Substantial genomic and methylation variability between MCF-7 sublines

Abstract Cancer cell lines have long been used as in vitro models for molecular assays in diagnostic and therapeutic development due to their accessibility as a well-controlled system. MCF-7 cell lines are the most widely studied cell lines in human breast cancer research, and its sublines have been reported to exhibit clonal, cytogenetic, and transcriptomic variability. However, allele-specific methylation alterations in cancer genomes remain inadequately explored, largely due to limitations in sequencing methods. Here, we applied nanopore sequencing technology to characterise the genomic and epigenomic landscapes of two MCF-7 sublines. We identified global and local DNA methylation differences as well as structural variants (SVs), and single-nucleotide variants (SNVs) between and within the sublines. Our analysis revealed substantial divergence in methylation patterns between the sublines, with ∼3% of the differentially methylated regions (DMRs) overlapping with known cancer driver genes. These DMRs overlap breast cancer-associated genes, including ERBB2, CDH1, SALL4, GATA2, GATA3, HMGA2, and FBLN2. We find that the majority of differentially methylated sites are explained by differential allelic methylation, and that allele-specific DMRs often overlap points where antisense non-coding RNAs overlap protein-coding genes. Transposable elements in both sublines also showed distinct methylation profiles, with one subline having hypomethylated L1 elements compared to the other, which correlated with the amount of apparent insertional mutagenesis attributable to L1 between the sublines. Our study demonstrates the utility of nanopore sequencing in providing novel insights into genomic and methylomic differences within cell lines, in addition to insight into the nature of differential allelic methylation. Competing Interest Statement A.E. has received reimbursement for travel, accommodation, and conference fees to speak at events organised by Oxford Nanopore Technologies. The remaining authors declare no competing interests. List of Abbreviations - 5mC/5mCpG/5mCG - 5-methyl cytosine, 5-methyl cytosine-guanine - AS - Antisense - ASE - Antisense expression - ATCC - American Type Culture Collection - CDH1 - Cadherin-1 / Epithelial Cadherin - CDK6 - Cyclin-dependent kinase 6 - COL2A1 - Collagen type II alpha 1 chain - CpG - Cytosine followed by Guanine in a DNA context - CRE - cis-regulatory element - DMR - Differentially Methylated Region - EBF1 - Early B-cell factor 1 - ECACC - European Collection of Authenticated Cell Cultures - ERBB2 - Erb-B2 Receptor Tyrosine Kinase 2, aka HER2 - FBLN2 - Fibulin-2 - FGFR2 - Fibroblast growth factor receptor 2 - GATA2 - GATA-binding protein 2 - GATA3 - GATA-binding protein 3 - HC - High-confidence - HMGA2 - High mobility group AT-hook 2 - HOTAIR - HOX Antisense Intergenic RNA - HOXC11 - Homeobox C11 - L1 / LINE-1 - Long INterspersed Element-1 - LEF1 - Lymphoid enhancer-binding factor 1 - mRNA - messenger RNA - MYCN - N-myc / basic helix-loop-helix protein 37 - ncRNA - non-coding RNA - NKX2-1 - NK2 homeobox 1 - PTEN - Phosphatase and tensin homolog - SALL4 - Spalt-like transcription factor 4 - SNV - Single-nucleotide variant - SV - Structural variant - TE - Transposable element - TF - Transcription factor - TFBS - Transcription factor binding site - VCF - Variant call format - VNTR - Variable number of tandem repeats - WT1 - Wilms’ tumour-1 - ZEB1 - Zinc finger E-box binding homeobox 1