epigenetic regulation by z-dna silencer function controls ......z-dna suppressor operates by...

Molecular and Cellular Pathobiology

Epigenetic Regulation by Z-DNA Silencer Function ControlsCancer-Associated ADAM-12 Expression in Breast Cancer:Cross-talk between MeCP2 and NF1 Transcription FactorFamily

Bimal K. Ray1, Srijita Dhar1, Carolyn Henry2, Alexander Rich3, and Alpana Ray1

AbstractA disintegrin and metalloprotease domain-containing protein 12 (ADAM-12) is upregulated in many human

cancers and promotes cancer metastasis. Increased urinary level of ADAM-12 in breast and bladder cancerscorrelates with disease progression. However, the mechanism of its induction in cancer remains less understood.Previously, we reported a Z-DNA–forming negative regulatory element (NRE) in ADAM-12 that functions as atranscriptional suppressor to maintain a low-level expression of ADAM-12 in most normal cells. We now reporthere that overexpression of ADAM-12 in triple-negative MDA-MB-231 breast cancer cells and breast cancertumors is likely due to amarked loss of this Z-DNA–mediated transcriptional suppression function.We show thatZ-DNA suppressor operates by interaction with methyl-CpG-binding protein, MeCP2, a prominent epigeneticregulator, and two members of the nuclear factor 1 family of transcription factors, NF1C and NF1X. While thistripartite interaction is highly prevalent in normal breast epithelial cells, both in vitro and in vivo, it issignificantly lower in breast cancer cells. Western blot analysis has revealed significant differences in thelevels of these 3 proteins between normal mammary epithelial and breast cancer cells. Furthermore, we show,by NRE mutation analysis, that interaction of these proteins with the NRE is necessary for effectivesuppressor function. Our findings unveil a new epigenetic regulatory process in which Z-DNA/MeCP2/NF1interaction leads to transcriptional suppression, loss of which results in ADAM-12 overexpression in breastcancer cells. Cancer Res; 73(2); 736–44. �2012 AACR.

IntroductionMetastatic spread of cancer is regarded as the greatest

hurdle to cancer cure. In the metastatic cascade, multipleinterrelated pathways are activated, which include proteolyticbreakdown of the tumor membrane and spreading of cancercells into the surrounding tissues, migration, and successfulattachment of the escaped cancer cells at new sites andcolonization and proliferation of cancer cells at secondarylocations. Advances in cancer research indicate that geneticmutations along with epigenetic alterations also contribute tometastasis-related gene expression. In many human cancers,markedly high-level expression of a multifunctional protein,

ADAM-12, is detected (1–7). In patients with breast andbladder cancer, increase of ADAM-12 is shown to correlatewith disease progression and tumor stage (2, 5, 8) and in animalmodels, ADAM-12 is found to be required for aggressive tumorprogression (3, 9).

ADAM-12 is capable of supporting several steps of thecancer-metastasis cascade. It proteolytically degrades severalcomponents of extracellular matrix (2), facilitates cell–cell andcell–extracellular matrix (ECM) attachments (10), and pro-motes cell proliferation by increasing bioavailability of growthfactors (2, 5). Incidentally, 3 somatic mutations in ADAM-12gene are frequently seen in breast cancers (11). These muta-tions cause mutant ADAM-12 proteins to be retained in theendoplasmic reticulum (ER) rather than at the cell surface (12).It is speculated that increased accumulation of ADAM-12 in theER may be linked to tumor growth, which is yet to be exper-imentally determined. Normal cellular expression of ADAM-12usually is very low and highly regulated. Previously, wereported a Z-DNA–forming negative regulatory element (NRE)that acts as transcriptional silencer of ADAM-12 expression(13). Here, we provide evidence thatmarked increase of ADAM-12 level in breast cancer cells is, at least in part, due to lossof NRE-silencer function. The results reveal a novel mode ofepigenetic regulation, which involves cross-talk betweenMeCP2, a prominent epigenetic regulator, and 2 members of

Authors' Affiliations: 1Departments of Veterinary Pathobiology and 2Vet-erinary Medicine and Surgery, University of Missouri, Columbia, Missouri;and 3Department of Biology, Massachusetts Institute of Technology,Cambridge, Massachusetts

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Alpana Ray, Department of Veterinary Pathobi-ology, University of Missouri, 126A Connaway Hall, Columbia, MO 65211;Phone: 573-882-6728; Fax: 573-884-5414; E-mail: [email protected],and Bimal K. Ray, E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-12-2601

�2012 American Association for Cancer Research.

CancerResearch

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the NF1 family of transcription factors and interaction ofMeCP2:NF1 complex with Z-DNA–forming dinucleotiderepeat sequences in regulating ADAM-12 expression.

Materials and MethodsCell lines and tissue samples, transfection assay, andcDNA library screeningMCF-10A, MDA-MB-231, MDA-MB-468, MCF-7, DU4475,

and Hs578T cells were obtained from American Type CultureCollection (ATCC) in 2010, cultured, and stored followingATCC protocol. The cells have been authenticated by shorttandem repeat DNA profiling method by using Cell ID system(Promega). PCR products of genomic DNA from each cell linewere detected on a capillary electrophoresis equipment. Theresults were analyzed by using GeneMapper 4.0 software. Cellswere tested at 4- to 5-month intervals and last tested in June2012. Normal and cancer human breast tissue lysates wereobtained from IMGENEX. Canine breast cancer tissues wereobtained from the University of Missouri Veterinary MedicalTeaching Hospital (Columbia, MO). Normal canine mammarytissues were obtained from cadavers. Histologic analysisconfirms adenocarcinoma in canine breast cancer tissues

(Supplementary Fig. S1). All procedures were approved by theAnimal Care and Use Committee.

Chloramphenicol acetyltransferase (CAT) assay was con-ducted following transfection of cells with reporter plasmidand pSVb-gal (Promega) DNA (for normalization of transfec-tion efficiency), as described (13).

A humanbreast cDNAexpression library inlgt11 (Clontech)was screened by ligand interactionmethod, using a 32P-labeledconcatenated ADAM-12 NRE (þ100/þ190) DNA, as describedearlier (14). Positive clones were analyzed by DNA sequencing.

RNA isolation and Northern blot analysisTotal RNA isolated by guanidinium thiocyanatemethodwas

fractionated in a 1% agarose gel and after transfer onto anitrocellulose membrane, hybridized to 32P-labeled ADAM-12and b-actin cDNA probes as described (13).

Preparation of ADAM-12 promoter-reporter constructsTwo ADAM-12–CAT reporters, wt ADAM-12 containing

ADAM-12 sequences from �1600 to þ190 and DNREADAM-12 with deletion of sequences from þ100 to þ190,were described earlier (13). Two additional ADAM-12

Figure 1. Induction of ADAM-12 and significant modulation of NRE-mediated transcriptional repression in breast cancer cells. A, total RNA (50 mg) fromMCF-10A, MDA-MB-231, andMDA-MB-468 cells was subjected to Northern blot analysis using an ADAM-12 cDNA probe. b-Actin is RNA loading control. B, totalprotein (70 mg) from MCF-10A, MDA-MB-231, and MDA-MB-468 cells was subjected to Western blot analysis using anti–ADAM-12 antibody. b-Actin is aprotein loading control. C, histograms summarize the Western blot results of 3 independent experiments. D, schematic of ADAM-12–CAT constructs.E, reporter activities following transfection of MCF-10A, MDA-MB-231, and MDA-MB-468 cells with plasmid DNAs (0.5 mg DNA). Relative CAT activity wasdeterminedby comparing the activities of transfectedplasmidswith that of pBLCAT3andcorrecting for transfection efficiency (b-gal). The results represent anaverage of 3 independent experiments (P < 0.05).

Epigenetic Regulation by Z-DNA/MeCP2/NF1 in Breast Cancer

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promoter constructs were generated by PCR amplification.The sequence of non Z-DNA element that replaced Z-DNA atþ124 to þ159 was: 50- GCATGCATTCAGGAACCATCGAACT-TAGTCAATCGG-30. The sequence of mutant NF1 oligonucle-otide that replaced NF-1 binding region at þ101 to þ122 was:50-GTCAAGCGGGGCTCGTCCAGAA-30. Underlined lettersrepresent altered nucleotides.

DNA-binding assay and Western blot analysisDNA-binding assays were conducted as described earlier

(13) using 10 mg of nuclear extracts (NE) prepared fromMCF10A, MDA-MB-231, canine cancer, and normal breasttissues. 32P-labeled ADAM-12 NRE DNA was used as probe.DNA probe was methylated by CpG methyltransferase (NewEngland BioLab) following manufacturer's protocol. In somebinding assays, 100-fold molar excess of unlabeled nonspecific(a 30-mer ds-DNA containing random sequences) or homol-ogous-specific (ds-DNA containing same sequence as theprobe or smaller fragment thereof as indicated in figurelegends) competitor DNA or antibodies against NF1-C(N. Tanese, New York University, Langone Medical Center,New York, NY), NF1-X (U.S. Singh, Uppsala University, Uppsala,Sweden) MeCP2 (P. L. Jones, Boston Biomedical ResearchInstitute, Watertown, MA), ADAR1, DAI/DLM1/ZBP1, andnormal IgG were added. Western blots were conducted using1:3,000 dilution of NF1-C, NF1-X, MeCP2, p-STAT3, and b-actin(Santa Cruz Biotechnology) antibodies. For dephosphorylationassay, protein extracts were treated with alkaline phosphatase(Fermentas).

Chromatin immunoprecipitation and re-ChIP assaysFor chromatin immunoprecipitation (ChIP) assays, lysed

solutions were incubated with NF1/C, NF1-X, MeCP2 anti-body or control IgG. For PCR, specific primers as describedearlier (13) that amplify the nucleotide position from þ68 toþ193 and as a negative control, sequences from�619 to�328,

were used. Re-ChIP assays were conducted by following amethod described earlier (15).

ResultsInduction of ADAM-12 and loss of transcriptionalrepression in breast cancer cells

Both mRNA and protein analyses revealed that ADAM-12expression is markedly (5–6 fold) increased in metastaticbreast cancer cells as compared with normal mammary epi-thelial cells (Fig. 1A–C). To evaluate possible role of regulatoryregion of the gene in its expression, we transfected these cellswith plasmids carrying a reporter gene, chloramphenicol acetyltransferase (CAT), whose expression was driven by either wild-type ADAM-12 promoter (�1600/þ190) or a truncated pro-moter (�1600/þ100) lacking the Z-DNA–forming NREsequences (Fig. 1D). Results showed that NRE caused about5-fold suppression of ADAM-12 expression in MCF-10A cells(Fig. 1E). However, the same NRE caused significantly lesstranscriptional suppression, 1.5- and 1.8-fold, respectively, inMDA-MB-231 and MDA-MB-468 cells. This finding suggestedthat the cancer cells probably lack some of the regulatoryfactors that normally bind to theNRE to suppress transcriptionof ADAM-12 in normal mammary epithelial cells or binding ofsome additional factors present in breast cancer cells that maycause reduction of suppression. To test these possibilities, weconducted an NRE-DNA–binding assay.

Loss of NRE-DNA–binding activity in breast cancer cellsDNA-binding assay revealed a lower interaction of protein(s)

inMDA-MB-231 cells as comparedwithMCF-10A cells (Fig. 2A,compare lanes 2 and 3). Similarly, proteins in canine breastcancer tissue, which expresses high level of ADAM-12 (Sup-plementary Fig. 1B), exhibited lower DNA-binding activity(Fig. 2A, compare lanes 7 and 8). These findings suggest thatinteraction of specific proteins to NRE may be necessary forsuppression of ADAM-12 expression, and breast cancer cells

Figure 2. Reduction of NRE-interacting DNA-binding activity inbreast cancer cells and tissues.A, 32P-labeled ADAM-12 DNA(þ100/þ190) was incubated withNE (10 mg each) from MCF-10A(lanes 2, 4, and 5) and MDA-MB-231 (lane 3) cells, normal caninemammary tissue (lanes 7 and 9),and canine breast cancer tissue(lane 8), as indicated. Forcompetition, 100-fold molarexcess of specific competitor (sp.comp.) or nonspecific competitor(nonsp. comp.) DNA was used. B,same probe as in A was incubatedwithMCF-10ANE in the absenceorin presence of ADAR1 and DAI/DLM1/ZBP1 antibodies or normalIgG.

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may have lost this regulatory process. Identification of NRE-binding proteins may, therefore, provide a valuable clueregarding this regulatory mechanism.As NRE of ADAM-12 forms Z-DNA structure (11), antibodies

against the 2 known mammalian Z-DNA–binding proteins,ADAR1 (16) and DAI/DLM-1/ZBP1 (17) were used in a DNA-binding assay to test whether these proteins are involved inbindingtoADAM-12NRE.Noeffectofeitherofthese2antibodieson the DNA–protein complex (Fig. 2B) suggested that theADAM-12NRE-specificcomplexdoesnot involvetheseproteins.

Identification of MeCP2 and NF1 as the interactingproteins to the Z-DNA–forming NRE

To search for NRE-binding proteins, we screened a humanbreast cDNA expression library with a 32P-labeled concatenat-ed ADAM-12 NRE DNA and isolated several clones. DNAsequencing revealed MeCP2, NF1-C, and NF1-X as NRE-DNAbinding factors.

MeCP2 is a methyl-CpG–binding protein (18–20) and NF1represents a family of transcription factors that function eitheras a transcriptional repressor or a transcriptional inducer (20).

Figure 3. MeCP2 and NF1 proteins interact with ADAM-12 NRE. A, schematic of ADAM-12 NRE. B, 32P-labeled ADAM-12 DNA (þ100/þ190) was incubatedwith MCF-10A (lanes 2–7), MDA-MB-231 (lanes 9–12), and mammary tissue (lanes 13–18) NE (10 mg each). For competition, 100-fold molar excess ofspecific competitor (sp. comp.) DNA was added. Super-shift (SS) was conducted with specific antibodies (MeCp2 Ab, NF1-C Ab, NF1-X Ab) with normalIgG as a control. C, effect of combination of antibodies. ADAM-12 DNA (þ100/þ190) was incubated with MCF-10A NE (10 mg) in presence of specificantibodies alone (MeCP2 Ab, NF1-C Ab, NF1-X Ab) or in combination. SS and higher SS (HSS) of DNA–protein complex are indicated. D, effect of ADAM-12DNA methylation on binding of proteins. Both methylated and unmethylated (þ100/þ190) DNA probes were used in the DNA-binding assay with MCF-10A(lanes 2 and 6) and MDA-MB-231 (lanes 3 and 7) NE (10 mg). Lanes 4 and 8 contain unmethylated and methylated probe, respectively, incubated with HhaI.Appearance of HhaI-cleaved probe in lane 4 (arrowhead) but not in lane 8 (arrow) indicates endonuclease resistance of methylated DNA and confirmsmethylation of DNA probe.






Interestingly, a conserved NF1-binding element TGGCT-TGTGCCA was located within nucleotide positions þ100 toþ122 of ADAM-12 (Fig. 3A). It is located adjacent to the Z-DNA–forming dinucleotide repeat element that is presentwithin sequences þ123 to þ160 (Fig. 3A). Given the long-known implication of MeCP2 as repressor of gene expressionvia recruitment of mSin3A, HDAC1 and histone methyltrans-ferase (18–20), andNF1 in chromatin-mediated transcriptionalcontrol (21, 22), we elected to address possible involvement ofMeCP2 and NF1 proteins in binding to ADAM-12 NRE.

In the DNA-binding assay, the NRE-specific DNA–proteincomplex was efficiently supershifted by MeCP2, NF1-C, andNF1-X antibodies (Fig. 3B). Specificity of DNA–protein inter-action was verified by competitor oligonucleotide and specificantibodies (Fig. 3B). Normal mammary tissue extracts fromcanine showed similar pattern indicating similarities betweenthe cultured cells andmammary tissues with regard toMeCP2,NF1-C, andNF1-X proteins (Fig. 3B, lanes 13–18). Super-shift of

the same DNA–protein complex by each of these antibodiessuggested that MeCP2, NF1-C, and NF1-X proteins coopera-tively interact with the ADAM-12 NRE DNA. This finding wasfurther substantiated by the combined antibodies that resultedin further or higher super-shift of the NRE-specific DNA–protein complex (Fig. 3C, compare between lanes 1–7).

Bindings of NF1 and MeCP2 to NRE are independent ofDNA methylation

As DNA methylation is prevalent in cancer, we testedwhether methylation of CpG element of ADAM-12 NRE wouldenhance the binding of cancer cell–derivedMeCP2.MethylatedDNA did not increase protein binding to ADAM-12 NRE ineither normal or cancer cells (Fig. 3D). Lack of increasedbinding suggested that function of MeCP2 for suppression ofADAM-12 is not altered by DNA methylation in breast cancercells, which may explain why ADAM-12 expression remainshigh in cancer cells.

Figure 4. The Z-DNA–formingsequences and NF1 DNA-bindingelement are both required for NRE-mediated function. A, ADAM-12DNA (þ100/þ122) that lacks the Z-DNA element was subjected toDNA-binding assay with NE (10 mg)from MCF-10A (lane 2) and MDA-MB-231 (lane 3) cells. Lanes 4–6represent 5-fold longer exposure.B, DNA-binding assay, same as A,was conducted with ADAM-12DNA (þ123/þ190), which containsthe Z-DNA but lacks the NF1-binding site. C, DNA-binding assaywas conducted with ADAM-12DNA (þ100/þ190), containing bothNF1-binding site and Z-DNA. Theradiolabeled DNA was incubatedwith MCF-10A NE (10 mg; lanes 2–6). For competition, 100-fold molarexcess of nonspecific DNA (nonsp.DNA), þ100/þ122, þ123/þ190, orþ100/þ190 DNAs were added. D,promoter function of the wild-typeand mutant ADAM-12–CATconstructs (0.5 mg of DNA) aftertransfection into MCF-10A cells.Relative CAT activity wasdetermined as described in Fig. 1.The results represent an average of3 independent experiments(P < 0.05).

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Mutual binding of MeCP2 and NF1 to ADAM-12 NREpromotes transcriptional repressionAs MeCP2 and NF-1–binding sites are present side-by-side

in the NRE of ADAM-12, to determine their relative contribu-tion, we divided NRE in 2 parts, þ100/þ122 and þ123/þ190,containing the NF1- and MeCP2-binding sites, respectively.Surprisingly, there was very little binding of both proteins eveninMCF-10A cells that showmore avid binding when both sitesare present (compare Figs. 4A and B with C). But, as compe-titors, these small DNA units inhibited DNA–protein complexformation by the full-length probe (Fig. 4C, lanes 4 and 5). Also,specific mutations of NF-1 (þ100/þ122) and MeCP2 (þ123/þ190) binding sites markedly affected NRE-mediated tran-scriptional suppression activity (Fig. 4D). Together, theseresults indicated that loss of either NF1 or Z-DNA (MeCP2-binding site) sequences severely compromises interactionof both NF1 and MeCP2 proteins. These data also suggestthat MeCP2 and NF1, after occupying their respective DNA-binding sites, most likely cooperatively stabilize NRE-specificDNA–protein complex. Consistent with these findings, ChIPassay revealed that both MeCP2 and NF1 proteins interactwith the ADAM-12 NRE in vivo (Fig. 5A). Simultaneouspresence of MeCP2 and NF1 proteins in the ADAM-12promoter was examined by re-ChIP assays, which showed thepresence of both proteins at the ADAM-12 NRE in MCF-10Acells (Fig. 5B).

MeCP2 and NF1 proteins levels are altered in breastcancer cellsReduced DNA–protein complex with NRE in breast cancer

cells (Figs. 2–4), raised the possibility that the levels of MeCP2and NF1 are altered in breast cancer cells. Western blotanalysis revealed 2 closely migrating MeCP2 bands in normalMCF-10A cells but only one in MDA-MB-231 cancer cells (Fig.6A). Further analysis showed only the upper MeCP2 band inseveral breast cancer cells (Fig. 6B). In human breast cancertissue,MeCP2 level is also very low comparedwith the adjacentnormal breast tissue, but unlike cell lines, only a single proteinband was detected (Fig. 6B, lanes 6 and 7). Quantitativeevaluation of MeCP2 levels revealed a significant reductionof the protein in cancer cells (Fig. 6C, MeCP2-b) and tissues(Fig. 6C, columns 6 and 7). Detection of different MeCP2proteins in normal and breast cancer cells, to the best of ourknowledge, has not been reported earlier. We investigatedwhether phosphorylation, which often generates multiplebands formany proteins in a PAGE, accounts for the differencein the protein pattern seen in Fig. 6A and B. Dephosphorylationreaction did not change MeCP2 migration (Fig. 6D) suggestingthat a mechanism, other than phosphorylation, might beinvolved for the difference of MeCP2 in MCF-10A and thebreast cancer cells and addressed in Discussion.A 50 kDa NF1-X protein was detected only in MCF-10A cells

and human normal breast tissue (Fig. 6E), whereas an approx-imately 74–75 kDa NF1-C protein was seen only in cancer cellsand human breast cancer tissue (Fig. 6F). Incidentally, reducedNF1-X level has been linked to increased ADAM-12 expressionduring heat-induced stress in U-251MG glioblastoma cells (23)and a 74 kDa NF1-C protein has been detected during early

mammary gland involution but not in lactating mammarygland (24).

DiscussionWe report a novel finding that delineates mechanism of

ADAM-12 overexpression in breast cancer cells. Our investi-gation has revealed a unique epigenetic regulatory process inwhich Z-DNA element plays an essential role. The loss of Z-DNA–mediated silencer function in breast cancer cells is amajor cause for marked increase of the expression of ADAM-12leading to cell proliferation and metastasis. Furthermore, theresults have revealed that MeCP2, a prominent epigeneticregulator, in association with NF1 family of transcriptionfactors, interacts with the Z-DNA element possibly in a meth-ylation-independent manner.

Z-DNA is an unusual left-handed conformation formed inthe DNA by a stretch of alternating purine-pyrimidine dinu-cleotide repeats, such as GC, TG, or TA repetitive sequences(n � 12 units; ref. 25). Z-DNA elements are predominantly

Figure5. MeCP2andNF1proteins interactwith theADAM-12NRE in vivo.A, semiquantitative ChIP assay. Cross-linked MCF-10A cells wereimmunoprecipitated with nonspecific (lane 3), MeCP2 (lane 4), NF1-C(lane 5), or NF1-X (lane 6) antibodies. Immunoprecipitated DNAwas usedfor PCR amplification of ADAM-12 NRE segment (þ68/þ193) or anupstream region (�619/�328), which was used as a negative control. Inlanes 1 and 2, the chromatin input was diluted 5 times at each step. B, re-ChIP assay. ChIP was conducted with MeCP2, NF1-C, or NF1-Xantibody. The eluent of each immunocomplex was furtherimmunoprecipitated using MeCP2, NF1/C, or NF1-X antibody, asindicated. The precipitated chromatin was subjected to PCRamplification as in A.






concentrated near the transcription start sites (26) and havebeen implicated in gene regulation (27–30), chromatin remo-deling (31), recombination (32, 33) and large-scale deletions(34). It is interesting to note that many cancer-associatedgenes, including EGFR, ER-a, Cyr61, ACCA, prolactin, MMP-9,heme oxygenase 1, and HMGA2, contain dinucleotide repeatelements that have been identified as regulatory elements(Supplementary Table S1); but how these sequences regulategene expression remained poorly understood. The findings,reported here, provide a regulatory mechanism that mayexplain how these cancer-associated genes could be regulatedin malignant cells.

MeCP2 generally acts as a transcriptional repressor. Twoglobal mechanisms of gene regulation, DNA methylation, andhistone deacetylation can be linked by MeCP2 (18, 19). It alsolinks histone methyltransferase and the DNA methyltransfer-ase DNMT1 (20, 35) and thus acts as a mechanistic bridgebetween DNA methylation, histone deacetylation, and histonemethylation. It also associates with the BAF/SWI/SNF chro-matin remodeling complex to repress gene expression (36).The NF1 family of transcription factors has been shown tointeract with hormone receptor (22), histones (37, 38), epige-neticmodifiers, such as histone deacetylase (HDAC; refs. 28, 39)and BAF/SWI/SNF (23) during regulation of gene expression.We show that binding ofMeCP2 at Z-DNA site and recruitmentof NF1 to the adjacent site (Fig. 3) are 2 events that lead to thesuppression of ADAM-12 expression.

Our finding of 2 MeCP2 bands in normal MCF-10A breastepithelial cells, but only the slower migrating MeCP2 band inseveral breast cancer cells, is intriguing. Phosphorylation ofMeCP2 does not seem to play any role in the appearance ofthese alternate forms (Fig. 6C). TwoMeCP2 bands could be thetranslation product of MeCP2_e1 and MeCP2_e2 splice var-iants, which differ by 12 amino acids (40). Although the longerMeCP2_e1 isoform is more abundant in brain (40), we find theshorter isoform seems to be predominant in normal breastepithelial cells (Fig. 6A). Similarly, in normal breast tissuelysate, only one MeCP2 band that comigrates with the shorterisoform (MeCP2-b) was detected. This isoform is markedlyreduced in cancer cells. Its low level in breast cancer cells offersan interesting possibility that this MeCP2 isoform might beinvolved in suppressing ADAM-12 expression in normal breastcells.

In summary, we have uncovered a novel interplay betweenZ-DNA, epigenetic regulator MeCP2, and NF1 family of tran-scription factors in regulating gene expression. A close asso-ciation betweenMeCP2 and NF1 proteins at Z-DNA element ofADAM-12 is found to be necessary for the suppression ofADAM-12 expression. MeCP2 deficiency in breast cancer cellsresults in the loss of this crucial suppression mechanismleading to overexpression of ADAM-12. Such a phenomenonhas not been reported earlier. Although Z-DNA–formingsequences have been identified in many prominent cancer-related genes (Supplementary Table S1), how these sequences

Figure 6. Differences in the level of MeCP2 and NF1 proteins in normal mammary epithelial and breast cancer cells and tissues. A, Western blot analysis ofMCF-10A and MDA-MB-231 cell extracts (70 mg) using MeCP2 and b-actin (loading control) antibody. Arrows indicate two (a, b) closely migratingMeCP2 bands. B, Western blot analysis of MCF-10A,MDA-MB-231, MCF-7, DU4475, Hs578T cell extracts, human normal breast, and human breast cancertissue lysates, as indicated, with MeCP2 and b-actin antibody. C, histogram summarizes the Western blot results. D, cell extracts were dephosphorylatedwith FastAP thermosensitive alkaline phosphatase (AP) for 1 hour before loading and immunoblotted with MeCP2 antibody. As control anti–phospho-STAT3antibody was used. Presence of phospho-STAT3 (lane 3) and absence (lane 4) confirms phosphatase action. E and F, MCF-10A and MDA-MB-231cell extracts, human normal, and human cancer breast tissue lysates were immunoblotted using NF1-X or NF1-C antibody, as indicated. G, histogramsummarizes the Western blot results.

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regulate gene expression remained unknown. Our results mayprovide amolecular basis for interpretation of Z-DNA–formingdinucleotide repeat length polymorphism-associated cancersusceptibility and unravel the ultimate relevance of this epi-genetic mechanism to cancer.

Disclosure of Potential conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: A. Ray, B.K. RayDevelopment of methodology: A. Ray, S. Dhar, B.K. Ray, A. RichAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): C.J. Henry, A. RayAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Ray, B.K. Ray, A. RichWriting, review, and/or revision of themanuscript: C.J. Henry, A. Ray, B.K. Ray

Administrative, technical, or material support (i.e., reporting or orga-nizing data, constructing databases): C.J. Henry, S. Dhar, A. Ray, B.K. RayStudy supervision: A. Ray, B.K. Ray

AcknowledgmentsThe authors thank N. Tanese, P.L. Jones and U.S. Singh for generous gift of

NF1/C, MeCP2, and NF1-X antibodies, respectively.

Grant SupportThis study was supported partly by grants from U.S. Army Medical Research

and Materiel Command, University of Missouri Research Board, and Universityof Missouri, College of Veterinary Medicine.

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

Received July 2, 2012; revised September 27, 2012; accepted October 23, 2012;published OnlineFirst November 7, 2012.

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Ray et al.





2013;73:736-744. Published OnlineFirst November 7, 2012.Cancer Res Bimal K. Ray, Srijita Dhar, Carolyn Henry, et al. Cross-talk between MeCP2 and NF1 Transcription Factor FamilyCancer-Associated ADAM-12 Expression in Breast Cancer: Epigenetic Regulation by Z-DNA Silencer Function Controls

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