Transcription Factor BORIS (Brother of the Regulator of Imprinted

Transcription Factor BORIS (Brother of the Regulatorof Imprinted Sites) Directly Induces Expression of aCancer-Testis Antigen, TSP50, through Regulated Binding ofBORIS to the Promoter*□S

Received for publication, March 29, 2011, and in revised form, June 6, 2011 Published, JBC Papers in Press, June 9, 2011, DOI 10.1074/jbc.M111.243576

Natsuki Kosaka-Suzuki1, Teruhiko Suzuki1,2, Elena M. Pugacheva, Alexander A. Vostrov3, Herbert C. Morse III,Dmitri Loukinov, and Victor Lobanenkov4

From the Laboratory of Immunopathology, NIAID, National Institutes of Health, Rockville, Maryland 20852

Cancer-testis antigens (CTAs) are normally expressed in tes-tis but are aberrantly expressed in a variety of cancers with vary-ing frequency. More than 100 proteins have been identifiedas CTA including testes-specific protease 50 (TSP50) and thetestis-specific paralogue of CCCTC-binding factor, BORIS(brother of the regulator of imprinted sites). Because manyCTAs are considered as excellent targets for tumor immuno-therapy, understanding the regulatory mechanisms governingtheir expression is important. In this study we demonstrate thatBORIS is directly responsible for the transcriptional activationof TSP50. We found two BORIS binding sites in the TSP50 pro-moter that are highly conserved between mouse and human.Mutations of the binding sites resulted in loss of BORIS bindingand the ability of BORIS to activate the promoter. However,although expression ofBORISwas essential, it was not sufficientfor high expression of TSP50 in cancer cells. Further studiesshowed that binding of BORIS to the target sites was methyla-tion-independent but was diminished by nucleosomal occu-pancy consistent with the findings that high expression ofTSP50 was associated with increased DNase I sensitivity andhigh BORIS occupancy of the promoter. These findings indicatethat BORIS-induced expression of TSP50 is governed by acces-sibility and binding of BORIS to the promoter. To our knowl-edge this is the first report of regulated expression of one CTAby another to be validated in a physiological context.

CTCF 5 is a versatile chromatin factor originally identified asa repressor of Myc transcription (1–4). CTCF recognizes DNAtarget sequences through its 11 zinc finger (11ZF) domain thatis highly conserved from Drosophila melanogaster to humans(5) and functions not only as a repressor but also as an activatorto regulate expression of various genes including APP, RB1,TP53, TERT, and ARF (6–11). In addition to functioning as aconventional transcription factor, CTCF is known to regulategene expression through organization of chromatin structure(12, 13). For instance, CTCF regulates expression of imprintedgenes H19/Igf2 through binding to H19 ICR, which is methyl-ated at the paternal allele and unmethylated at the maternalallele, in a CpG methylation-dependent manner (14, 15). Thebinding of CTCF to theH19 ICR regulates promoter activity ofH19/Igf2 through formation of intrachromosomal loops, whichresults in allele-specific expression (16–18). Furthermore,recent reports identified additional unique functions of CTCFsuch as regulated stability of trinucleotide repeats (19) andlatency of various viruses (20–22).In mammals, there is a single CTCF paralogous gene named

BORIS (brother of the regulator of imprinted sites), also known asCTCFL andCT27 (cancer-testis antigen 27; see CTDatabase) (23,24). BORIS contains an 11ZF domain that is highly similar to the11ZF domain of CTCF. We recently reported that BORIS playsimportant physiological functions in progression of spermatogen-esis by regulating expression of a testis-specific splicing variant ofcerebroside sulfotransferase (Cst),Cst form FTS (25).CTAs such as BORIS are encoded by genes that show

restricted expression in testis and tumors. For the restrictedexpression, CTAs are considered as candidate targets of cancervaccines (26, 27). Immunization against BORIS was shown tobe effective in treating mice injected with BORIS-expressingcancer cells (28, 29). Furthermore, it has been reported thatBORIS regulates expression of genes encoding other CTAsincluding MAGE-A1, SPANX, and NY-ESO-1 (30, 31). Recentpublications suggested BORIS was involved in DNA hypo-methylation in cancers that is important for derepression of

* This work was supported, in whole or in part, by the National Institutes ofHealth Intramural Research Program of the NIAID. This work was also sup-ported by the Japan Society for the Promotion of Science Research Fellow-ship for Japanese Biomedical and Behavioral Researchers at the NationalInstitutes of Health (to T. S.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Table 1.

1 Both authors contributed equally to this work.2 A Japan Society for the Promotion of Science Research Fellow in Biomed-

ical and Behavioral Research at NIH. Present address: ChromosomeEngineering Research Center, Tottori University, Tottori 683-8503,Japan.

3 Present address: Dept. of Psychiatry and Behavioral Science, State Universityof New York at Stony Brook, Stony Brook, NY 11794.

4 To whom correspondence should be addressed: Molecular PathologySection, Laboratory of Immunopathology, NIAID, NIH, Twinbrook I, Rm.1417, MSC-8152, 5640 Fishers Lane, Rockville, MD 20852. Tel.: 301-435-1690; Fax: 301-402-0077; E-mail: [email protected].

5 The abbreviations used are: CTCF, CCCTC-binding factor; 11ZF domain, 11zinc finger domain of CTCF; BORIS, brother of the regulator of imprintedsites; CTA, cancer-testis antigen; Cst, cerebroside sulfotransferase; TSS,transcription start site; NHDF, normal human dermal fibroblasts; Fr, frag-ment; qPCR, quantitative PCR.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 31, pp. 27378 –27388, August 5, 2011Printed in the U.S.A.

27378 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 286 • NUMBER 31 • AUGUST 5, 2011

by guest on January 30, 2018http://w

ww

.jbc.org/D

ownloaded from

http://www.jbc.org/cgi/content/full/M111.243576/DC1

http://www.jbc.org/

CTAs expression (32, 33). These studies suggest that BORIS isan important regulator of CTA expression in cancer cells.PRSS50, also known asTSP50 (testes-specific protease 50) or

CT20, encodes a protease originally found as a gene with a pro-moter that is hypomethylated in tumors (34). Like BORIS,TSP50 is known as aCTA.Although the physiologic function ofTSP50 is not clarified, the characteristic of its expressionmakesit interesting as a potential target for cancer immunotherapy.Recent publications suggested that expression of TSP50 is reg-ulated by CpG methylation of the promoter (35), similar tomany other CTAs, and by various transcription factors includ-ing SP1, CCAAT/enhancer binding protein (C/EBP), and TP53(36, 37). However, none of these factors was shown to regulateexpression of TSP50 in a physiological context, which make itunclear which factors are involved in vivo. Given the possibleutility of TSP50 as a target for tumor immunotherapy, we feltthat clarification of the regulatory mechanisms was an impor-tant issue. We recently reported that BORIS is involved in reg-ulating Tsp50 expression in testis, although it was not clearwhether reduced expression of Tsp50 in testis of Boris knock-out (KO) mice was a primary or secondary effect of the loss ofBORIS (25).Here we report that BORIS is directly involved in activation

of TSP50 expression by binding to the promoter region. Weidentified two BORIS target sites in the TSP50 promoter thatare highly conserved among species. We found that BORIS-induced expression ofTSP50was dependent on regulated bind-ing of BORIS to the promoter. The promoter of TSP50 wasDNase I-hypersensitive in the cell lineDelta47, which expressesTSP50 at high levels, but not in other cell lines with little or noexpression. This suggested that accessibility to the promoter isthe limiting factor for binding of BORIS and downstreamexpression of TSP50. To our knowledge, this is the first reportdemonstrating direct regulation of a CTA by BORIS in a phys-iological context.

EXPERIMENTAL PROCEDURES

Cell Culture—NIH3T3 cells were cultured in Dulbecco’smodified Eagle’s medium supplemented with 10% FBS andpenicillin-streptomycin. Normal human dermal fibroblasts(NHDF) and other human cancer cell lines, Delta47, EKVX,OVCAR8, K562,MM-S1, 8226, IGRov1, UO31, A498, andARKwere cultured in RPMI medium 1640 supplemented with 10%FBS and penicillin-streptomycin.Mice—Boris knock-out mice were generated as described

previously (25). All animal experiments were conducted incompliance with the Animal Care and Use Committee of theNIAID, National Institutes of Health.Antibodies—The antibody against mouse BORIS used for

chromatin immunoprecipitation (ChIP) analysis was describedpreviously (25). Mouse monoclonal antibodies specific forhuman BORIS were produced using recombinant humanBORIS protein prepared from larvae ofTrichoplusia ni infectedwith baculovirus expressing full-length human BORIS.6 Nineantibody-producing clones that produced antibodies active in

mobility super-shift assay were chosen, and ChIP assays werecarried out using a mixture of these antibodies.Quantitative PCR (qPCR)—Total RNAwas isolated from tis-

sues and cells using RNeasy Mini kit (Qiagen, Valencia, CA).cDNA was prepared with oligo-dT primers using SuperScriptIII First-strand Synthesis System (Invitrogen) according to themanufacturer’s protocol. qPCR was performed using PowerSYBRGreenPCRMasterMix (Applied Biosystems, FosterCity,CA) and the 7900HT sequence detection system (Applied Bio-systems). The expression of TSP50 and BORIS in primary mel-anomas was analyzed using TissueScan Melanoma TissueqPCR Panel I (Origene, Rockville, MD) and Mx3000p (AgilentTechnologies, Santa Clara, CA). Fisher’s exact test was con-ducted to analyze statistical significance of correlation betweenexpression of BORIS and TSP50 in melanomas. Primers forqPCR were designed using Primer Express software (AppliedBiosystems). Primer sequences are listed in supplemental Table1 or described in a previous publication (25) Expression levelswere normalized against the housekeeping gene GAPDHexcept for the melanoma panel, which was pre-normalizedagainst ACTB. Relative expression levels were determined bystandard curvemethod or comparative Ctmethod according tothe manufacturer’s protocol. Each sample was analyzed in trip-licate. Student’s t test was performed to evaluate statistical sig-nificance. Data shown are the means � S.D.Purification of Spermatocytes and Round Spermatids—Sper-

matocytes and round spermatids were purified as previouslydescribed (25). Briefly, dissociated testicular cells were sepa-rated by centrifugal elutriation. Partially purified spermatocyteand round spermatid fractions were incubated with 10 �M

Vybrant DyeCycle Green (Invitrogen) for 30 min at 32 °C fol-lowed by staining with 4,6-diamidino-2-phenylindole (DAPI).Spermatocytes and round spermatids were sorted according toDNA content to improve purity using FACSAria (BD Biosci-ences). DAPI-positive dead cells were eliminated.Electrophoretic Mobility Shift Assay (EMSA) and Methyla-

tion Interference Assay—EMSA and methylation interferenceassays were performed as described previously (25). Mouse andhuman TSP50 promoters were analyzed with BORIS protein ofmouse and human origin, respectively. Because the amino acidsequences of human andmouseCTCF are highly conserved, weused mouse CTCF for analyses of both the mouse and humanTSP50 promoters. Partially purified recombinant humanBORIS protein prepared from larva of T. ni infected with bacu-lovirus expressing full-length human BORIS was used forEMSA.6 Briefly, larvae were homogenized in buffer containing40 mM HEPES (pH 7.6), 100 mM NaCl, 2 mM MgSO4, 10 �M

ZnSO4, and 4� Complete protease inhibitor mixture withoutEDTA (Roche Applied Science). Homogenate was centrifugedat 10,000 � g for 10 min. The bulk of the BORIS protein wasdetected in the pellet using antibodies against the V5 tag. Thepellet was washed sequentially with homogenization buffercontaining 1, 2, and 4 M urea. After final washing, BORIS wasextracted using homogenization buffer containing 6 M urea and500 mM NaCl. The DNA binding ability and specificity of therecombinant proteinwas confirmed in EMSA tests using a vari-ety of knownBORIS bindingDNAsequences aswell as negativecontrols. The binding specificity was further confirmed by6 A. A. Vostrov, manuscript in preparation.

BORIS Induces Expression of TSP50

AUGUST 5, 2011 • VOLUME 286 • NUMBER 31 JOURNAL OF BIOLOGICAL CHEMISTRY 27379


ww

.jbc.org/D

ownloaded from



http://www.jbc.org/

mobility supershift assay using antibodies against humanBORIS (Abcam, Cambridge, MA). Partially purified recombi-nant mouse BORIS and CTCF proteins were prepared fromEscherichia coli transformed with pET16b-mouse Boris andmouse Ctcf, respectively, following the procedure describedabove. Bacteria were disrupted by sonication. Samples pre-pared from E. coli transformedwith pET16b empty vector wereused as a negative control. For EMSA with nucleosomal DNAfragments, nucleosomes were reconstituted using a ChromatinAssembly kit (Active Motif, Carlsbad, CA) according to themanufacturer’s protocol. Luciferase and control lysate fromlarva ofT. niwere used as negative controls in EMSA for in vitrotranslated protein and recombinant protein prepared withbaculovirus, respectively. Primers to prepareDNA fragments ofhuman TSP50 promoter for EMSA are listed in supplementalTable 1.Luciferase Assay—Fragments containing the entire 5�-un-

translated region (5�-UTR) sequences of Tsp50 and its pro-moter sequences starting from position �150, �300, or �600were cloned into pGL3-basic vector. NIH3T3 cells werecotransfected using FuGENE 6 (Roche Applied Science) withluciferase constructs together with pRL-CMV vector as aninternal control and expression vectors for BORIS or CTCF inpCIneo according to the manufacture’s protocol. Cells werecultured for 48 h at 37 °C with 5% CO2. Luciferase assays werecarried out with the Dual-Luciferase Reporter Assay System(Promega, Madison, WI) according to the manufacturer’s pro-tocol. The luciferase activities were normalized to the emptyvector-transfected samples for each luciferase construct. Stu-dent’s t test was performed to evaluate statistical significance.Data shown are the means � S.D.ChIP assay—ChIP assays were carried out as previously

described (25) with slight modifications. Briefly, fixed cells ortissue lysates corresponding to 1� 106 cells or 20mgof tissue in100 �l of SDS lysis buffer (20 mM Tris-HCl (pH 8), 0.1% SDS, 2mM EDTA, 150 mM NaCl, 1% Triton X-100) were incubatedwith Dynabeads M-280 (Invitrogen) preconjugated with anti-bodies for 1.5 h at 4 °C with rotation. DNA fragments bound tobeads were purified and subjected for qPCR. Primers used forqPCR are listed in supplemental Table 1. Student’s t test wasperformed to evaluate statistical significance. Data shown arethe means � S.D.Bisulfite Sequencing—Bisulfite modification of genomic

DNA was carried out using the Imprint DNA Modification kit(Sigma) according to the manufacturer’s protocol. The bisul-fite-modified TSP50 promoter region from �198 to �32was amplified using 5�-GGGTTAAGGAGGGAAGTTAGT-3�and 5�-CTAATAAACTAAATAAAAATACTCCTAATA-3�as primers with PCR Platinum Taq Polymerase (Invitrogen)under the following conditions: 94 °C for 2 min; 35 cycles of94 °C for 30 s, 52 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min. ACpG island from �65 to �159 was amplified using 5�-GGG-GTATTAGGAGTATTTTTATTTAGTTTATT-3� and 5�-ACCTCAACAACAAAAACAACAACAA-3� as primers underthe following conditions: 94 °C for 2 min; 35 cycles of 94 °C for30 s, 55 °C for 30 s, 72 °C for 30 s; 72 °C for 5 min. Ampliconswere purified and cloned into pGEM-T vector (Promega). Aftertransformation, plasmids from individual bacterial colonies

were extracted and subjected to sequencing (GenomicsResearch Facility, Rocky Mountain Laboratories, NIAID,National Institutes of Health).Methylation-specific PCR—Methylation status was analyzed

by PCR using bisulfite-treated DNA as templates with primersspecific for methylated or unmethylated sequences withdesigns based on the promoter region or the CpG island insidethe transcribed region of TSP50. Primers used for these exper-iments are shown in supplemental Table 1. PCRwas performedunder the following conditions: 94 °C for 2 min; 35 cycles of94 °C for 30 s, annealing temperature of 57 °C (unmethylatedpromoter)/57 °C (methylated promoter)/53 °C (unmethylatedtranscribed region)/60 °C (methylated transcribed region) for30 s, 72 °C for 30 s; 72 °C for 5 min. PCR products were electro-phoresed on 3% agarose gels and visualized by ethidium bro-mide staining.DNase IHypersensitiveAssay—DNase I hypersensitive assays

were carried out as previously described with slight modifica-tions (38). Briefly, 2 � 107 cells homogenized with a Douncehomogenizer were split into four samples and treated with 0,80, 160, or 320 units/ml of DNase I for 3 min at 25 °C. Sampleswere then treated with RNase A followed by treatment withproteinase K. 70 �g of purified DNA digested with NheI andKpnI was separated on 0.8% agarose gels. The probe forhybridization was generated by PCR using 5�-CAG-TCTTTTCAGTGTCCCACCG-3� and 5�-CCCTTGGAGTC-TGTATGTTGGC-3� as primers.Nucleosome Phasing—Nucleosome positioning was analyzed

as described previously with modifications (39, 40). 1.5 � 107cells were harvested and washed with cold PBS. Cells were sus-pended in solution A (10mMTris-HCl (pH 7.4), 10 mMNaCl, 3mMMgCl2, 0.4%Nonidet P-40, protease inhibitormixture) andincubated for 10min on ice followed by treatmentwith solutionB (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 0.4%Nonidet P-40, 1 mM CaCl2, protease inhibitor mixture). Sam-pleswere treatedwith 120 units/mlmicrococcal nuclease for 10min at 37 °C, and the reaction was terminated by adding solu-tion C (100 mM EDTA, 4% SDS). After treatment with 0.3mg/ml RNase A for 30min at 37 °C, samples were treated over-night at 50 °C with 20 mg/ml proteinase K. Purified DNA wasseparated on 1.5% agarose gels, and the band corresponding tomononucleosomes was excised from the gel followed by purifi-cation withQIAquick Gel Extraction kit (Qiagen). Nucleosomepositioning was identified by tiling qPCR using mononucleo-somal DNA as a template. Primers were designed by Primer3from the Whitehead Institute to produce overlapping ampli-cons covering the promoter region of TSP50. Primers used forqPCR are shown in supplemental Table 1. Data were normal-ized to the signal of the highest peak.

RESULTS

Characterization of Tsp50 Expression inWild Type (WT) andBoris KO Mice—As a first step in evaluating the role played byBORIS in expression of Tsp50, we quantified levels of Tsp50transcripts in WT and Boris KO mice by qPCR (Fig. 1A). Asreported previously (34),Tsp50 transcripts were present at highlevels only in testis andwere significantly lower in testis ofBorisKO mice (25). To determine if Tsp50 was differentially ex-




ww

.jbc.org/D

ownloaded from






http://www.jbc.org/

pressed among subsets of spermatogenic cells, we comparedthe levels of transcripts in purified spermatocytes and roundspermatids. We found that transcript levels in round sperma-tids were 16-fold higher than in SC (Fig. 1B, left panel). Thedifferential distribution of Tsp50 expression between thesesubsets of spermatogenic cells was quite similar to thatdescribed previously forCst form F, a reported target of BORIS,and Boris as illustrated in Fig. 1B (middle and right panels,respectively) (25).The Mouse Tsp50 Promoter Contains Two BORIS Binding

Sites—The above findings prompted us to determine if BORISwas directly involved in governingTsp50 expression by bindingto the promoter region. To examine this issue, we generatedfive DNA fragments (Fr), designated Fr1 to Fr5, that span thepromoter region (Fig. 2A) and tested them for BORIS bindingby EMSA. BORIS bound uniquely and specifically to Fr2 (Fig.2B), which includes sequences around transcription start site(TSS). We next examined the functional relevance of theseresults by generating a series of promoter truncations (Fig. 2A)for use in luciferase reporter assays.NIH3T3 cells were cotrans-fected with each of the promoter constructs together withexpression vectors for BORIS or CTCF. The results of thesestudies demonstrated that all three reporters were significantlyactivated by BORIS but not by CTCF (Fig. 2C). These resultsindicated that the activation of Tsp50 promoter was directlydependent on binding of BORIS to sequences downstream of�150 of the Tsp50 promoter. To more precisely define the tar-

get sites for BORIS on the Tsp50 promoter, we conductedmethylation interference assays using the Fr2 sequence as aprobe (Figs. 3, A–C). Because CTCF is known to share targetsequences with BORIS in vitro for the highly conserved DNArecognition domain, we first probed the Fr2 sequence using the11ZF domain of CTCF that efficiently binds to target sequencesto identify contact nucleotides (Fig. 3B). We identified twobinding sites in the TSP50 promoter that we designated as B1and B2. To validate BORIS would also recognize these residues,we carried out methylation interference assays using mouseBORIS protein and found that BORIS bound exactly the sameguanine residues as 11ZF domain of CTCF (Fig. 3C). This resultclearly shows that BORIS and CTCF recognize the same targetsequences in the Tsp50 promoter. Furthermore, we conductedEMSA using eitherWT Fr2 (Fr2-wt) or Fr2 carrying mutationsat contact nucleotides within B1 and/or B2 (Fig. 3, A and D).BORIS was bound to Fr2-wt as well as Fr2 with mutations

FIGURE 1. Expression of Tsp50. A, expression of Tsp50 in various tissues of WTand Boris knock-out mice (KO) were analyzed by qPCR (n � 3). Expressionlevels are shown as the ratio to the level in testes of WT mice. An asteriskindicates significant difference versus WT testis (p � 0.005). B, expression ofTsp50, Cst form F, and Boris in liver, spermatocytes (SC), and round spermatids(RS) was analyzed by qPCR (n � 3). Expression levels are shown as the ratio tothe level in SC. Asterisks indicate statistical significance versus SC (p � 0.005).

FIGURE 2. Binding of BORIS to mouse Tsp50 promoter. A, shown is a sche-matic diagram of the mouse Tsp50 promoter region. Open box, open readingframe; filled box, 5�-UTR. DNA fragments used for EMSA and constructs usedfor luciferase assay are shown. Numbering is relative to the TSS of Tsp50.B, BORIS binding to the mouse Tsp50 promoter region was analyzed by EMSA.DNA fragments used for experiments are shown in A. An arrowhead indicatesa major shifted band. N, negative control; B, BORIS. C, activity of the mouseTsp50 promoter was analyzed by luciferase assay (n � 3). Each constructshown in A was cotransfected with empty, BORIS, or CTCF expression vectorin NIH3T3 cells. Luciferase activities were normalized against the empty vec-tor-transfected sample. Asterisks denote statistical significance versus theempty vector-transfected sample of each construct (p � 0.05).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

within either B1 (Fr2-B1mut) or B2 (Fr2-B2mut). In contrast,almost no band-shift was seen with Fr2-carrying mutations inbothB1 andB2 (Fr2-B1/B2mut).We then tested the Fr2-wt andmutated Fr2 sequences in EMSA for binding of either full-length CTCF or the 11ZF domain (Fig. 3E). The results of thesestudies showed that, like BORIS, both CTCF and the 11ZFdomain bound to Fr2-wt, B1mut, and B2mut but that bindingto Fr2-B1/B2mut was markedly reduced or undetectable. It isnoteworthy that EMSA with BORIS, CTCF, and 11ZF domainshowed the slowermigrating shifted bands due to double occu-

pancy of Fr2-wt (Fig. 3D,arrow1; Fig. 3E,arrow1; Fig. 3E,arrow3, respectively) and the more rapidly migrating shifted banddue to single occupancy of the single mutants, Fr2-B1mut andFr2-B2mut (Fig. 3D, arrow 2; Fig. 3E, arrow 2; Fig. 3E, arrow 4,respectively).To assess the functional relevance of these findings, we per-

formed luciferase assays inNIH3T3 cells usingTsp50 promoterconstructs starting from �600 that wereWT or mutated at theB1, B2, or both the B1 and B2 sites. BORIS-induced activationof the promoterwas reduced bymutations in either site and lost

FIGURE 3. Identification of BORIS binding sites on mouse Tsp50 promoter. A, shown is the DNA sequence of mouse Tsp50 promoter. The numbering isrelative to the TSS of Tsp50, which is marked by an arrow. The first ATG shown in bold is boxed. The solid line denotes Fr2. Contacting guanine residues of B1 andB2 BORIS binding sites are shown with asterisks. Residues marked with squares were converted to adenine in mutant constructs. B, methylation interferenceassay of the Fr2 fragment using 11ZF protein is shown. Partial sequences of the fragment around B1 and B2 sites are shown. Only the top strand is shown, asthe bottom strand did not show any differences. Left lane, unbound fragments (Free); right lane, bound fragments (Bound). Asterisks denote contacting guanineresidues. C, methylation interference assay of the Fr2 fragment using BORIS is shown. Partial sequences of the fragment around B1 and B2 sites are shown. Onlythe top strand is shown, as the bottom strand did not show any differences. Left lane, unbound fragments (Free); right lane, bound fragments (Bound). Asterisksdenote contacting guanine residues. D, binding of BORIS to the mutated fragments was analyzed by EMSA. N, negative control; B, BORIS; Wt, Fr2-wt; B1,Fr2-B1mut; B2, Fr2-B2mut; B1/B2, Fr2-B1/B2mut. Arrow 1, shifted band for simultaneous binding of BORIS to both B1 and B2 site; arrow 2, shifted band forbinding of BORIS to one site. E, binding of CTCF and 11ZF to the mutated fragments was analyzed by EMSA as in D. N, negative control; ZF, 11ZF; C, CTCF. Arrow1, shifted band for simultaneous binding of CTCF to both B1 and B2 site; arrow 2, shifted band for binding of CTCF to one site; arrow 3, shifted band forsimultaneous binding of 11ZF to both B1 and B2 site; arrow 4, shifted band for binding of 11ZF to one site. F, luciferase assays were performed using reporterconstructs of the mutated TSP50 promoter (n � 3). Empty vector, BORIS, or CTCF expression vectors were cotransfected with each construct. Luciferaseactivities were normalized against the empty vector-transfected sample. An asterisk denotes statistical significance (p � 0.005). G, binding of BORIS on 1.9 kbupstream from TSS (5�) and mouse Tsp50 promoter region (Pr) in testes of WT and Boris KO mice was examined by ChIP-qPCR using anti-mouse BORISantibodies (n � 3). An asterisk denotes statistical significance (p � 0.005).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

almost completely with a construct bearing mutations in bothsites (Fig. 3F). Taken together, these observations indicated thatrecognition of theTsp50promoter byBORIS, involving bindingto both the B1 and B2 sites, was responsible for the promoteractivation.These findings prompted us to determine if BORIS bound to

Tsp50 promoter sequences in vivo. To do this we performedChIP-qPCR analyses using BORIS antibodies with testis tissuefrom WT and Boris KO mice (Fig. 3G). The results showedmarked binding of BORIS to sequences in the Tsp50 promoterregion in WT but not in Boris KO testis, whereas enrichmentwas not found in the 5�negative control region of either sample.From this, we conclude that BORIS binds directly to specificsequences within the Tsp50 promoter and is responsible fortranscriptional activation of the gene in testis.BORIS Binding Sites in the TSP50 Promoter Are Conserved

between Mice and Humans—Because some CTAs are recog-nized to be important targets for immunotherapy of cancer andothers, such as BORIS, to have potential for preventative vac-cines (28, 41–43), we felt it is important to characterize mech-

anisms governing expression of TSP50 in human cells. A com-parison of the TSP50 promoter regions of various mammalianspecies revealed areas of strong conservation around the B1 andB2BORIS target sites defined formice (Fig. 4A), suggesting thatsimilar regulatory mechanisms might be operative across spe-cies lines. This suggestion was strengthened by the results ofmethylation interference assays of the human TSP50 promoterregion (Fig. 4B). These studies identified two BORIS bindingregions with contact guanines lying in the regions of homologyto the mouse B1 and B2 binding sites. We validated the impor-tance of these two sites for binding of BORIS by EMSA usingprobes with WT sequences and sequences mutated at contactresidues in B1, B2, or both sites (Fig. 4C). Studies performedwith a WT probe revealed two shifted bands with the slowermigrating band (Fig. 4C, arrowhead), corresponding to theoccupancy of both target sites and the more rapidly migratingband (Fig. 4C, arrow), corresponding to the occupancy of singlesites as seen with probes mutated at only B1 or B2. No bandshifts were observed with a probe mutated at both B1 and B2,paralleling similar studies of themouseTSP50 promoter. Addi-

FIGURE 4. Binding of BORIS to human TSP50 promoter. A, alignment of sequences around B1 and B2 sites of TSP50 promoter from various species is shown.Gray boxes indicate conserved residues among species. The TSS is marked by an arrow. Asterisks denote contacting guanine residues of human or mouse.Residues marked with squares were converted to adenine in mutant constructs. B, shown is a methylation interference assay using DNA fragment containingB1 and B2 sites of human TSP50 promoter. Partial sequences of the fragment around B1 and B2 sites are shown. Only the top strand is shown, as the bottomstrand did not show any differences. Left lane, unbound fragments (Free); right lane, bound fragments (Bound). Asterisks denote contacting guanine residues.C, binding of BORIS to the mutated fragments of human TSP50 promoter was analyzed by EMSA as Fig. 3D. Arrowhead, shifted band for simultaneous bindingof BORIS to both B1 and B2 site; arrow, shifted band for binding of BORIS to one site. An asterisk indicates a shifted band most likely for binding of degradatedBORIS protein. D, binding of CTCF and 11ZF to the mutated fragments of human TSP50 promoter was analyzed by EMSA as Fig. 3E. Arrow 1, shifted band forsimultaneous binding of CTCF to both B1 and B2 site; arrow 2, shifted band for binding of CTCF to one site; arrow 3, shifted band for simultaneous binding of11ZF to both B1 and B2 site; arrow 4, shifted band for binding of 11ZF to one site. N, negative control; ZF, 11ZF; C, CTCF.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

tional studies of theWT andmutant probes were performed inEMSAusing CTCF and 11ZF domain (Fig. 4D). In keeping withthe observations made with the WT probe and BORIS, weobserved two shifted bands in the presence of CTCF. Consis-tently, the patterns of band shifts seen with 11ZF proteinresembled those seen with CTCF. Taken together, these resultsindicated that the mechanisms governing regulation of TSP50expression by BORIS are conserved betweenmice and humans.Expression of BORIS Is Essential but Not Sufficient for Expres-

sion of TSP50 in Human Cancer Cell Lines—To analyze theregulation of TSP50 expression by BORIS in human cells, wequantified expression of both BORIS and TSP50 in five BORIS-positive cell lines, five BORIS-negative cell lines, and NHDF,which are supposed to be negative for both BORIS and TSP50(Fig. 5A). The results showed that TSP50 transcripts wereexpressed by three BORIS-positive cell lines of different origins(Delta47, a multiple myeloma cell line; EKVX, a non-small celllung carcinoma line; OVCAR8, an ovarian tumor cell line) butwere almost completely negative in two other BORIS-positivelines (K562, a myelogenous leukemia cell line; MM-S1, a mul-tiple myeloma cell line). It is noteworthy that the levels ofTSP50 transcripts in Delta47 cells were more than 17-foldhigher than in the other expressing lines and that there was nodirect correlation between the levels of transcripts for TSP50and BORIS in the cell lines expressing both genes. None of theBORIS-negative cancer cell lines including NHDF expressedTSP50. To investigate the relationship between the expressionof BORIS and TSP50 in primary cancers, we analyzed theirexpression in melanomas, which are known to express variousCTAs (Fig. 5B). Among 40 melanoma samples analyzed, nineshowed higher expression of TSP50 than the backgrounddetected in normal skin samples (Fig. 5B, 1–9). Of these ninecases, five melanomas, including samples showing high expres-sion of TSP50 (Fig. 5B, 1–3 and 5) expressed BORIS. There wasa statistically significant correlation between the expression ofBORIS and TSP50 (p � 0.016), consistent with the idea that

BORIS has a crucial function in activation of theTSP50 expres-sion. On the other hand,BORIS expressionwas not sufficient toinduce expression of TSP50 in melanomas as four cases wereBORIS-positive butTSP50-negative (Fig. 5B, 13, 17, 36, and 37),similar to the observations made with cancer cell lines. Theseresults suggest that the mechanisms involved in regulation ofTSP50 expression by BORIS in cancer cell lines as well as pri-mary melanomas are not completely congruent with thoseoperative in normal testis.Our studies about mouse Tsp50 promoter indicated that

BORIS-induced expression of Tsp50was controlled by bindingof BORIS to the promoter. To determine if this mechanismwasalso operative in cancer cells, we performed ChIP-qPCR assaysof theTSP50 promoter and an irrelevant site 3� to the promoterwith antibodies against human BORIS for each of the cancercell lines (Fig. 5C). We identified high enrichment of BORIS atthe TSP50 promoter in Delta47 cells but markedly less enrich-ment for the other BORIS-positive cell lines. Taken together,these results indicate that expression of TSP50 in cancer cellsmay be controlled by regulating access of BORIS protein to thepromoter rather than simply by the levels of BORIS expressionas determined by transcript levels.Methylation of the Body of TSP50 Gene Rather Than of the

Promoter Region Correlates with Expression—It is known thatbinding of CTCF and BORIS to their target sequences isaffected by DNA methylation. Moreover, other analyses dem-onstrated that theTSP50 promoter wasmethylated in a TSP50-negative testicular embryonic carcinoma cell line and in tissuenegative for TSP50, whereas the promoter was hypomethylatedin testis and spermatogenic cells that expressed TSP50 (35).These data prompted us to analyze the cell lines describedabove for themethylation status of theTSP50 promoter, first bymethylation-specific PCR (Fig. 6A, left column). Although theTSP50 promoter of Delta47 cells was unmethylated, the pro-moter of several TSP50-negative cell lines, including K562 andMM-S1 cells that were BORIS-positive, was also unmethylated.

FIGURE 5. Correlation between expression of TSP50 and binding of BORIS to TSP50 promoter. A, expression of TSP50 and BORIS in various human cell lineswas analyzed by qPCR (n � 3). Expression levels are shown as the ratio to NHDF. B, expression of TSP50 and BORIS in melanoma samples was analyzed by qPCR.Expression levels are shown as the ratio to testis. Asterisks shown on the top panel indicate BORIS-positive samples. C, binding of BORIS to TSP50 promoterregion (Pr) and 10 kb downstream from TSS (3�) in each of the cell lines was examined by ChIP-qPCR using anti-human BORIS antibodies (n � 3). An asteriskdenotes statistical significance against enrichment in other cell lines (p � 0.005).




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

These results suggested that demethylation of the promoterwas not sufficient to recruit BORIS to the promoter and induceexpression ofTSP50. Because the CpG island of theTSP50 pro-moter extends into the body of the gene, we also analyzed themethylation status of CpGs downstream of the TSS. Interest-ingly, CpGs inside the transcribed region of TSP50 were com-pletely unmethylated in Delta47 cells, whereas they were fullymethylated in other cell lines (Fig. 6A, right column). Parallelstudies of the same CpGs in NHDF also showed relatively highmethylation of the sequences. To extend our understandings ofthe relations between the methylation status of CpG islands inthe TSP50 promoter region and the body of the gene to TSP50expression, we analyzed theirmethylation status in selected celllines as well as normal liver and NHDF by bisulfite sequencing(Fig. 6B). The TSP50 promoter region in TSP50-negative K562cells, 8226 cells, NHDF, and liver was hypomethylated, validat-ing that methylation of the promoter does not contribute in amajor way to suppression of TSP50 expression in cancer andnormal cells. Notably, CpGs flanking the B1 and B2 sites in thepromoter region of the K562 cell line were not methylated,

indicating that demethylation is not sufficient to induce bind-ing of BORIS to the promoter. Moreover, EMSA using in vitromethylated fragments demonstrated that binding of BORIS tothese sequences is methylation-independent (Figs. 6, C andD).Taken together, these results indicate that BORIS binding tothe TSP50 promoter is not regulated by methylation of DNA.Binding of BORIS to Nucleosome-free Target Sites—Areas of

chromatin associated with active gene transcription frequentlyexhibit nucleosome-free status that results in the generation ofDNase I hypersensitive site. Because the removal of nucleo-some enables access of transcription factors to the targetsequence, we performed DNase I hypersensitive assays of theTSP50 promoter region to analyze the status of chromatin inrelation to binding of BORIS using cells that expressed bothTSP50 and BORIS (Delta47), cells that are BORIS-positive butTSP50-negative (K562), and cells that were negative for theexpression of both genes (8226) (Figs. 7,A andB). The results ofthese studies showed that Delta47 alone had a DNase I hyper-sensitive site in theTSP50 promoter region. To precisely deter-mine the DNase I hypersensitive site in the promoter, we stud-

FIGURE 6. Methylation of TSP50 promoter. A, methylation status around human TSP50 promoter in various human cell lines was analyzed by MethylationSpecific PCR. Primer sets specific for methylated (M) and unmethylated (U) sequences were designed to analyze methylation status of promoter region (Pr) ortranscribed region (TR). Amplicons were shown with arrows. B, schematic representation of human TSP50 promoter region is shown on the top. Positions of B1and B2 BORIS binding sites are shown with a solid line. Open box, ORF; filled box, 5�-UTR. Methylation status was examined by bisulfite sequencing. Open circles,unmethylated cytosine; filled circles, methylated cytosine. C, shown are EMSA using fragments containing the B1 and B2 site of human TSP50 promoter as Fig.4C. Fragments treated with or without SssI were used for experiments. Arrowhead, shifted band for simultaneous binding of BORIS to both B1 and B2 site; arrow,shifted band for binding of BORIS to one site. N, negative control; B, BORIS. D, DNA fragments were digested with methyl-sensitive restriction enzyme HhaI toconfirm methylation status of fragments used for EMSA.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

ied nucleosome phasing of the TSP50 promoter region in theDelta47 and K562 cells (Fig. 7C). Strikingly, the promoterregion from approximately �200 to the TSS that encompassesthe BORIS binding sites was nucleosome-free in Delta47 cellsbut nucleosome-occupied in K562 cells. These data suggestedthat nucleosome occupancy might govern the ability of BORISto bind to target sites in the TSP50 promoter. To analyze this

possibility, TSP50 promoter sequences containing the twoBORIS binding sites were used as free or mononucleosomalDNA in EMSA with BORIS (Fig. 7D). Nucleosome modifica-tion resulted in a distinct shifted band in the absence of addedBORIS (Fig. 7D, arrow 3). The addition of increasing amountsof BORIS resulted in progressive reductions in the amount ofunshifted free probe (Fig. 7D, arrow 4), whereas the intensity ofthe nucleosomal band was unchanged (Fig. 7D, arrow 3). Theseresults suggested that BORIS is not able to bind the TSP50promoter region when it is occupied by nucleosomes.Histone Modifications around BORIS Binding Sites in the

TSP50 Promoter—Recent studies showed that CTCF bindingsites provide an anchor point for the positioning of flankingnucleosomes that are highly enriched for the specific histonemarks such as H2A.Z, whereas there is no enrichment for othermodifications including H3K27me3 (44). To explore in a pre-liminaryway if BORIS target sites in theTSP50 promotermightbe similarly structured, we performed ChIP-qPCR analyses ofH2A.Z andH3K27me3 occupancy in the cell lines examined forbinding of BORIS to TSP50 promoter (Fig. 8). The resultsshowed that therewas significant enrichment forH2A.Z only inDelta47 cells, whereas the levels of H3K27me3 occupancy werethe lowest in the cells. These data implied that BORIS bindingmight, like CTCF, be associated with occupancy of H2A.Z andcanonical histone modifications, although genome-wide stud-ies of BORIS binding sites are clearly required to determine ifthis model can be generalized.

DISCUSSION

Previous studies from our laboratory demonstrated thatTsp50, a gene encoding CTA, was differentially expressed intestis fromWT and BorisKOmice, suggesting that BORISmaybe directly or indirectly involved in governing its expression(25). The results of the current study provide definitive evi-dence that TSP50 is a direct transcriptional target of BORIS inboth mice and humans. It was recently reported that TSP50 isexpressed at higher levels in conjunction with BORIS in sper-matocytic seminomas than in seminoma/dysgerminomas (45).Our findings suggest that the high expression of TSP50 in sper-matocytic seminoma can be ascribed to expression of BORIS.

Transcriptional activation of TSP50 by BORIS was shown tobe dependent on occupancy of two targets sites in the promoterregion that were conserved between mice and humans. Bothbinding sites share sequence similarity with the BORIS bindingsequenceswe found in the promoter ofCst formFTS (25) as well

FIGURE 7. Regulation of BORIS binding to TSP50 promoter. A, shown is arestriction map of human TSP50 promoter region. The probe used for DNaseI hypersensitive assay is denoted. B, genomic DNA prepared from each of thecell lines was treated with DNase I followed by restriction digestion of NheIand KpnI. Samples were hybridized using the probe shown in A. An arrowindicates a hypersensitive site. An arrowhead denotes intact fragments gen-erated by NheI and KpnI digestion. An asterisk shows nonspecific band.C, nucleosomal positioning around the TSS of human TSP50 promoter inDelta47 cells and K562 cells was analyzed by tilling qPCR using micrococcalnuclease-treated genomic DNA as templates. D, BORIS binding to the free(Free) or nucleosomal (Nuc) DNA fragment containing both the B1 and B2sites of human TSP50 promoter was analyzed by EMSA. Arrow 1, shifted bandfor simultaneous binding of BORIS to both B1 and B2 site; arrow 2, shiftedband for binding of BORIS to one site; arrow 3, shifted band for nucleosome;arrow 4, free fragment. N, negative control.

FIGURE 8. Histone marks around BORIS binding sites of human TSP50promoter. H2A.Z occupancy and H3K27me3 modification around BORISbinding sites in each of the cell lines were examined by ChIP-qPCR (n � 3).Asterisks denote statistical significance against other cell lines (p � 0.005).NCT, control IgG for a negative control.




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

as CTCF recognition sequences (46). EMSA analyses indicatedthat both binding sites of mouse and human TSP50 promotercould be occupied simultaneously by BORIS or byCTCF. Intro-duction of mutations to either B1 or B2 sites resulted in com-promised activation of the mouse Tsp50 promoter by BORIS(Fig. 3F), indicating a requirement for both binding sites toinduce full activation of Tsp50 promoter.Analyses of human cancer cell lines failed to show a direct

correlation between expression of BORIS and TSP50 tran-scripts. This indicated that mechanisms other than thoseresponsible for expression ofBORISwere required to drive highlevel expression of TSP50. ChIP analyses showed that BORISwas highly enriched at the TSP50 promoter in the Delta47 cellsthat expressed bothBORIS andTSP50, but not in other BORIS-positive cell lines, including K562 cells that express �2-foldhigher levels of BORIS transcripts than Delta47 cells but arenegative for TSP50 expression. Although it is well establishedthat binding of CTCF to its target sequences is regulated byCpG methylation (14, 47), we found that the differences inBORIS binding to the promoter could not be attributed to dif-ferences in methylation as the promoter was shown to beunmethylated even in BORIS-positive but TSP50-negative cellsincluding K562 cells and binding of BORIS to the TSP50 pro-moter was methylation-independent. Although previous stud-ies by others suggested that regulation ofTSP50 expressionwasgoverned by promoter methylation (35), our results indicatedthat this is not the primary mechanism of regulation eventhough the methylation status may affect the total extent ofactivation. Our data suggest instead that it is the methylationstatus of the CpG island inside the transcribed region ratherthan the promoter that governs high expression ofTSP50 as theCpG island inside the gene body was highly methylated inTSP50-low or -negative cells, whereas both the promoter andgene body were demethylated in Delta47 cells. Consistently, arecent study showed that methylation downstream of TSSs istightly linked to gene silencing (48). On the other hand,although TSP50 was not expressed in NHDF and liver, theirCpG island inside the transcribed region was not fully methyl-ated like others, indicating partial demethylation is not enoughto induce expression ofTSP50without BORIS. Further analyseswill be required to clarify the effect of CpG methylation inTSP50 expression.We found that the BORIS target sites in the TSP50 promoter

were nucleosome-free in Delta47 but nucleosome-occupied inK562 (Figs. 7, B and C). In vitro studies strongly suggested thatbinding of BORIS to the TSP50 promoter was dependent onnucleosome-free sequences (Fig. 7D). Analysis of nucleosomepositioning also revealed that nucleosomes on the TSP50 pro-moter were well phased in Delta47 but not in K562 cells (Fig.7C). In view of the reported activity of CTCF in phasing nucleo-somes around its target sites (44), it seems quite possible thatBORIS could have similar functional attributes. Another poten-tially important parallel between BORIS and CTCF biding sitesare the features of flanking nucleosomes that appear to be sim-ilarly marked by enrichment for H2A.Z and relative lack ofH3K27me3 (Fig. 8). Genome-wide analysis of BORIS bindingsites and histone marks are needed to clarify this possibility.

Finally, it was reported that down-regulation of BORIS (49)or TSP50 (50) expression results in apoptosis of cancer celllines, suggesting that induction of TSP50 expression by BORISmay contribute to the survival of cancer cells. Although thepotential clinical relevance of these observations is at presentonly conjectural, there is increasing evidence that BORIS andother CTAs may be important targets for immunotherapy (28,41). Further studies are clearly required to explore thesepossibilities.

REFERENCES1. Filippova, G. N., Fagerlie, S., Klenova, E. M., Myers, C., Dehner, Y., Good-

win, G., Neiman, P. E., Collins, S. J., and Lobanenkov, V. V. (1996) Mol.Cell. Biol. 16, 2802–2813

2. Klenova, E. M., Nicolas, R. H., Paterson, H. F., Carne, A. F., Heath, C. M.,Goodwin, G. H., Neiman, P. E., and Lobanenkov, V. V. (1993) Mol. Cell.Biol. 13, 7612–7624

3. Lobanenkov, V. V., Nicolas, R. H., Adler, V. V., Paterson, H., Klenova,E. M., Polotskaja, A. V., and Goodwin, G. H. (1990) Oncogene 5,1743–1753

4. Ohlsson, R., Renkawitz, R., and Lobanenkov, V. (2001) Trends Genet. 17,520–527

5. Moon, H., Filippova, G., Loukinov, D., Pugacheva, E., Chen, Q., Smith,S. T., Munhall, A., Grewe, B., Bartkuhn, M., Arnold, R., Burke, L. J., Ren-kawitz-Pohl, R., Ohlsson, R., Zhou, J., Renkawitz, R., and Lobanenkov, V.(2005) EMBO Rep. 6, 165–170

6. De La Rosa-Velazquez, I. A., Rincon-Arano, H., Benítez-Bribiesca, L., andRecillas-Targa, F. (2007) Cancer Res. 67, 2577–2585

7. Filippova, G. N., Qi, C. F., Ulmer, J. E., Moore, J. M.,Ward,M. D., Hu, Y. J.,Loukinov, D. I., Pugacheva, E. M., Klenova, E. M., Grundy, P. E., Feinberg,A. P., Cleton-Jansen, A. M., Moerland, E. W., Cornelisse, C. J., Suzuki, H.,Komiya, A., Lindblom, A., Dorion-Bonnet, F., Neiman, P. E., Morse, H. C.,3rd, Collins, S. J., and Lobanenkov, V. V. (2002) Cancer Res. 62, 48–52

8. Renaud, S., Loukinov, D., Bosman, F. T., Lobanenkov, V., and Benhattar, J.(2005) Nucleic Acids Res. 33, 6850–6860

9. Soto-Reyes, E., and Recillas-Targa, F. (2010) Oncogene 29, 2217–222710. Vostrov, A. A., and Quitschke, W. W. (1997) J. Biol. Chem. 272,

33353–3335911. Yang, Y., Quitschke,W.W., Vostrov, A. A., and Brewer, G. J. (1999) J. Neu-

rochem. 73, 2286–229812. Phillips, J. E., and Corces, V. G. (2009) Cell 137, 1194–121113. Ohlsson, R., Lobanenkov, V., and Klenova, E. (2010) Bioessays 32, 37–5014. Kanduri, C., Pant, V., Loukinov, D., Pugacheva, E., Qi, C. F., Wolffe, A.,

Ohlsson, R., and Lobanenkov, V. V. (2000) Curr. Biol. 10, 853–85615. Han, L., Lee, D. H., and Szabo, P. E. (2008)Mol. Cell. Biol. 28, 1124–113516. Kurukuti, S., Tiwari, V. K., Tavoosidana, G., Pugacheva, E., Murrell, A.,

Zhao, Z., Lobanenkov, V., Reik, W., and Ohlsson, R. (2006) Proc. Natl.Acad. Sci. U.S.A. 103, 10684–10689

17. Nativio, R., Sparago, A., Ito, Y., Weksberg, R., Riccio, A., and Murrell, A.(2011) Hum. Mol. Genet. 20, 1363–1374

18. Li, T., Hu, J. F., Qiu, X., Ling, J., Chen, H.,Wang, S., Hou, A., Vu, T. H., andHoffman, A. R. (2008)Mol. Cell. Biol. 28, 6473–6482

19. Libby, R. T., Hagerman, K. A., Pineda, V. V., Lau, R., Cho, D. H., Baccam,S. L., Axford,M.M., Cleary, J. D.,Moore, J.M., Sopher, B. L., Tapscott, S. J.,Filippova, G. N., Pearson, C. E., and La Spada, A. R. (2008) PLoS Genet. 4,e1000257

20. Amelio, A. L., McAnany, P. K., and Bloom, D. C. (2006) J. Virol. 80,2358–2368

21. Chau, C. M., Zhang, X. Y., McMahon, S. B., and Lieberman, P. M. (2006)J. Virol. 80, 5723–5732

22. Stedman, W., Kang, H., Lin, S., Kissil, J. L., Bartolomei, M. S., and Lieber-man, P. M. (2008) EMBO J. 27, 654–666

23. Klenova, E. M., Morse, H. C., 3rd, Ohlsson, R., and Lobanenkov, V. V.(2002) Semin. Cancer Biol. 12, 399–414

24. Loukinov, D. I., Pugacheva, E., Vatolin, S., Pack, S. D., Moon, H., Cher-nukhin, I., Mannan, P., Larsson, E., Kanduri, C., Vostrov, A. A., Cui, H.,




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Niemitz, E. L., Rasko, J. E., Docquier, F.M., Kistler,M., Breen, J. J., Zhuang,Z., Quitschke, W. W., Renkawitz, R., Klenova, E. M., Feinberg, A. P.,Ohlsson, R., Morse, H. C., 3rd, and Lobanenkov, V. V. (2002) Proc. Natl.Acad. Sci. U.S.A. 99, 6806–6811

25. Suzuki, T., Kosaka-Suzuki, N., Pack, S., Shin, D.M., Yoon, J., Abdullaev, Z.,Pugacheva, E.,Morse, H. C., 3rd, Loukinov, D., and Lobanenkov, V. (2010)Mol. Cell. Biol. 30, 2473–2484

26. Akers, S. N., Odunsi, K., and Karpf, A. R. (2010) Future Oncol. 6, 717–73227. Kirkin, A. F., Dzhandzhugazyan, K. N., and Zeuthen, J. (2002) Cancer

Invest. 20, 222–23628. Loukinov, D., Ghochikyan, A., Mkrtichyan, M., Ichim, T. E., Lobanenkov,

V. V., Cribbs, D. H., and Agadjanyan, M. G. (2006) J. Cell Biochem. 98,1037–1043

29. Mkrtichyan, M., Ghochikyan, A., Loukinov, D., Davtyan, H., Ichim, T. E.,Cribbs, D. H., Lobanenkov, V. V., and Agadjanyan, M. G. (2008) GeneTher. 15, 61–64

30. Vatolin, S., Abdullaev, Z., Pack, S. D., Flanagan, P. T., Custer, M., Louki-nov, D. I., Pugacheva, E., Hong, J. A., Morse, H., 3rd, Schrump, D. S.,Risinger, J. I., Barrett, J. C., and Lobanenkov, V. V. (2005) Cancer Res. 65,7751–7762

31. Hong, J. A., Kang, Y., Abdullaev, Z., Flanagan, P. T., Pack, S. D., Fischette,M. R., Adnani, M. T., Loukinov, D. I., Vatolin, S., Risinger, J. I., Custer, M.,Chen, G. A., Zhao, M., Nguyen, D. M., Barrett, J. C., Lobanenkov, V. V.,and Schrump, D. S. (2005) Cancer Res. 65, 7763–7774

32. Smith, I. M., Glazer, C. A., Mithani, S. K., Ochs, M. F., Sun, W., Bhan, S.,Vostrov, A., Abdullaev, Z., Lobanenkov, V., Gray, A., Liu, C., Chang, S. S.,Ostrow, K. L.,Westra,W. H., Begum, S., Dhara,M., and Califano, J. (2009)PLoS ONE 4, e4961

33. Woloszynska-Read, A., Zhang,W., Yu, J., Link, P. A.,Mhawech-Fauceglia,P., Collamat, G., Akers, S. N., Ostler, K. R., Godley, L. A., Odunsi, K., andKarpf, A. R. (2011) Clin. Cancer Res. 17, 2170–2180

34. Yuan, L., Shan, J., De Risi, D., Broome, J., Lovecchio, J., Gal, D.,Vinciguerra, V., and Xu, H. P. (1999) Cancer Res. 59, 3215–3221

35. Huang, Y.,Wang, Y.,Wang,M., Sun, B., Li, Y., Bao, Y., Tian, K., andXu, H.(2008) Biochem. Biophys. Res. Commun. 374, 658–661

36. Xu, H., Shan, J., Jurukovski, V., Yuan, L., Li, J., and Tian, K. (2007) CancerRes. 67, 1239–1245

37. Wang, M., Bao, Y. L., Wu, Y., Yu, C. L., Meng, X., Xu, H. P., and Li, Y. X.(2008) DNA Cell Biol. 27, 307–314

38. Lu, Q., and Richardson, B. (2004)Methods Mol. Biol. 287, 77–8639. Lam, F. H., Steger, D. J., and O’Shea, E. K. (2008) Nature 453, 246–25040. Jin, C., Zang, C., Wei, G., Cui, K., Peng, W., Zhao, K., and Felsenfeld, G.

(2009) Nat. Genet. 41, 941–94541. Caballero, O. L., and Chen, Y. T. (2009) Cancer Sci. 100, 2014–202142. Jager, E., Karbach, J., Gnjatic, S., Neumann, A., Bender, A., Valmori, D.,

Ayyoub, M., Ritter, E., Ritter, G., Jager, D., Panicali, D., Hoffman, E., Pan,L., Oettgen, H., Old, L. J., andKnuth, A. (2006) Proc. Natl. Acad. Sci. U.S.A.103, 14453–14458

43. Marchand, M., van Baren, N., Weynants, P., Brichard, V., Dreno, B., Tes-sier,M. H., Rankin, E., Parmiani, G., Arienti, F., Humblet, Y., Bourlond, A.,Vanwijck, R., Lienard, D., Beauduin, M., Dietrich, P. Y., Russo, V., Kerger,J., Masucci, G., Jager, E., De Greve, J., Atzpodien, J., Brasseur, F., Coulie,P. G., van der Bruggen, P., and Boon, T. (1999) Int. J. Cancer 80, 219–230

44. Fu, Y., Sinha, M., Peterson, C. L., and Weng, Z. (2008) PLoS Genet. 4,e1000138

45. Looijenga, L.H.,Hersmus, R., Gillis, A. J., Pfundt, R., Stoop,H. J., vanGurp,R. J., Veltman, J., Beverloo, H. B., van Drunen, E., van Kessel, A. G., Pera,R. R., Schneider, D. T., Summersgill, B., Shipley, J., McIntyre, A., van derSpek, P., Schoenmakers, E., and Oosterhuis, J. W. (2006) Cancer Res. 66,290–302

46. Kim, T. H., Abdullaev, Z. K., Smith, A. D., Ching, K. A., Loukinov, D. I.,Green, R. D., Zhang, M. Q., Lobanenkov, V. V., and Ren, B. (2007) Cell128, 1231–1245

47. Filippova, G. N., Thienes, C. P., Penn, B. H., Cho, D. H., Hu, Y. J., Moore,J. M., Klesert, T. R., Lobanenkov, V. V., and Tapscott, S. J. (2001) Nat.Genet. 28, 335–343

48. Brenet, F., Moh, M., Funk, P., Feierstein, E., Viale, A. J., Socci, N. D., andScandura, J. M. (2011) PLoS ONE 6, e14524

49. Dougherty, C. J., Ichim, T. E., Liu, L., Reznik, G., Min, W. P., Ghochikyan,A., Agadjanyan, M. G., and Reznik, B. N. (2008) Biochem. Biophys. Res.Commun. 370, 109–112

50. Zhou, L., Bao, Y. L., Zhang, Y., Wu, Y., Yu, C. L., Huang, Y. X., Sun, Y.,Zheng, L. H., and Li, Y. X. (2010) IUBMB life 62, 825–832




ww

.jbc.org/D

ownloaded from

http://www.jbc.org/

Herbert C. Morse III, Dmitri Loukinov and Victor LobanenkovNatsuki Kosaka-Suzuki, Teruhiko Suzuki, Elena M. Pugacheva, Alexander A. Vostrov,

Regulated Binding of BORIS to the PromoterDirectly Induces Expression of a Cancer-Testis Antigen, TSP50, through

Transcription Factor BORIS (Brother of the Regulator of Imprinted Sites)

doi: 10.1074/jbc.M111.243576 originally published online June 9, 20112011, 286:27378-27388.J. Biol. Chem.

10.1074/jbc.M111.243576Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

When this article is cited•

to choose from all of JBC's e-mail alertsClick here

Supplemental material:

http://www.jbc.org/content/suppl/2011/06/09/M111.243576.DC1

http://www.jbc.org/content/286/31/27378.full.html#ref-list-1

This article cites 50 references, 19 of which can be accessed free at


ww

.jbc.org/D

ownloaded from

http://www.jbc.org/lookup/doi/10.1074/jbc.M111.243576

http://www.jbc.org/cgi/alerts?alertType=citedby&addAlert=cited_by&cited_by_criteria_resid=jbc;286/31/27378&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/286/31/27378

http://www.jbc.org/cgi/alerts?alertType=correction&addAlert=correction&correction_criteria_value=286/31/27378&saveAlert=no&return-type=article&return_url=http://www.jbc.org/content/286/31/27378

http://www.jbc.org/cgi/alerts/etoc

http://www.jbc.org/content/suppl/2011/06/09/M111.243576.DC1

http://www.jbc.org/content/286/31/27378.full.html#ref-list-1

http://www.jbc.org/

Documents

Transcription Factor BORIS (Brother of the Regulator of Imprinted