5-hydroxymethylcytosine marks promoters in colon that resist hypermethylation in cancer [ExpressionArray]

The discovery of cytosine hydroxymethylation (5-hmC) as a mechanism that potentially controls DNA methylation changes typical of neoplasia prompted us to investigate its behavior in colon cancer. 5-hmC is globally reduced in proliferating cells such as colon tumors and the gut crypt progenitors, from which tumors can arise. Here, we show that colorectal tumors and cancer cells express Ten-Eleven Translocation (TET) transcripts at levels similar to normal tissues. Genome-wide analyses show that promoters marked by 5-hmC in normal tissue, and those identified as TET2 targets in colorectal cancer cells, are resistant to methylation gain in cancer. In vitro studies of TET2 in cancer cells confirm that these promoters are resistant to methylation gain independently of sustained TET2 expression. We also find that a considerable number of the methylation gain-resistant promoters marked by 5-hmC in normal colon overlap with those that are marked with poised bivalent histone modifications in embryonic stem cells. Together our results indicate that promoters that acquire 5-hmC upon normal colon differentiation are innately resistant to neoplastic hypermethylation by mechanisms that do not require high levels of 5-hmC in tumors. Our study highlights the potential of cytosine modifications as biomarkers of cancerous cell proliferation. messenger RNA levels were measured in total RNA extracted from primary colon tissues. Normal away are at least 20cm from tumors.

胞嘧啶羟甲基化（cytosine hydroxymethylation，5-hmC）作为一种潜在调控肿瘤典型DNA甲基化改变的机制被发现后，我们着手探究其在结肠癌中的表达行为。5-hmC在增殖细胞（如结肠肿瘤及肿瘤起源的肠道隐窝祖细胞）中呈全局水平降低的状态。本研究发现，结直肠肿瘤及癌细胞中十-十一易位（Ten-Eleven Translocation，TET）转录本的表达水平与正常组织相当。全基因组分析显示，在正常组织中被5-hmC标记的启动子，以及在结直肠癌细胞中被鉴定为TET2靶标的启动子，在肿瘤发生发展过程中均不易发生甲基化获得。针对癌细胞中TET2的体外实验证实，此类启动子的甲基化获得抗性并不依赖于TET2的持续表达。我们还发现，正常结肠组织中被5-hmC标记的甲基化获得抗性启动子中，有相当比例与胚胎干细胞中带有poised二价组蛋白修饰的启动子存在重叠。综合以上结果，我们认为：在正常结肠分化过程中获得5-hmC的启动子，天生具有抵抗肿瘤性高甲基化的特性，其发挥作用的机制并不依赖于肿瘤组织中高水平的5-hmC。本研究凸显了胞嘧啶修饰作为癌细胞增殖生物标志物的应用潜力。本研究对提取自原发性结肠组织的总RNA进行了信使RNA水平检测，其中正常组织样本距肿瘤至少20cm。