Published OnlineFirst May 1, 2014; DOI: 10.1158/0008-5472.CAN-13-3147 Cancer Tumor and Stem Cell Biology Research Loss of the Polycomb Mark from Bivalent Promoters Leads to Activation of Cancer-Promoting Genes in Colorectal Tumors Maria A. Hahn1, Arthur X. Li2, Xiwei Wu3, Richard Yang1, David A. Drew4, Daniel W. Rosenberg4, and Gerd P. Pfeifer1 Abstract In colon tumors, the transcription of many genes becomes deregulated by poorly defined epigenetic mechanisms that have been studied mainly in established cell lines. In this study, we used frozen human colon tissues to analyze patterns of histone modification and DNA cytosine methylation in cancer and matched normal mucosa specimens. DNA methylation is strongly targeted to bivalent H3K4me3- and H3K27me3-associated promoters, which lose both histone marks and acquire DNA methylation. However, we found that loss of the Polycomb mark H3K27me3 from bivalent promoters was accompanied often by activation of genes associated with cancer progression, including numerous stem cell regulators, oncogenes, and proliferation-associated genes. Indeed, we found many of these same genes were also activated in patients with ulcerative colitis where chronic inflammation predisposes them to colon cancer. Based on our findings, we propose that a loss of Polycomb repression at bivalent genes combined with an ensuing selection for tumor-driving events plays a major role in cancer progression. Cancer Res; 74(13); 3617–29. Ó2014 AACR. Introduction cells (21, 22). The acquisition of the more permanent silencing Tumorigenesis is a complex process that is driven by a mark, 5mC, at the promoters of Polycomb target genes does number of genetic and epigenetic alterations, which often not fundamentally change their expression levels although result in aberrant gene expression (1–3). Mutations are gen- plasticity of expression will be reduced. For these reasons, it erally considered to be the primary drivers of tumorigenesis (4, has remained unclear whether methylation of Polycomb target 5). However, dysregulation of epigenetic regulatory mechan- genes plays an essential role in tumor promotion and is in fact a isms also contributes to malignant transformation. Methyla- tumor-driving event (23). fi tion of cytosine at CpG sequences in DNA leading to the Recently, genome sequencing has identi ed a high frequen- formation of 5-methylcytosine (5mC) is one of the most stable cy of mutations in epigenetic regulatory factors or chromatin and most widely studied epigenetic modifications (6). In most structural elements in human malignancies (24, 25). However, fi human tumors, widespread hypermethylation of CpG-rich technical limitations have made it dif cult to directly examine sequences (CpG islands) is observed along with genome-wide chromatin changes in normal and malignant tissues, with most in vitro DNA hypomethylation (1, 7–14). DNA hypermethylation in studies being limited to cell culture models. To deter- fi cancer is not a random event; it commonly affects specific mine the role of histone modi cations in targeting DNA gene classes and is seen most frequently at targets of the methylation and in altering gene expression patterns in human fi Polycomb repression complex (15–18) including numerous primary tumors, we have conducted the rst comprehensive homeobox genes (19, 20). However, many of the genes marked analysis of frozen tissues from patients with colorectal cancer by Polycomb in normal tissues and cell types, for example focusing on two histone methylation marks, the activating embryonic stem cells, are expressed at very low levels in these histone H3 lysine 4 trimethylation (H3K4me3) and the repres- sive histone H3 lysine 27 trimethylation (H3K27me3), which are thought to be critical for gene regulation. We found that Authors' Affiliations: Departments of 1Cancer Biology, 2Information genes carrying both histone modifications (bivalent genes) in 3 Sciences, and Molecular Medicine, Beckman Research Institute, City of normal tissue are characterized by substantial variability and Hope, Duarte, California; and 4Center for Molecular Medicine, University of Connecticut Health Center, Farmington, Connecticut undergo reorganization of these modifications in colorectal cancer, leading to activation of cancer-promoting genes as one Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). important outcome that can confer tumor-driving properties onto an emerging malignant cell population. M.A. Hahn and A.X. Li contributed equally to this work. Corresponding Author: Gerd P. Pfeifer. Department of Cancer Biology, Materials and Methods Beckman Research Institute, 1450 East Duarte Road, City of Hope, Duarte, CA 91010. Phone: 626-301-8853; Fax: 626-358-7703; E-mail: Human tissue samples [email protected] Human Duke's stage II colon cancers and matching normal doi: 10.1158/0008-5472.CAN-13-3147 mucosa were obtained from the Cooperative Human Tissue Ó2014 American Association for Cancer Research. Network (CHTN). www.aacrjournals.org 3617 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2014 American Association for Cancer Research. Published OnlineFirst May 1, 2014; DOI: 10.1158/0008-5472.CAN-13-3147 Hahn et al. Gene expression analysis 300 mmol/L NaCl at 65C overnight. Primer sequences for Total RNA from patient samples was purified by using the qPCR of individual genes are available upon request. Analysis mirVana Kit (Ambion; Life Technologies). For whole genome was done with tissues from patient no. 14. Results are for expression analysis, GeneChip Human Gene 2.0 ST arrays triplicate experiments (ÆSD). (Affymetrix) were used. Validation of gene expression changes by real-time reverse transcription-PCR was performed as Bioinformatics analysis described previously (18). All expression data were normalized All analysis was performed using R statistical language, to GAPDH in the same sample. except for gene ontology analysis, which was performed using DAVID annotation tools and Ingenuity Pathway Analysis. The Analysis of 5mC patterns heat maps were generated with Cluster v3.0 and Java Treeview For analysis of genomic 5mC patterns, the methylated CpG v2.0. Refseq genes were downloaded from the UCSC hg18 fi À þ island recovery assay (MIRA) was used as described previously annotation database. Promoter was de ned as 1.4 to 0.6 (26). After genome amplification, the methylated DNA fraction kb relative to the transcription start site (TSS). For heat maps was hybridized versus input DNA on human CpG island/ with epigenetic marks, only genes with gene body length promoter microarrays (NimbleGen). The observed MIRA pat- greater than 2 kb were included. Genes that were differentially fi terns for single CpG islands were validated by combined expressed during colorectal cancer formation were identi ed > bisulfite restriction analysis (COBRA) as described previously as those with a log2-fold change 1. Probes were considered (18). Primer sequences are available upon request. positive if their normalized log2 ratios were above 1.5-fold. Peaks in each sample were defined as four or more consecutive Chromatin immunoprecipitation positive probes with either one or no gaps. A promoter was fi For chromatin immunoprecipitation (ChIP), frozen tissues de ned as bivalent if it contained overlapping H3K4me3 and À < < were crushed with a plastic pestle in ice-cold phosphate- H3K27me3 peaks at expanded promoter areas ( 2.4 kb TSS buffered saline (PBS) and fixed for 10 minutes at room 0.6 kb). For heat-map analysis, genes were assumed activated temperature in 1% formaldehyde. ChIP and genome ampli- in all tumors and bivalent in mucosa if those genes had a fication protocols were performed as described previously promoter bivalent status in at least 3 of 4 normal colon samples (26). The following antibodies were used: anti-H3K4me3 and tumor-associated gene activation of log2 0.4 in four (39159; Active Motif), and anti-H3K27me3 (07-449; Millipore). patient sample pairs. For obtaining the H3K4me3 profile, H3K4me3 antibodies Affymetrix microarray data were processed by Expression fi were preblocked with an H3K9me3 peptide (Abcam) to Console v 2.0 with default settings. Transcription pro les of remove minor cross-reactivity of this antibody. After genome unique genes were obtained by averaging the transcriptional amplification, immunoprecipitated DNA was hybridized ver- levels of all different transcript isoforms of the same gene sus input DNA on human CpG island/promoter microarrays according to Affymetrix's annotation. Genes with log2 intensity < (NimbleGen). 5 in more than 10% of biopsies were considered uninforma- For sequential ChIP (ReChIP), the first immunoprecipita- tive and were removed. Affymetrix expression array data were tion was performed as described above except for the elution used to generate heat maps as described previously (27). All step, which was performed with SDS lysis buffer (1% SDS, 10 array data were deposited in the GEO database (accession mmol/L EDTA, 50 mmol/L Tris-HCl, pH 8.1, 1Â cOmplete number GSE47076). Protease Inhibitor Cocktail; Roche Applied Science) for 10 minutes at 68C on a shaker at 1,000 rpm. After removal of Results beads, the samples were diluted with 1:10 ChIP dilution buffer Chromatin mapping studies with frozen tissue (0.01% SDS, 1.1% Triton X-100, 1.2 mmol/L EDTA, 16.7 mmol/L Previous correlative studies, mostly performed with cul- Tris-HCl, pH 8.1, 167 mmol/L NaCl, 22 mg/mL BSA, 1Â cOm- tured cells (e.g., embryonic stem cells), indicated