RRBS analysis of Braf-activated MEFs. Mus musculus

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 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.

我们采用简化代表性亚硫酸氢盐测序（RRBS）方法，探究了使用源自BRAF敲入小鼠的小鼠胚胎成纤维细胞（MEFs）进行致癌性BRAF激活后的DNA甲基化状态。基于窗口的分析显示，相较于野生型MEFs，BRAF激活后转录起始位点（TSS）两侧5000 bp的区域甲基化水平更低。人类癌症基因组的典型特征是全基因组DNA低甲基化伴随局灶性DNA高甲基化。尽管目前已阐明癌细胞中介导DNA高甲基化的因子与通路，但DNA低甲基化的分子基础仍有待明确。本研究证实，激活型BRAF致癌蛋白BRAFV600E的表达足以诱导小鼠胚胎成纤维细胞发生全基因组DNA低甲基化。将5-甲基胞嘧啶（5-mC）转化为其氧化产物5-羟甲基胞嘧啶（5-hmC）被认为是完整DNA去甲基化通路的第一步。我们发现，BRAFV600E诱导的全基因组DNA低甲基化是通过TET家族蛋白成员TET3将5-mC转化为5-hmC实现的。在正常细胞中，TET3会被含有已知肿瘤抑制因子F框蛋白FBXW7的SKP1-CUL1-F-box（SCF）型泛素连接酶泛素化，随后经蛋白酶体介导降解，因此TET3维持在低水平。BRAFV600E通过抑制糖原合成激酶3β（GSK3β）提升TET3的水平——GSK3β可磷酸化TET3，是SCFFBXW7介导的泛素化过程所必需的激酶。与细胞培养实验结果一致，我们在实验诱导的BRAFV600E阳性小鼠肺腺瘤以及源自患者的BRAFV600E阳性结直肠腺瘤中，均检测到TET3与5-hmC水平升高。此外，我们发现通过RNA干扰介导的Tet3敲低可削弱BRAFV600E诱导的肿瘤发生。最后，为验证研究结果的普适性，我们使用KRASG12D致癌蛋白替代BRAFV600E重复了关键实验，得到了相似的结果。综上，本研究阐明了致癌性BRAF与KRAS驱动的全基因组DNA低甲基化机制，并证实TET3在肿瘤发生早期发挥关键作用。整体实验设计：将Rosa26-CreERT2; Braf+/+雌性MEFs（对照组）与Rosa26-CreERT2; Braf+/LSL雌性MEFs用4-羟基他莫昔芬（4-OHT）处理以诱导Cre重组酶表达。分别在4-OHT处理后0天与21天收集MEFs。我们使用不同克隆重复实验3次，总样本量为12份。