Epigenetic enigma: reinstating normalcy at a naïve chromosomal locus by suppressing an oncogenic lncRNA transgene [WGS]

Maintaining genome integrity is crucial for the proper functioning and development of organisms. One intriguing aspect of genome integrity is the formation and function of neocentromeres at non-centromeric sites. The centromere-specific protein CENP-A plays a vital role in centromere identification and function. However, in many cancers, CENP-A is often found to be ectopically misplaced when overexpressed. Moreover, the deposition of CENP-A at the centromere depends on the transcription of centromeric non-coding RNAs. Consequently, ectopic CENP-A is found at transcriptionally active and frequent breakpoint regions. To further explore the ectopic CENP-A localization, we engineered a stable ectopic CENP-A site at a naïve chromosome by overexpressing a non-centromeric oncogenic lncRNA capable of recruiting CENP-A to its transcription site. We tracked cells carrying the stable transgene to understand the stability and longevity of the induced ectopic CENP-A site and the harboring chromosome. Our findings revealed that the induced epigenetic memory was eventually lost by suppression of the transgene through competing epigenetic silencing mechanisms. This epigenetic restoration naturally reversed the ectopic CENP-A level to its previous levels at the engineered site. Our observations suggest that cells may have evolved failsafe mechanisms to prevent neocentromere formation at ectopic sites unless otherwise favored by selection involving multiple components. The metastatic colorectal cancer cell line SW480 wild type and stable SW480 cells expressing the lncRNA PCAT2 transgene cassette were employed in this work. ChIP, immunoblotting, quantitative real-time PCR, immunofluorescence, and DNA-FISH analysis were used on these two cell lines to investigate the levels of various epigenetic marks, lncRNAs,and gene expression, and the localization of CENP-A nucleosomes in non-centromeric regions. The DNA methylation profile ofthe same two cell lines wasalso studied using next-generation sequencing.