Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis

Abstract

The conversion of 5-methylcytosine to 5-hydroxymethylcytosine relaxes the repressive marks of gene expression, and their imbalance leads to cancer development. Global loss of 5-hmC is found to be associated with the downregulation of TET1 and TET3 genes in breast cancer. Genome-wide analysis revealed 4809 differentially methylated regions and 4841 differentially hydroxymethylated regions in breast cancer. The abundance of 5-mC was observed at gene promoter regions while the 5-hmC was profusely distributed at the distal regulatory regions of the breast cancer genome. The accumulation of 5-hmC at distal regulatory sites can potentially enhance gene transcription. Alteration in the 5-hmC levels was positively associated with the respective gene expression. Novel 5-hmC candidates such as TXNL1, CNIH3, BNIPL, and CHODL were found to be promising diagnostic and therapeutic markers for breast cancer. Highlights: An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>−5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.