Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes

February Hierarchical Clustering of Breast Cancer Methylomes Revealed Differentially Methylated and Expressed Breast Cancer Genes I-Hsuan Lin 0 1 Dow-Tien Chen 0 1 Yi-Feng Chang 0 1 Yu-Ling Lee 0 1 Chia-Hsin Su 0 1 Ching Cheng 0 1 Yi-Chien Tsai 0 1 Swee-Chuan Ng 0 1 Hsiao-Tan Chen 0 1 Mei-Chen Lee 0 1 Hong-Wei Chen 0 1 Shih-Hui Suen 0 1 Yu-Cheng Chen 0 1 Tze-Tze Liu 0 1 Chuan-Hsiung Chang 0 1 Ming-Ta Hsu 0 1 0 1 VGH-YM Genome Center, National Yang-Ming University , Taipei, Taiwan , 2 Institute of Biochemistry and Molecular Biology, National Yang-Ming University , Taipei, Taiwan , 3 Center for Systems and Synthetic Biology, National Yang-Ming University , Taipei, Taiwan , 4 Institute of Biomedical Informatics, National Yang- Ming University , Taipei , Taiwan 1 Academic Editor: Osman El-Maarri, University of Bonn, Institut of experimental hematology and transfusion medicine , GERMANY Oncogenic transformation of normal cells often involves epigenetic alterations, including histone modification and DNA methylation. We conducted whole-genome bisulfite sequencing to determine the DNA methylomes of normal breast, fibroadenoma, invasive ductal carcinomas and MCF7. The emergence, disappearance, expansion and contraction of kilobase-sized hypomethylated regions (HMRs) and the hypomethylation of the megabasesized partially methylated domains (PMDs) are the major forms of methylation changes observed in breast tumor samples. Hierarchical clustering of HMR revealed tumor-specific hypermethylated clusters and differential methylated enhancers specific to normal or breast cancer cell lines. Joint analysis of gene expression and DNA methylation data of normal breast and breast cancer cells identified differentially methylated and expressed genes associated with breast and/or ovarian cancers in cancer-specific HMR clusters. Furthermore, aberrant patterns of X-chromosome inactivation (XCI) was found in breast cancer cell lines as well as breast tumor samples in the TCGA BRCA (breast invasive carcinoma) dataset. They were characterized with differentially hypermethylated XIST promoter, reduced expression of XIST, and over-expression of hypomethylated X-linked genes. High expressions of these genes were significantly associated with lower survival rates in breast cancer patients. Comprehensive analysis of the normal and breast tumor methylomes suggests selective targeting of DNA methylation changes during breast cancer progression. The weak causal relationship between DNA methylation and gene expression observed in this study is evident of more complex role of DNA methylation in the regulation of gene expression in human epigenetics that deserves further investigation. - Data Availability Statement: All data from this study have been submitted to the ArrayExpress database (https://www.ebi.ac.uk/arrayexpress/). WBGS data for normal breast (NB), BT089, BT126, BT198 and MCF7 are under accession number E-MTAB-2014. RNA-seq data for NB and MCF7 are under accession number E-MTAB-1961. ChIP-seq data for MCF7 PolII and input controls are under accession number E-MTAB-1952. DNA-seq data for MCF7 HpaII and MNase hypersensitive assays are under accession number E-MTAB-1958. ChIP-chip data for H3K4Me1, Competing Interests: The authors have declared that no competing interests exist. Breast cancer is the most common cancer in women in the world. Since 2008, breast cancer incidence has increased by more than 20% and mortality increased by 14%. Apart from the genetic and hormonal risk factors that predisposing women to breast cancer, other factors such as life-styles, environmental and nutritional also seemed to play a part in this complex, multifactorial disease. Like many cancers, epigenetic dysregulation has been implicated to play a role in breast cancer development [13]. DNA methylation is one of the major epigenetic regulatory mechanisms in higher organisms. It plays significant roles in many biological processes, including genomic imprinting, embryonic development, X-chromosome inactivation (XCI), genome stability, suppression of repetitive sequences and tumorigenesis [48] DNA methylation involves the addition of a methyl group to the carbon-5 position of cytosine residues at CpG dinucleotides by the DNA methyltransferase enzymes. Of the 28 million CpG sites in the human genome, 70 to 80% are methylated in most cell types [9]. The CpG sites are unevenly distributed in the genome whereby clusters of CpG sites, termed CpG islands (CGIs), are often found at the promoter regions. The regulation of gene expression through differential methylation of the CpG sites within promoter CGIs has been extensively studied [1012]. Promoter CGIs are often found unmethylated and this state is associated with gene activation whereas gene silencing is often associated with promoter CGIs methylation. DNA methylation is a relatively stable epigenetic trait, hence aberrant promoter (de-)methylation often leads to adverse alteration in gene expression, and such event is one of the major hallmarks of tumor progression [1317]. DNA methylation changes may occur at regions immediately adjacent to CGIs (CGI shores), and at CpG sites far away from CGIs and/or promoters in cancer cells [1822]. The term hypomethylated region (HMR) was used to describe small genomic loci (usually less than 50 Kb) that were lowly methylated or unmethylated. HMRs found at non-promoter regions may mark cryptic promoters and enhancers associated with tissue-specificity [2325]. There are currently four genome-wide methylation profiling technologies to study DNA methylation in a high-throughput manner, namely whole-genome bisulfite sequencing (WGBS), enrichment-based sequencing, reduced representation bisulfite sequencing and the Infinium HumanMethylation BeadChip which is a low-cost alternative to the sequencingbased methods [26]. Past array-based DNA methylation studies have concluded that the unusual hypermethylation of a number of CpG loci were receptor specific in breast tumors [2730]. Aberrant hypermethylation of certain genes have been significantly associated with worse outcome, and some were associated with increased risk of developing metastases [28, 29, 31]. While at a higher cost, the WGBS provides the whole-genome coverage at a singlenucleotide resolution and is considered the gold-standard approach for quantitative measurement of methylation level. Since 2009, several human WGBS studies have been conducted to explore the DNA methylation landscapes in various tissue types and cell lines, at different age, as well as between normal and diseased states [3241]. With regards to the breast cancer research, Hon et al decoded the methylomes of HMEC and HCC1954, cell lines derived from breast epithelium and breast carcinoma respectively [38]. In this work, the authors confirmed the presence of extensive DNA hypomethylation at the partially methylated domains (PMDs), a term used to describe large genomic blocks with abnormal hypomethylat (...truncated)