Hu Caroline Y, Mohtat Davoud, Yu Yiting, Ko Yi-An, Shenoy Niraj, Bhattacharya Sanchari, Izquierdo Maria C, Park Ae Seo Deok, Giricz Orsolya, Vallumsetla Nishanth, Gundabolu Krishna, Ware Kristin, Bhagat Tushar D, Suzuki Masako, Pullman James, Liu X Shirley, Greally John M, Susztak Katalin, Verma Amit
Albert Einstein College of Medicine, Bronx, New York.
Renal Electrolyte and Hypertension Division, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania.
Clin Cancer Res. 2014 Aug 15;20(16):4349-60. doi: 10.1158/1078-0432.CCR-14-0494. Epub 2014 Jun 10.
Even though recent studies have shown that genetic changes at enhancers can influence carcinogenesis, most methylomic studies have focused on changes at promoters. We used renal cell carcinoma (RCC), an incurable malignancy associated with mutations in epigenetic regulators, as a model to study genome-wide patterns of DNA methylation at a high resolution.
Analysis of cytosine methylation status of 1.3 million CpGs was determined by the HELP assay in RCC and healthy microdissected renal tubular controls.
We observed that the RCC samples were characterized by widespread hypermethylation that preferentially affected gene bodies. Aberrant methylation was particularly enriched in kidney-specific enhancer regions associated with H3K4Me1 marks. Various important underexpressed genes, such as SMAD6, were associated with aberrantly methylated, intronic enhancers, and these changes were validated in an independent cohort. MOTIF analysis of aberrantly hypermethylated regions revealed enrichment for binding sites of AP2a, AHR, HAIRY, ARNT, and HIF1 transcription factors, reflecting contributions of dysregulated hypoxia signaling pathways in RCC. The functional importance of this aberrant hypermethylation was demonstrated by selective sensitivity of RCC cells to low levels of decitabine. Most importantly, methylation of enhancers was predictive of adverse prognosis in 405 cases of RCC in multivariate analysis. In addition, parallel copy-number analysis from MspI representations demonstrated novel copy-number variations that were validated in an independent cohort of patients.
Our study is the first high-resolution methylome analysis of RCC, demonstrates that many kidney-specific enhancers are targeted by aberrant hypermethylation, and reveals the prognostic importance of these epigenetic changes in an independent cohort.
尽管最近的研究表明增强子处的基因变化可影响致癌作用，但大多数甲基化组研究都集中在启动子处的变化。我们使用肾细胞癌（RCC），一种与表观遗传调节因子突变相关的无法治愈的恶性肿瘤，作为模型来高分辨率研究全基因组DNA甲基化模式。
通过HELP分析确定了RCC和健康显微切割肾小管对照中130万个CpG的胞嘧啶甲基化状态。
我们观察到RCC样本的特征是广泛的高甲基化，优先影响基因体。异常甲基化在与H3K4Me1标记相关的肾脏特异性增强子区域中尤为富集。各种重要的低表达基因，如SMAD6，与异常甲基化的内含子增强子相关，并且这些变化在一个独立队列中得到了验证。对异常高甲基化区域的基序分析揭示了AP2a、AHR、HAIRY、ARNT和HIF1转录因子结合位点的富集，反映了RCC中缺氧信号通路失调的作用。RCC细胞对低水平地西他滨的选择性敏感性证明了这种异常高甲基化的功能重要性。最重要的是，在多变量分析中，增强子的甲基化可预测405例RCC患者的不良预后。此外，来自MspI代表性的平行拷贝数分析显示了新的拷贝数变异，这些变异在一个独立的患者队列中得到了验证。
我们的研究是首次对RCC进行高分辨率甲基化组分析，证明许多肾脏特异性增强子是异常高甲基化的靶点，并揭示了这些表观遗传变化在一个独立队列中的预后重要性。
