Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

Integrative ChIP-seq/Microarray Analysis Identifies a CTNNB1 Target Signature Enriched in Intestinal Stem Cells and Colon Cancer Kazuhide Watanabe 1 , Jacob Biesinger 2,3 , Michael L. Salmans 1 , Brian S. Roberts 4 , William T. Arthur 4 , Michele Cleary 4 , Bogi Andersen 1 , Xiaohui Xie 2,3 , Xing Dai 1,2 * 1 Department of Biological Chemistry, School of Medicine, University of California Irvine, Irvine, California, United States of America, 2 Institute for Genomics and Bioinformatics, University of California Irvine, Irvine, California, United States of America, 3 Department of Computer Science, University of California Irvine, Irvine, California, United States of America, 4 Rosetta Inpharmatics, LLC, Merck & Co Inc., Seattle, Washington, United States of America Abstract Background: Deregulation of canonical Wnt/CTNNB1 (beta-catenin) pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. Results: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5 + intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. Conclusion: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells. Citation: Watanabe K, Biesinger J, Salmans ML, Roberts BS, Arthur WT, et al. (2014) Integrative ChIP-seq/Microarray Analysis Identifies a CTNNB1 Target Signature Enriched in Intestinal Stem Cells and Colon Cancer. PLoS ONE 9(3): e92317. doi:10.1371/journal.pone.0092317 Editor: Ted S. Acott, Casey Eye Institute, United States of America Received October 30, 2013; Accepted February 20, 2014; Published March 20, 2014 Copyright: s 2014 Watanabe et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding from Susan G. Komen grant KG110897 (XD), a U.S. Department of Defense BCRP Postdoctoral Fellowship (K.W. W81XWH-10-1-0383), and National Institutes of Health grant R01HG006870 (to XX). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Brian S. Roberts and William T. Arthur are former employees of Rosetta Inpharmatics, LLC, Merck & Co Inc. Michele Cleary is currently an employee of Merck & Co., Inc. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: xdai@uci.edu leads to abnormal proliferation of mutated cells through activation of target genes such as MYC and CCND1 [7]. However, given the divergent cellular roles of Wnt/CTNNB1 signaling, other target genes may also contribute to the pathogenesis of CRCs. Thus, identification and characterization of CTNNB1 target genes genome-wide have been an important pursuit in CRC studies. Transcriptional profiling was used to identify Wnt/ CTNNB1 target genes in colon cancer cells [8]. However, analysis of gene expression alone is limited due to inability to distinguish between primary and secondary effects of Wnt pathway activation. More recently, chromatin immunoprecipitation (ChIP) followed by large-scale DNA analysis such as DNA-chip (ChIP-chip) or high-throughput sequencing (ChIP-seq) [9,10] was used to identify the genomic loci to which CTNNB1 or TCF factors directly bind [11–14]. However, ChIP-based studies of various transcription factors suggest that not all binding events identified correlate with Background Wnt/CTNNB1 signaling is a conserved pathway that plays fundamental roles in embryonic development, tissue homeostasis and maintenance of stem cells. In normal intestine, this pathway is essential for the development and maintenance of intestinal stem cells [1,2]. Activation of canonical Wnt signaling involves stabilization of cytoplasmic CTNNB1, which is otherwise degrad- ed by the proteasome through a degradation complex composed of tumor suppressor protein APC, serine/threonine kinase GSK-3, and Axin. Stabilized CTNNB1 translocates into the nucleus where it binds to TCF/LEF transcription factors and activates target gene expression [2]. A key initiation event of colorectal cancers (CRCs) is CTNNB1 stabilization through loss of the APC gene or activating mutations in the CTNNB1 gene [3]. This genetic event occurs primarily in the intestinal stem cell population [4–6] and PLOS ONE | www.plosone.org March 2014 | Volume 9 | Issue 3 | e92317