Abstract A029: Transposable elements exhibit loss of DNA methylation and increases in transcription in a model of tumorigenesis
Authors: Kanholm Tomas , Rentia Uzma , Beaty Melissa Hadley , McDonald James I. , Xie Wenbing , Baylin Stephen B. , Burns Kathleen H. , Easwaran Hari , Chiappinelli Katherine B. 
Published: December 2022

Abstract
Transposable elements (TEs), including LINEs, SINEs, and LTRs, are typically silenced by epigenetic mechanisms in differentiated healthy tissues. However, TE expression increases in a wide range of cancer types, likely due to global hypomethylation of cancer genomes. We explored expression and DNA methylation of TEs in the Weinberg transformation model, which models tumorigenesis from fibroblasts. Although this model has been well characterized, analysis of TE expression and the associated epigenetic changes during transformation has never been performed. How TEs are turned on during tumorigenesis and the functional consequences of such activation remain unknown. BJ fibroblast cells were serially transduced with hTERT, SV40, and HRASR24C to immortalize and then transform the cells. RNA-sequencing and whole-genome bisulfite sequencing (WGBS) were performed at each stage of transformation. We used TEtranscripts and Telescope to analyze RNA-seq data and determine differential expression of all TEs at the subfamily level, as well as a subset of LINE and LTR elements in a locus-specific manner. RNA-sequencing revealed an increase in TE expression as cells progressed through transformation, with the largest increase in expression after the final stage of transformation, consistent with data from human tumors. Expression of individual LINEs and LTRs was more evenly distributed between up and down regulation, indicating that overall, TE upregulation may be driven by a subset of individual TE loci. In general, upregulated TEs were dominated by LTRs or endogenous retroviruses, and transcriptional upregulation of LTRs was correlated with increases in ERV3 and SYN-1 Envelope proteins. We performed WGBS on each stage of the model and determined differentially methylated regions (DMRs) between stages using DSS. The majority of DMRs in all stages were hypomethylated, with further hypomethylation in the final stage of transformation. A majority of DMRs overlapped TEs from the RepeatMasker database, with the most overlaps in LINE, LTR, and SINE elements, indicating that TEs are preferentially demethylated. Many hypomethylated TEs displayed a concordant expression increase. Interestingly, we found that demethylation began during immortalization and continued into transformation, while upregulation of TE transcription occurred in transformation. We thus conclude that transposable elements, specifically LTRs/endogenous retroviruses, are demethylated and transcribed during transformation. Current experiments manipulating specific candidate TEs in this model during transformation will determine the effect of expression of these TEs on tumorigenesis.
Citation Format: Tomas Kanholm, Uzma Rentia, Melissa Hadley Beaty, James I. McDonald, Wenbing Xie, Stephen B. Baylin, Kathleen H. Burns, Hari Easwaran, Katherine B. Chiappinelli. Transposable elements exhibit loss of DNA methylation and increases in transcription in a model of tumorigenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A029.