Abstract
DNA methylation is important for establishing and maintaining cell identity and for genomic stability. This is achieved by regulating the accessibility of regulatory and transcriptional elements and the compaction of subtelomeric, centromeric, and other inactive genomic regions. Carcinogenesis is accompanied by a global loss in DNA methylation, which facilitates the transformation of cells. Cancer hypomethylation may also cause genomic instability, for example through interference with the protective function of telomeres and centromeres. However, understanding the role(s) of hypomethylation in tumor evolution is incomplete because the precise mutational consequences of global hypomethylation have thus far not been systematically assessed. Here we made genome-wide inventories of all possible genetic variation that accumulates in single cells upon the long-term global hypomethylation by CRISPR interference-mediated conditional knockdown of DNMT1. Depletion of DNMT1 resulted in a genomewide reduction in DNA methylation. The degree of DNA methylation loss was similar to that observed in many cancer types. Hypomethylated cells showed reduced proliferation rates, increased transcription of genes, reactivation of the inactive X-chromosome and abnormal nuclear morphologies. Prolonged hypomethylation was accompanied by increased chromosomal instability. However, there was no increase in mutational burden, enrichment for certain mutational signatures or accumulation of structural variation to the genome. In conclusion, the primary consequence of hypomethylation is genomic instability, which in cancer leads to increased tumor heterogeneity and thereby fuels cancer evolution.