728 MOLECULAR MEDICINE REPORTS 12: 728-736, 2015 Regulation of the expression of claudin 23 by the enhancer of zeste 2 polycomb group protein in colorectal cancer NATALIA MARYAN1*, MALGORZATA STATKIEWICZ2*, MICHAL MIKULA2, KRZYSZTOF GORYCA2, AGNIESZKA PAZIEWSKA1, ADRIANA STRZAŁKOWSKA2, MICHALINA DABROWSKA2, MATEUSZ BUJKO3 and JERZY OSTROWSKI1,2 1Department of Gastroenterology and Hepatology, Medical Center for Postgraduate Education; Departments of 2Genetics, and 3Molecular and Translational Oncology, Maria Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Warsaw 02-781, Poland Received April 27, 2014; Accepted January 21, 2015 DOI: 10.3892/mmr.2015.3378 Abstract. Altered epigenetic mechanisms, similar to gene the GSK126-treated cell lines. Furthermore, ChIP analysis mutations, contribute to the pathogenesis and molecular of these samples detected histone H3 lysine 4 trimethylation heterogeneity of neoplasms, including colorectal cancer (H3K4me3) at the CLDN23 promoter, demonstrating that the (CRC). Enhancer of zeste 2 (EZH2) is a histone methyltrans- balance between H3K27me3 and H3K4me3 may underlie ferase, which is involved in epigenetic gene silencing and the regulation of the expression of CLDN23. The present is aberrantly expressed in CRC. Therefore, the identifica- study demonstrated an epigenetic link between the activity tion of the genes regulated by EZH2 in CRC is important of the EZH2 methyltransferase at the CLDN23 locus and the to improve current understanding of its role in cancer expression of CLDN23 in CRC tissue. epigenetics. The present study used chromatin immuno- precipitation (ChIP) followed by deep sequencing to assess Introduction genome-wide EZH2-DNA interactions in healthy or CRC mucosa samples. In total, 86.9/61.6 and 92.5/62.6 million Gene expression is regulated at different hierarchical levels, tags were sequenced/mapped in healthy and CRC mucosa one of which is the accessibility of genes and their cis-regu- samples, respectively. The EZH2-binding densities were latory elements to the transcriptional complex. This can be correlated with transcriptomic datasets and this demon- effected by the degree of chromatin compaction, which is strated that the claudin-23 (CLDN23) gene, which encodes controlled, in part, by epigenetic regulators belonging to the a component of cell-cell adhesion structures, was occupied polycomb group of proteins (1). The histone methyltrans- by EZH2 and significantly silenced in CRC tissue. The ferase, enhancer of zeste 2 (EZH2), is a component of the measurement of DNA methylation at the CLDN23 promoter polycomb-repressive complex (PRC) 2, which catalyzes the using pyrosequencing excluded the possibility that silencing methylation of histone H3 lysine 27 residues and mediates of this gene in CRC patient samples was a result of DNA the epigenetic silencing of target genes. Identification of the hypermethylation. Following treatment of the Colo205 and genes that PRC2 regulates is important as EZH2 is often HT-29 CRC cell lines, with the EZH2 inhibitor, GSK126, aberrantly expressed in neoplasms, including colorectal the level of histone H3 lysine 27 trimethylation (H3K27me3) cancer (CRC) (2). was reduced and the mRNA and protein expression levels of Cell-to-cell adhesion in the epithelial cell monolayer is CLDN23 were increased. ChIP analysis confirmed that the mediated by four types of junctions: Tight junctions, adhe- level of H3K27m3 along the CLDN23 gene was decreased in rens junctions, gap junctions and desmosomes (3). Claudins (CLDNs) constitute the backbone of tight junctions and are comprised of a multigene family of 24 protein members with molecular masses ranging between 20 and 27 kDa. The CLDN proteins create paracellular barriers, which are Correspondence to: Professor Jerzy Ostrowski, Maria critical for sealing epithelial sheets, maintaining cell polarity Sklodowska-Curie Memorial Cancer Center and Institute of Oncology, Roentgena 5, Warsaw 02-781, Poland and controlling the flux of ions and small moleculesbetween E-mail: [email protected] epithelial cells (4). These proteins also regulate intercellular signaling and cell morphology (5). CLDNs are expressed *Contributed equally in a tissue-specific manner and the majority of tissues express multiple CLDNs (6). Aberrant expression of CLDNs Key words: colorectal cancer, ChIP-Seq, enhancer of zeste 2, has been demonstrated in several malignancies and this is claudin 23, GSK126 dependent on the tumor localization, stage and grade (4,7). However, tumors exhibit changes in the expression levels of a limited number of CLDNs. Furthermore, previous studies MARYAN et al: EPIGENETIC REGULATION OF CLAUDIN-23 EXPRESSION IN COLORECTAL CANCER 729 have investigated the epigenetic mechanism underlying the 3‑(4,5‑Dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium brom deregulation of CLDNs in certain types of tumor (7,8). ide (MTT) assay. Following incubation with 0.5, 1, 2 and 5 µM One of the characteristics of cancer is disruption of GSK126 for 72 h in a humidified 5% CO2 atmosphere at 37˚C, the attachment of epithelial cells to other cells and to the MTT solution (Sigma‑Aldrich) was added at a final concentra- extracellular matrix, followed by local invasion and distant tion of 0.5 mg/ml from a stock solution of 5 mg/ml, prepared metastasis (9). E-cadherin is the predominant and most using phosphate-buffered saline (PBS; Sigma-Aldrich), and extensively investigated example of a junction component, the cells were incubated for 3 h in a humidified 5% CO2 atmo- which is involved in cancer. This protein has been impli- sphere at 37˚C. The cells were subsequently lysed in acidified cated as a tumor suppressor in various types of cancer via 2-propanol (POCH S.A., Gliwice, Poland) and the absorbance its negative regulation of cancer cell invasion (10,11). The was subsequently measured at 550-560 nm using a Victor3 importance of other protein components of intracellular junc- microplate reader (Perkin Elmer, Waltham, MA, USA). tions, including CLDNs, in tumorigenesis is also emerging. However, the molecular mechanisms underlying their altered ChIP assay. The tissue cryostat section (50 mg) was immersed expression in cancer and the biological significance of this in 2 ml hypotonic buffer A, containing 10 mM HEPES remain to be elucidated (4). This is important for developing (pH 7.9), 2 mM MgCl2 and 2 mM KCl, supplemented with therapeutic interventions, which may restore normal intercel- protease and phosphatase inhibitors (78441) (Thermo Fisher lular attachment. Scientific, Co., Ltd., Waltham, MA, USA). The samples were The present study demonstrated that the expression of ground using a loose (10 times) and tight (30 times) dounce CLDN23 is significantly reduced in CRC tissue. A chro- pestle (Wheaton Industries, Inc., Millville, NJ, US) and matin immunoprecipitation followed by deep sequencing subsequently centrifuged at 700 x g for 5 min in 4˚C. The (ChIP-Seq) assay revealed that a high level of EZH2 is nuclei in the pellet were cross-linked in 1 ml PBS, containing bound to the CLDN23 locus in CRC tissue, suggesting that 1% formaldehyde (Sigma‑Aldrich) for 10 min at room temper- EZH2-mediated histone methylation was responsible for the ature, following which the reaction was quenched at room downregulation of the expression of CLDN23. The present temperature for 5 min by adding glycine (Sigma-Aldrich) at study suggested a functional link between the binding and a final concentration of 125 mM (from a stock solution of activity of EZH2 at the CLDN23 locus and the repression of 2 M). Following centrifugation at 700 x g for 5 min at room CLDN23 expression during the development of CRC. temperature, the nuclei were washed once with 1 ml of PBS and subsequently stored at -80˚C. Materials and methods Cross-linking of the cell lines to isolate chromatin was performed, as described previously (14). Pelleted nuclei were Tissue samples. Chromatin was isolated from 12 adenocarci- resuspended in lysis buffer, containing 12.5 mM Tris-HCl noma (AC) samples and their healthy colonic mucosa (NC) (pH 8.0), 2.5 mM EDTA and 0.25% sodum dodecyl sulphate counterparts, which were collected in our previous study (12). (SDS) (Sigma-Aldrich), supplemented with protease and phos- Cryostat sections were prepared from each sample using a phatase inhibitors (no. 78441; Thermo Fisher Scientific). Microm HM 505E cryostat (Zeiss, Gӧttingen, Germany). Chromatin was sheared in a Bioruptor Plus (Diagenode, Upper and lower sections from each cryosection were evalu- Denville, NJ, USA) using a 30 sec on-off cycle for 15 min ated by a pathologist to confirm whether they contained the at high intensity. ChIP assays were performed using the expected cell type. Furthermore, individual RNA and DNA Matrix‑ChIP method (15). Alternatively, a modified Fast‑ChIP samples from 24 pairs of healthy colon and AC specimens, method was used for the ChIP-Seq libraries (16). ChIP DNA which were extracted in our previous study (13) were used for data are expressed as the percentage of input DNA, as described reverse transcription quantitative polymerase chain reaction previously (14). For sequencing, ChIP was performed using (RT-qPCR) and DNA methylation analyses. a rabbit polyclonal anti-EZH2 antibody (ab3748; Abcam, Cambridge, UK) and non‑specific rabbit immunoglobulin G Cell lines. All cell lines were obtained from American (IgG; I-1000; Vector Laboratories, Burlingame, CA, USA). Type Culture Collection (Rockville, MD, USA). A total of The following antibodies were also