Alternative methylation of intron motifs is associated with cancer-related gene expression in both canine mammary tumor and human breast cancer

Abstract

Background: Canine mammary tumor (CMT) has long been considered as a good animal model for human breast cancer (HBC) due to their pathological and biological similarities. However, only a few aspects of the epigenome have been explored in both HBC and CMT. Moreover, DNA methylation studies have mainly been limited to the promoter regions of genes. Results: Genome-wide methylation analysis was performed in CMT and adjacent normal tissues and focused on the intron regions as potential targets for epigenetic regulation. As expected, many tumor suppressors and oncogenes were identified. Of note, most cancer-associated biological processes were enriched in differentially methylated genes (DMGs) that included intron DMRs (differentially methylated regions). Interestingly, two PAX motifs, PAX5 (tumor suppressive) and PAX6 (oncogenic), were frequently found in hyper-and hypomethylated intron DMRs, respectively. Hypermethylation at the PAX5 motifs in the intron regions of CDH5 and LRIG1 genes were found to be anti-correlated with gene expression, while CDH2 and ADAM19 genes harboring hypomethylated PAX6 motifs in their intron region were upregulated. These results were validated from the specimens originally MBD-sequenced as well as additional clinical samples. We also comparatively investigated the intron methylation and downstream gene expression of these genes using human breast invasive carcinoma (BRCA) datasets in TCGA (The Cancer Genome Atlas) public database. Regional alteration of methylation was conserved in the corresponding intron regions and, consequently, gene expression was also altered in HBC. Conclusions: This study provides good evidence for the conservation of epigenetic regulation in CMT and HBC, and suggests that intronic methylation can be an important factor in better understanding gene regulation in both CMT and HBC.

ANK2 Hypermethylation in Canine Mammary Tumors and Human Breast Cancer

“…Other regions on ANK2 that contain conserved CpG islands could potentially be more successfully analyzed via the qMSP method and could potentially prove more influential in the downregulation of expression seen in Figure 5 B. Pyrosequencing could be another alternative to pursue, as opposed to cloning and Sanger sequencing. We did not observe any methylation difference on the promoter region in our previous MBD-seq data [ 26 ]. This orthologous human region should be further studied in the future in tissue and cell culture to further investigate the nature and role of these hypermethylated CpGs play a significant role in HBC.…”

“…Target hypermethylated DMRs were identified using MBD-seq and corresponding RNA-seq according to the scheme in Figure 1 A. From our previous MBD-seq data obtained for 11 pairs of CMT and adjacent normal tissue from NCBI BioProject database (accession number PRJNA601533) [ 26 ], 16,061 differentially methylated genes (DMGs) were identified based on the presence of a differentially methylated region (DMR) within the gene body. To develop DNA methylation-based biomarkers applicable to CMT and HBC, we decided to narrow the scope to regions that contained a hypermethylated CpG island in CMT.…”

“…Tissue samples used in this study consisted of surgically removed CMT tissue and paired adjacent normal tissue obtained from a variety of dog breeds. Tumors identified as carcinomas were selected, yet the selection was not subtype specific (simple, complex, and ductal carcinomas) seeing as the MBD-seq data (accession number PRJNA601533) [ 26 ] did not show any subtype specific differences in methylation patterns for our targets. Canine blood samples were taken from dogs diagnosed with mammary carcinoma and normal samples were obtained from healthy dogs without cancer.…”

“…We performed an integrative analysis of MBD-seq (accession number PRJNA601533) [ 26 ] and RNA-seq data (SRA accession number: SRR8741587-SRR8741602) [ 25 ] from 8 overlapped tissue samples to identify canine mammary gland DNA methylation markers. We first selected DMRs which are located in a CpG island on the gene body and examined the correlation between DNA methylation and gene expression for each sample.…”

Key Macrophage Responses to Infection With Mycobacterium tuberculosis Are Co-Regulated by microRNAs and DNA Methylation

“…A growing body of evidence suggests that methylation changes in non-promoter regions are relevant to epigenetic regulation of gene expression. Previously, methylation of introns was thought to serve little function, but has recently been shown to exhibit an inverse relationship with gene expression and may be implicated in progression of cancer (36,37). Functional enrichment GO : BP analysis revealed that hypermethylated CG DMRs were most associated with pathways involved in immune cell activation, while hypomethylated DMRs were most associated with alteration of positive regulation of metabolic processes FIGURE 4 | Pathway analysis for significantly dysregulated targets of candidate miRNAs.…”