TET2-mediated deprogramming of breast cancer cells. TET2-mediated deprogramming of breast cancer cells

Methylation and demethylation of cytosines in DNA are believed to act as keystones of cell-specific gene expression through controlling chromatin structure and accessibility to transcription factors. Cancer cells have their own transcriptional programs and we sought to alter such a cancer-specific program by enforcing expression of the catalytic domain (CD) of the methylcytosine dioxygenase TET2 in breast cancer cells. TET2 CD decreased the tumorigenic potential of cancer cells through both activation and repression of a repertoire of genes. In addition to promoting the establishment of an antiviral state, TET2 activated 5mC turnover at thousands of MYC binding motifs and down-regulated a panel of known MYC-repressed genes involved in lysosome biogenesis and function. Overall design: MCF-7 cells stably transfected with an empty vector or with plasmids encoding either wild type mouse TET2 catalytic domain (CD - aa916-1921, pcDNA3-Flag-TET2 CD, addgene #72219) or the catalytically dead mutant H1304Y, D1306A mutant (pcDNA3-Flag-TET2 mCD, addgene #72220) were analyzed for gene expression by RNA-seq and for chromatin marks by ChIP-seq (H3K4me3 and H3K27me3) and selective chemical labeling (5hmC).

DNA胞嘧啶的甲基化与去甲基化过程，被认为是通过调控染色质结构与转录因子结合可及性，从而介导细胞特异性基因表达的核心枢纽。癌细胞拥有独特的转录调控程序，本研究通过在乳腺癌细胞中强制表达甲基胞嘧啶双加氧酶TET2的催化结构域（catalytic domain, CD），以期改写这一癌症特异性转录程序。TET2催化结构域可通过激活与抑制一系列基因，降低癌细胞的致瘤潜能。除了诱导细胞建立抗病毒状态外，TET2还可在数千个MYC结合基序处激活5-甲基胞嘧啶（5mC）的周转过程，并下调一组已知的受MYC抑制的、参与溶酶体生物发生与功能调控的基因。整体实验设计：将MCF-7细胞分别稳定转染空载体、编码野生型小鼠TET2催化结构域的质粒（CD，氨基酸位点916-1921，pcDNA3-Flag-TET2 CD，Addgene #72219），或催化失活突变体H1304Y、D1306A的质粒（pcDNA3-Flag-TET2 mCD，Addgene #72220），随后通过RNA测序（RNA-seq）检测基因表达水平，通过染色质免疫沉淀测序（ChIP-seq，检测H3K4me3与H3K27me3两种组蛋白修饰）以及选择性化学标记法检测5-羟甲基胞嘧啶（5hmC），以分析染色质修饰状态。