Deficiency of the Polycomb protein RYBP and TET methylcytosine oxidases promotes extensive CpG island hypermethylation and malignant transformation.

Hypermethylation of CpG islands is a common feature of cancer cells and predominantly affects Polycomb-associated genomic regions. Elucidating the underlying mechanisms leading to DNA hypermethylation in human cancer could help identify chemoprevention strategies. Here, we evaluated the role of Polycomb complexes and 5-methylcytosine oxidases in protecting CpG islands from DNA methylation and observed that four genes coding for components of Polycomb repressive complex 1 (PRC1) are downregulated in tumors. Inactivation of RYBP, a key activator of variant PRC1 complexes, in combination with all three 5-methylcytosine oxidases (TET proteins) in nontumorigenic bronchial epithelial cells led to widespread hypermethylation of Polycomb-marked CpG islands affecting almost 4,000 target genes, which closely resembled the DNA hypermethylation landscape observed in human squamous cell lung tumors. The RYBP- and TET-deficient cells showed methylation-associated aberrant regulation of cancer-relevant pathways, including defects in the Hippo tumor suppressor network. Notably, the quadruple knockout cells acquired a transformed phenotype, including anchorage-independent growth and formation of squamous cell carcinomas in mice. This work provides a mechanism promoting hypermethylation of CpG islands and shows that such hypermethylation can lead to cell transformation. The breakdown of a two-pronged protection mechanism can be a route towards genome-wide hypermethylation of CpG islands in tumors.