RRBS analysis of Braf-activated MEFs

We investigated DNA methylation status by RRBS method after oncogenic Braf activation using MEFs isolated from Braf knockin mice. Window-based analysis made it clear that the regions <500 bp from TSS were hypermethylated after Braf activation, whereas those >5000 bp from TSS were less methylated after Braf activation compared with wild-type MEFs. A hallmark of human cancer genomes is global DNA hypomethylation accompanied by focal DNA hypermethylation. Although factors and pathways that mediate DNA hypermethylation in cancer cells have been described, the basis of DNA hypomethylation remains to be determined. Here we show that expression of an activated BRAF oncoprotein, BRAFV600E, is sufficient to promote global DNA hypomethylation in mouse embryonic fibroblasts (MEFs). Conversion of 5-methylcytosine (5-mC) to its oxidation product 5-hydroxymethylcytosine (5-hmC) is thought to be the first step in a pathway leading to complete DNA demethylation. We find that BRAFV600E-directed global DNA hypomethylation occurs through conversion of 5-mC to 5-hmC by TET3, a member of the ten-eleven translocation (TET) family of proteins. In normal cells, TET3 is maintained at low levels resulting from ubiquitination by a SKP1–CUL1–F-box (SCF)-type ubiquitin ligase containing the F-box protein FBXW7, a known tumor suppressor, and subsequent proteasome-mediated degradation. BRAFV600E increases TET3 levels through inhibition of glycogen synthase kinase-3b (GSK3b), a protein kinase that phosphorylates TET3 and is required for SCFFBXW7-mediated ubiquitination. Consistent with these cell culture results, we find increased levels of TET3 and 5-hmC in experimentally induced mouse lung adenomas and patient-derived human colorectal adenomas that contain BRAFV600E. Moreover, we show that RNAi-mediated knockdown of Tet3 results in decreased BRAFV600E-induced tumorigenesis. Finally, to test the generality of our findings, we repeated several key experiments using a KRASG12D oncoprotein rather than BRAFV600E and obtained similar results. Collectively, our results elucidate a mechanism of global DNA hypomethylation promoted by oncogenic BRAF and KRAS, and establish an essential role for TET3 at an early stage of oncogenesis. Overall design: Rosa26-CreERT2; Braf+/+ female MEFs (Control) and Rosa26-CreERT2; Braf+/LSL female MEFs were treated with 4-OHT to induce Cre recombinase. MEFs were harvested 0 or 21 days after 4-OHT treatments. We repeated the experiments 3 times with different clones. Total samples are 12.