ORIGINAL ARTICLE

Aberrant epigenetic regulation of bromodomain Brd4in human colon cancer

R. M. Rodriguez & C. Huidobro & R. G. Urdinguio &

C. Mangas & B. Soldevilla & G. Domínguez & F. Bonilla &

A. F. Fernandez & M. F. Fraga

Received: 31 March 2011 /Revised: 6 October 2011 /Accepted: 11 November 2011 /Published online: 27 November 2011# Springer-Verlag 2011

Abstract The bromodomain protein BRD4 is involved incell proliferation and cell cycle progression, primarilythrough its role in acetylated chromatin-dependent regula-tion of transcription at targeted loci. Here, we show thatBRD4 is frequently downregulated by aberrant promoterhypermethylation in human colon cancer cell lines andprimary tumors. Ectopic re-expression of BRD4 in thesecolon cancer cell lines markedly reduced in vivo tumorgrowth, suggesting a role of BRD4 in human colon cancer.

Keywords Colon cancer . DNA methylation . BRD4 .

Tumor suppressor gene

Introduction

The bromodomain and extra terminal (BET) familyconstitutes a group of proteins characterized by twobromodomains able to bind acetylated lysine [1, 2].BRD4 is highly expressed in dividing cells and isinvolved in cell growth regulation, although the under-lying molecular mechanisms have not been fully eluci-dated yet. During mitosis, BRD4 is associated tochromosomes, which is necessary for G1/S and G2/Mtransition [2, 3] and for the expression of many G1 geneproducts [4, 5]. The ubiquitous positive transcriptionelongation factor b (P-TEFb) has been implicated in theBRD4-mediated expression regulation. BRD4 binding toacetylated histones H3 and H4 thus leads to BRD4-dependent recruitment of P-TEFb, allowing de novo M/G1 gene transcription [4, 5]. BRD4 overexpression [6] ordownregulation [7–9] in cultured cells leads to reducedproliferation, indicating an important role in cell growth.BRD4−/−mouse embryos die shortly after implantation[7], and mouse BRD4 knockout embryonic stem cells donot grow in culture [10].

BRD4 is expressed ubiquitously in healthy prolifer-ating cells [2], and its deregulation is linked to cancer. Intestis tumors, BRD4 participates in a translocation leadingto a fusion oncoprotein with the nuclear protein in testis[11]. In breast cancer, BRD4 expression is reported toreduce invasiveness, tumor growth, and metastasis bymodulating extracellular matrix gene expression [12].Here, we show that BRD4 is frequently downregulated byaberrant promoter hypermethylation in human coloncancer and that its re-expression impairs tumor growthin vivo.

Electronic supplementary material The online version of this article(doi:10.1007/s00109-011-0837-0) contains supplementary material,which is available to authorized users.

R. M. Rodriguez : C. Huidobro : R. G. Urdinguio : C. Mangas :A. F. Fernandez :M. F. Fraga (*)Cancer Epigenetics Laboratory,Instituto Universitario de Oncología del Principado deAsturias (IUOPA), HUCA, Universidad de Oviedo,Oviedo, Spaine-mail: mffraga@cnb.csic.es

B. Soldevilla :G. Domínguez : F. BonillaDepartment of Medical Oncology,Hospital Universitario Puerta de Hierro,Universidad Autónoma de Madrid,C/Manuel de Falla 1, Majadahonda,Madrid, Spain

M. F. FragaDepartment of Immunology and Oncology,Centro Nacional de Biotecnología (CNB/CSIC),Cantoblanco,Madrid, Spain

J Mol Med (2012) 90:587–595DOI 10.1007/s00109-011-0837-0

Methods

Human colon cancer cell lines and primary tumor samples

The nine human colon cancer cell lines (HCT116, Caco2,Co115, HT29, Colo205, SW48, SW480, SW620, andDLD1) used in this study were obtained from the AmericanType Culture Collection. The cell line lacking DNAmethyltransferase 3b (DNMT3b) and DNA methyltransfer-ase 1 (DNMT1) was derived from HCT116 cells. All celllines were maintained in DMEM supplemented with 10%fetal calf serum and nonessential amino acids. Nineteenpairs of healthy and tumor colon tissues from the samepatients, peripheral blood lymphocytes, and 40 coloncancer samples paired with normal tissue from the samepatients and mounted on a paraffin-embedded tissue micro-array were obtained from the Institute of Oncology ofAsturias Tumor Bank. Finally, a DNA collection of 48colon cancer samples paired with healthy colon epitheliumwas obtained from Hospital Puerta de Hierro in Madrid.

RNA extraction and quantitative real-time PCR

Total RNA was isolated using Trizol (Invitrogen) accordingto manufacturer’s instructions. Residual genomic DNA wasremoved by DNAse I digestion with an RNase-Free DNaseSet (Qiagen). Reverse transcription was performed usingSuperscript II (Invitrogen) following manufacturer’s proto-cols for oligo dT primers. Quantitative PCR reactionscontained 10 ng cDNA, Taqman Master Mix, and BRD4Taqman probe hs_00293232_m1 (Applied Biosystems).Each sample was tested in triplicate in an ABI 7900sequence detection system. Data were analyzed usingsystem software. Expression of genes of interest wasnormalized to GAPDH in all cases. Significant differenceswere assessed by two-tailed independent samples t test.

Genomic DNA extraction and pyrosequencing analysis

Genomic DNA was obtained with Wizard Genomic DNAPurification Kit (Promega) following manufacturer’sinstructions. Sodium bisulfite modification of 500 ngDNA was carried out with the EZ DNA methylation kit(D5002, Zymo Research, CA) following the manufacturer’sprotocol. Pyrosequencing for BRD4 was performed usingthe PyroMark kit (Qiagen). Primers used were: F1 (5′-GTGAAGGAGGATTAAGGTTTTTAAG-3′), R1 biotiny-lated (5′-CACAAA TAAAATTACTTTTCCATCTAA-3′),and the sequencing primer S1 (5′-GTGAAGGAGGATTAAGG-3′). These primers amplify a region 607 bp up-stream of the transcription initiation site containing sixCpG. The PCR condition was 50 cycles at 95°C for 60 s,60°C for 30 s, and 72°C for 30 s, followed by 72°C for

5 min. The biotinylated PCR product was purified andmade single-stranded to act as a template in a pyrosequenc-ing reaction using the Pyrosequencing Vacuum Prep Tool(Qiagen). Methylation density was quantified using Pyro-sequencing Analysis and the PyroMark Q24 system(Biotage).

Chromatin immunopreciputation assay

Chromatin immunopreciputation (ChIP) assays (0.5–1×106

cells per sample) were performed with AcH4K16 (Active-Motive), AcH3 (Millipore), H3 (Upstate Biotechnologies),and H3K9m3 (Upstate Biotechnologies) antibodies. NormalIgG was used as negative control (Abcam). In brief, cellsfixed with 1% formaldehyde were lysed in sodium dodecylsulfate (SDS) lysis buffer [1% SDS, 10 mM ethylenedia-minetetraacetic acid (EDTA), and 50 mM Tris–HCl pH 8.1]and sonicated. The shared chromatin was diluted in a ChIPdilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mMEDTA, 16.7 mM Tris–HCl pH 8.1, and 167 mM NaCl) andincubated with antibody overnight at 4°C. Antibody–chromatin complexes were precipitated with Salmon SpermDNA/Protein A-Agarose beads (Upstate Biotechnologies),washed, and eluted from beads using an elution buffer (1%SDS, 0.1 M NaHCO3). After cross-link reversal, DNA wasextracted with phenol–chloroform and was ethanol-precipitated. Immunoprecipitated DNA was analyzed intriplicate by real-time PCR from 1 μl eluted DNA. Primersused were 5′-GTGATTTCATGAGGGGCTGT-3′ (sense)and 5′-GCTGAAGTCATGA GGTGTGG-3′ (antisense),amplifying a 158-bp region 110 bp upstream of the BRD4transcriptional start site. Unbound fractions were analyzedas input control. Results are presented as x-fold enrichmentof precipitated DNA associated with a given histonemodification, relative to a 1:200 dilution of input chromatin.

Cell treatment

Cells were treated with 2 μmol/L 5-aza-2′-deoxycytidine(AZA; A3656, Sigma) for 2 days to achieve demethylation.For trichostatin A (TSA) treatment, cells were incubated(24 h) at a concentration of 300 μM. For combined TSA+AZA treatments, cells were incubated with AZA 2 μmol/Lfor 2 days and then treated with 300 μM TSA for 24 h.

Immunofluorescence

Cells were fixed with 4% paraformaldehyde, then washedin Dulbecco's phosphate-buffered saline (DPBS) containing0.1% Triton X-100 (Sigma), and permeabilized with 1%Triton X-100 (15 min, room temperature). Slides wereblocked with 10% donkey serum (2 h) and incubated withanti-BRD4 (Abcam) antibody overnight at 4°C. After

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washing, the slides were incubated with Alexa 594-conjugated secondary antibody (Invitrogen). Cells werevisualized with an epifluorescence system on a Leicamicroscope. Paraffin-embedded tissue microarray slideswere pretreated with Tris–EDTA buffer pH 9 for 3 min at100°C. Immunofluorescence quantification was performedonly if healthy colon epithelium, adenomatous polyp, andcolonic adenocarcinoma from the same patient were presenton the tissue microarray in triplicate (seven cases). Theremaining samples in the tissue microarray were excludedfrom this study. Each nucleus was delineated in the bluechannel (nuclear staining). A total of 100 regions wereselected in each replicate and transferred to the red channel(BRD4 staining). Average pixel intensity was calculatedwith ImageJ (http://rsbweb.nih.gov/ij/) for each region. Foranalysis, each region’s fluorescence intensity in the redchannel was divided by average pixel intensity in the bluechannel to normalize data, and the mean signal for allnuclei in the three replicates was calculated. Significantdifferences were assessed by two-tailed independentsamples t test.

Brd4 transfection

The pcDNA4c hBrd4 full-length construct was obtainedfrom Addgene (addgene plasmid 14441). HCT116 cellswere then transfected with the pcDNA4c hBrd4 full-lengthplasmid using Lipofectamine 2000 (Invitrogen). Stabletransfectants were obtained after selection with zeocin(Invitrogen; 200 μg/ml, 2 weeks). BRD4 expression wasconfirmed by immunostaining with anti-BRD4 antibody(Abcam).

Cell viability assay

Cell viability was assayed on BRD4 full length-transfectedHCT116 cells and mock controls. Cells (2×103) wereplated onto 96-well microdilution plates, allowed to attach,and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT; 50 μg) was added to medium (100 μl/well;3 h, 37°C, 5% CO2). MTT was removed and MTT-formazan crystals dissolved in DMSO (100 μl/well).Absorbance at 595 nm was determined in an automatedmicrotiter plate reader. Optical density was directlyproportional to cell number up to the density attainedby the end of the assay. Results are expressed as themean±SD (n=3).

Colony formation assay

Colony formation was assayed on BRD4 full length-transfected HCT116 cells and mock controls. The colono-genic assay was as described [13]. After 10 days in culture,

cells were fixed with 5% glutaraldehyde (Sigma) and 0.5%crystal violet (30 min). Plating efficiency is expressed asthe ratio between cells seeded and number of coloniesobserved.

Mouse xenograft model

Six-week-old athymic nude-Foxn1nu/nu mice were used fortumor xenograft experiments with hBRD4- and mock-transfected HCT116 cells. The experiments were carriedout with three different amounts of cells (1.5, 2, and 2.5×106 cells in 200 μl Ca2+/Mg2+-free DPBS; Gibco). Eachdifferent amount of cells was injected in six independentnude mice. A total of 18 nude mice were used in this study.Previously to injection, cell viability was checked byTrypan blue staining (0.1% in DPBS). In all cases, viabilitywas over 95%. Cells were injected subcutaneously intoboth flanks of each mouse (right flank, human Brd4-transfected cells; left flank, mock cells). Tumor width (W)and length (L) were measured at 14, 21, and 28 days; tumorvolume was estimated as V=0.4×L×W2. Mean volumeand tumor mass±SEM were calculated for each group.Significant differences were assessed by the Wilcoxonsigned-rank test.

Promoter DNA methylation profiling using bead arrays

Microarray-based DNA methylation profiling of BRD4 wasperformed in several samples with the HumanMethyla-tion27 DNA Analysis BeadChip (Illumina, San Diego,CA). The Infinium Methylation Assay combines bisulfiteconversion of genomic DNA and whole-genome amplifi-cation sample preparation with direct, array-based captureand enzymatic scoring of the CpG loci. Bisulphiteconversion of DNA was performed using the EZ DNAMethylation Kit (Zymo Research) according to manufac-turer’s procedures. Processed DNA samples were hybrid-ized to the BeadChip (Illumina). The assay interrogates thechemically differentiated loci using two site-specificprobes, one designed for the methylated locus (M beadtype) and another for the unmethylated locus (U bead type).Single-base extension of the probes incorporates a labeledddNTP, which is subsequently stained with a fluorescencereagent. The methylation level for the interrogated locus isdetermined by calculating the ratio of the fluorescentsignals from the methylated vs. unmethylated sites. Theratio of fluorescent signals was then computed from the twoalleles according to the following formula: Beta=Max(M,0)/Max(U,0)+Max(M,0)+100. This beta value (β) is aquantitative measure of DNA methylation levels of specificCpG sites and ranges from 0 for completely unmethylatedto 1 for completely methylated [14]. The arrays werescanned on the Illumina iScan system, and the raw data

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were imported and analyzed with the BeadStudio software(version 3.1.3.0 Illumina, Inc).

Results

Human bromodomain BRD4 is frequently aberrantlyhypermethylated in colon cancer

To study the possible aberrant epigenetic regulation ofBRD4 in colon cancer, we used bisulfite pyrosequencing todetermine the methylation status of a CpG cluster located607 bp from the BRD4 transcription start site in healthycolon epithelium and nine colon cancer cell lines (HCT116,Caco2, Co115, HT29, Colo205, SW48, SW480, SW620,and DLD1). This analysis showed that the BRD4 promoterwas completely unmethylated in non-tumorigenic primary

colon tissue, while it was densely hypermethylated in mostcancer cell lines analyzed (HCT116, Caco2, Co115, HT29,Colo205, SW48, and SW480; Fig. 1a). We confirmed theseresults using the 27K Illumina Infinium methylationplatform to study the DNA methylation status of two CpGpositions (665 bp upstream and 138 bp downstream theBRD4 transcription start site, respectively) within theBRD4 CpG island (Fig. 1b).

To determine whether BRD4 promoter hypermethylationis also a frequent event in vivo, we analyzed BRD4promoter methylation by bisulfite pyrosequencing in 19primary colon adenocarcinoma tissue samples and com-pared them to healthy colon epithelium from the samepatient. Tumors with an average percent of methylation atleast 10% higher than healthy epithelium were consideredhypermethylated. We found hypermethylation in 9 of the 19sample pairs analyzed (47%), confirming that human BRD4

Fig. 1 BRD4 aberrant promoter hypermethylation in colon cancercell lines and primary colon adenocarcinomas. a Pyrosequencinganalysis of DNA methylation in the BRD4 promoter region. Six CpGsites located approximately 600 bp upstream the transcriptioninitiation site were analyzed. Illumina array probes (asterisk) and theregion analyzed by chromatin immunoprecipitation (white bar) arehighlighted. The percentage of methylation for each CpG site isshown. b Illumina methylation array data indicating the percentage of

methylation for two CpG sites (665 bp upstream and 138 bpdownstream the BRD4 transcription start site). c BRD4 methylationin normal colon epithelium and paired colon cancer tissues (left).Percentages represent the average percent of methylation of the sixCpG sites analyzed. Hypermethylated tumors are marked with anasterisk. Percentages of unmethylated (U) tumors and tumorsdisplaying BRD4 promoter hypermethylation (M) are shown in theright panel

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hypermethylation is a frequent phenomenon in humancolon cancer (Fig. 1c). In addition, another DNA collectionfrom 48 paired colon cancer samples with detailedhistopathological data was analyzed to evaluate whetherBRD4 hypermethylation was associated with stage andhistological grade. We found hypermethylation in 15 of 48tumors (31%). Hypermethylated BRD4 was enriched inhigh histological grade and advanced Dukes stage tumors(Fig. S1 in the Electronic supplementary material). How-ever, the associations were not statistically significant (χ2

exact test, p>0.05). In the case of the histological grade,lack of statistical significance (χ2 exact test, p=0.060)could be due to the low number of samples in some ofthe tumor types analyzed (Fig. S1 in the Electronicsupplementary material). Further studies with a greaternumber of tumors should determine the associationbetween BRD4 promoter hypermethylation and tumorhistopathological and clinical status.

Promoter DNA methylation, histone deacetylation,and histone methylation mediate BRD4 repressionin colon cancer

We studied the role of BRD4 hypermethylation in geneexpression to compare relative mRNA levels in healthycolon epithelium and the SW620 and DLD1 cell lines,which show no BRD4 promoter hypermethylation, with theHCT116, Caco2, Co115, Colo205, SW48, HT29, andSW480, which show dense DNA methylation at theBRD4 promoter. These experiments revealed that, indepen-dently of methylation status, BRD4 mRNA was down-regulated in all cancer cell lines compared to healthy tissue(Fig. 2a). To determine whether promoter methylationstatus is linked to BRD4 expression, we incubated fourcolon cancer cell lines with the demethylating drug 5-aza-2-deoxycytidine (Fig. 2b). We treated two hypermethylatedcell lines (HCT116 and Colo205) and two unmethylatedlines (DLD1 and SW620). The drug induced BRD4 geneexpression in hypermethylated cells but not in the unme-thylated ones, indicating that BRD4 expression is associat-ed with promoter DNA methylation. To further explore therole of DNA methylation in BRD4 expression, we analyzedgene expression and DNA methylation in a knockout cellline lacking DNA methyltransferase 3b (DNMT3b) andDNA methyltrasnferase 1 (DNMT1) (DKO) derivedfrom the colon cancer cell line HCT116. A decreasein methylation in cells lacking the DNA methyltrans-ferases was associated to BRD4 expression upregulation(Fig. 2c).

To study the relationship between promoter hyper-methylation and BRD4 expression in vivo, we analyzedBRD4 expression at mRNA level in 19 paired tumorand normal colon tissues from the same patients. BRD4

was downregulated in 15 of the 19 samples; meanBRD4 expression was 32% lower in colon adenocarci-noma (p<0.01; Fig. 3a). To study BRD4 at the proteinlevel, we used a tissue microarray containing colon cancersamples from seven patients with healthy colon epitheli-um, adenomatous polyp, and colonic adenocarcinomafrom the same patient; quantitative analysis of BRD4

Fig. 2 Promoter methylation-dependent BRD4 repression in coloncancer-derived cell lines. a Relative BRD4 expression in colon cancercell lines compared to normal colon epithelium. b BRD4 expressionafter 5-aza-2′-deoxycytidine treatment of unmethylated and hyper-methylated colon cancer-derived cell lines. c Relative BRD4 expres-sion (left) and percentage of promoter methylation (right) in HCT116control and double DNMT3b and DNMT1 knockout cells (DKO)

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immunofluorescence confirmed a significant decrease inBRD4 protein levels in colon adenocarcinoma (−22%,p<0.01) relative to healthy epithelium from the samepatient (Fig. 3b).

We compared methylation status with BRD4 expressionin colon cancer and observed that eight of the ninehypermethylated samples (89%) showed BRD4 down-regulation, confirming the correlation between DNA hyper-

Fig. 3 Epigenetic silencing ofBRD4 in primary colon cancer.a Average BRD4 expression in19 paired samples of healthycolon epithelium and coloncancer (**p<0.01). b Immuno-fluorescence staining for BRD4in healthy colon epithelium,adenomatous polyp, and colonadenocarcinoma; Brd4 in red.Arrowheads indicate epithelialnuclei in colonic crypts.Fluorescence signal is expressedas the relative average pixelintensity per nucleus comparedto healthy colon from thesame individual (**p<0.01).c Relative percentage of DNAmethylation (top) and relativegene expression (bottom) of fiverepresentative tumor samplescompared to normal tissue.Hypermethylation samples showBRD4 downregulation (T13 andT14), but not unmethylatedtumors (T10 and T11). In somecases, unmethylated samplesshowed BRD4 downregulationas well (T17). d BRD4reactivation by 5-aza-2′-deoxycytidine (AZA) andtrichostatin A (TSA) inunmethylated and hypermethy-lated colon cancer-derived celllines. e AcH4K16 and AcH3enrichment at the BRD4promoter region analyzed bychromatin immunoprecipitation(ChIP) in HCT116 and DLD1cells after TSA treatment. f ChIPanalysis of trimethylated lysine9 of histone H3 (H3K9m3) inHCT116 and DLD1 cells

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methylation and gene repression in vivo. Some lowmethylated tumors nonetheless showed BRD4 downregu-lation (see representative samples in Fig. 3c). All cancercell lines tested showed low BRD4 levels compared tonormal tissues, regardless of methylation status (Fig. 2a).The results suggest that additional mechanisms might beinvolved in BRD4 repression in colon cancer. To test thishypothesis, we treated two cell lines (HCT116 and DLD1)with trichostatin A (TSA), which is a histone deacetylaseinhibitor. After a 24-h treatment, HCT116 cells upregulatedBRD4 in response to TSA. On the other hand, DLD1response was very weak (Fig. 3d). We used a chromatinimmunoprecipitation assay (ChIP) to confirm that TSAtreatment increased BRD4 expression through inhibition ofhistone deacetylation in the BRD4 promoter and notthrough an indirect pleiotropic mechanism (Fig. 3e).AcH3 and AcH4K16 increased in response to TSAtreatment in HCT116 (hypermethylated) and DLD1 cells(unmethylated), although the response was higher inHCT116. Since DLD1 response to TSA was weak, wewanted to analyze other epigenetic marks that could beinvolved in BRD4 regulation. By ChIP analysis, weobserved that the repressive mark H3K9m3 becomes highlyenriched in DLD1 but not in HCT116 cells (Fig. 3f). On theother hand, H3K9m3 levels were not altered after treatmentwith TSA or TSA and AZA (Fig. S2 in the Electronicsupplementary material). Enrichment of the repressiveepigenetic mark H3K9m3 in DLD1 cells could explainthe absence of response to TSA treatment and suggests thatBRD4 expression is regulated not only by DNA methyla-tion but also by a complex epigenetic mechanism that mayinclude histone acetylation and histone methylation.

BRD4 restoration reduces tumorigenesis of colon cancercells in vivo

To evaluate the functional role of BRD4 repression bypromoter hypermethylation in colon cancer, we transfectedBRD4 into the HCT116 colon cancer cell line, which showsDNA methylation-dependent BRD4 inactivation (Fig. 4a).MTT analysis, used to measure the proliferation potential ofHCT116 cells after BRD4 restoration, showed no signifi-cant differences compared to control HCT116 cells(Fig. 4b), nor was colony formation potential affected(Fig. 4c). To further characterize the role of BRD4 in vivo,we injected BRD4-overexpressing HCT116 cells andcontrol cells transfected with empty vector subcutaneouslyinto immunodeficient nude mice. BRD4 overexpressionresulted in a reduction of, at least, 75% of tumor volumeusing three different amounts of cells (1.5, 2, and 2.5 x 106

cells; Fig. 4d). Retention of BRD4 expression in the tumorxenografts was confirmed by real-time PCR (Fig. S3 in theElectronic supplementary material).

Discussion

The double bromodomain in BRD4 is a functional unit thatrecognizes histone acetylation codes and is involved in cellcycle regulation [3]. The role of this protein in cancer isnonetheless largely unknown. BRD4 expression appears tobe essential for normal cell biology since BRD4 knock-down leads to cell growth arrest and mitotic catastrophe [7,9, 12], and its overexpression is associated to cell growtharrest [6]. Alteration of BRD4 in cancer could thus result ina different outcome, depending on initial BRD4 status in aspecific cancer cell since either up- or downregulation ofthis protein can exert a negative effect on proliferation. Anearlier report proposed BRD4 as a tumor suppressor genesince its overexpression leads to reduction in tumor growthand metastasis of a mouse mammary tumor cell line [12].Here, we observed that BRD4 is often epigeneticallydownregulated in human colon cancer. To study thefunctional role of BRD4 epigenetic downregulation, werestored BRD4 expression in a human colon cancer cellline. Ectopic BRD4 expression did not exert a clear effecton cell proliferation or clonogenic potential in vitro, butrather resulted in a marked reduction of tumor growth afterxenograft injection. These results are in agreement with aprevious study in which BRD4 was overexpressed in abreast cancer cell line [12], with no effect on in vitroproliferation but with a marked reduction in tumor growthand metastatic potential. Overall, the data suggest thatBRD4 behaves as a characteristic aberrantly hypermethy-lated gene in colon cancer. Nevertheless, our data did notprovide a mechanistic explanation that would allow us toelucidate the link between BRD4 repression and coloncancer.

Our findings show that, in some cases, BRD4 can bedownregulated in the absence of promoter hypermethyla-tion, suggesting that this gene might be also regulated byother mechanisms. Histone acetylation is a posttranslationalmodification associated to active gene expression, producedby histone acetyltransferases (HAT) and eliminated byhistone deacetylase enzymes (HDAC) [15]. Balanced HATand HDAC recruitment to the transcription initiation site isoften involved in gene regulation [14]. Response to aHDAC inhibitor in spite of methylation status can thus beindicative of the concurrence of an additional regulationmechanism. Treatment of several colon cancer-derived celllines with the HDAC inhibitor TSA resulted in BRD4overexpression, suggesting that BRD4 expression is alsoregulated by an alternative mechanism that affects histoneacetylation. Nevertheless, DLD1 cells responded weakly toTSA but showed enrichment of the repressive epigeneticmark H3K9m3 at BRD4 promoter region. All together,these results indicate that BRD4 is subject to a complexepigenetic regulation.

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As BRD4 is important in proliferation and cell cycleprogression, the epigenetic-dependent downregulation of thisgene in colon cancer could require a compensation mecha-nism that supplies BRD4 function. Other bromodomain-containing proteins (BET) might be upregulated, although the

mechanism by which this compensation could take place isunknown.

In conclusion, our data provide evidence of aberrantepigenetic silencing of BRD4 in colon cancer. Mousexenograft experiments may suggest a role of BRD4 in

Fig. 4 Ectopic BRD4 expressionimpairs tumor growth in vivo. aImmunofluorescence stainingshowing BRD4 overexpressionin HCT116 after transfectionwith the pcDNA4c hBrd4 full-length plasmid. b Proliferation offour BRD4-transfected HCT116cell clones compared tomock-transfected cells, analyzedby MTT assay. c Colonyformation assay of HCT116-transfected cell lines. Platingefficiency is measured as thepercentage of colonies relative tonumber of cells seeded after10 days in culture. Data representthe mean value of threetransfected clones. d Mousexenograft model. Mock- andBRD4-transfected HCT116 cellswere injected subcutaneouslyinto athymic nude mice. Arepresentative image of miceinjected with 2 x 106 cells28 days post-injection is shownin the left panel. Quantificationof tumor volume (cubiccentimeters) from threeindependent experimentsusing three different amountsof cells per inoculum (1.5, 2, and2.5 x 106 cells) is shown at theright. *p<0.05, statisticallysignificant

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colon cancer, but further studies are needed to elucidate themolecular mechanisms involved.

Acknowledgments We thank OIB (FYCIT), M. Santirso, and C.Mark for editorial assistance. CH received support from FIS FI07/00380. AFF, RGU, RMU, and CM are supported by the IUOPA. Thiswork was supported by grants from the Spanish Ministry of Health(PI061267, PS09/02454), the Spanish National Research Council(CSIC 200820I172 to MFF), and the Community of Asturias (FICYTIB09-106). The IUOPA is supported by the Obra Social Cajastur, Spain.

Conflict of interest The authors declare that they have no conflict ofinterests.

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