated DNA. Electromobility shift assays, using both whole-cell extracts and in vitro translated CTCF and BORIS protein, and methylation-specific ChIP PCR showed that BORIS is a methylation-independent DNA-binding protein. Finally, experiments in murine hybrid cells containing either the maternal or paternal human chromosome 11 showed that BORIS preferentially binds to the methylated paternal H19 differentially methylated region, suggesting a mechanism in which the affinity of CTCF for the unmethylated maternal allele directs the DNA binding of BORIS toward the paternal allele.

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Figures

Figure 1
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 (Supplementary Fig. S2) antibody. DNA was eluted and purified before analysis by real-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 by PCR (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.
Figure 2
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(-/-)/DNMT3B(-/-) 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 by ChIP analysis with either an anti-BORIS (left) or anti-CTCF (right) antibodyto determine relative levels of CTCF and BORIS binding to the H19 DMR. C, BORIS preferentially binds to methylated DNA. MS-ChIP-PCR was done using HCT116 cells. In these experiments, cells were harvested followed by ChIP 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 purity checked, 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-β-gal and results are presented as luciferase/β-galactosidase relative units. 1.0 represents luciferase activity in DKO cells transfected with DMR-tk-Luc. All results are the mean of at least three experiments, all done in duplicate. The results of individual transfections varied by <25%. Columns, mean; bars, SD. Statistical significance was established by Student's t test. *, P < 0.05.
Figure 3
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)or CTCF siRNA (see Supplementary Fig. S5 for decreased CTCF levels). ChIP was done followed by either (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 by Student's t test. *, P < 0.05.
Figure 4
Figure 4
BORIS preferentially binds to the paternal H19 DMR. A, BORIS binds preferentially to the paternal allele. A911M and A911P mouse hybrid cells 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).
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