Epigenetics Research Laboratory, Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Duke University Medical Center , Durham, NC , USA.
Front Oncol. 2013 May 24;3:131. doi: 10.3389/fonc.2013.00131. eCollection 2013.
Overexpression of insulin-like growth factor-II (IGF2) is a prominent characteristic of many epithelial ovarian malignancies. IGF2 imprinting and transcription are regulated in part through DNA methylation, which in turn regulates binding of the insulator protein CTCF within the IGF2/H19 imprint center. We have shown that IGF2 overexpression in ovarian cancer is associated with hypermethylation of CTCF binding sites within the IGF2/H19 imprint center. The aim of this study was to investigate the methylation and binding capacity of a novel putative CTCF binding motif located intragenic to IGF2 and determine how this relates to IGF2 expression. Among 35 primary serous epithelial ovarian cancer specimens, methylation of two CpGs, including one within the core binding motif and another adjacent to this motif, was higher in the 18 cancers with elevated IGF2 expression versus 10 with low expression (average 68.2 versus 38.5%; p < 0.0001). We also found that the CpG site within the CTCF binding motif is hypermethylated in male gametes (>92%; average 93.2%; N = 16). We confirmed binding of CTCF to this region in ovarian cancer cells, as well as the paralog of CTCF, Brother Of the Regulator of Imprinted Sites (BORIS), which is frequently overexpressed in cancers. The unmethylated CTCF binding motif has insulator activity in cells that express CTCF or BORIS, but not in cells that express both CTCF and BORIS. These intragenic CpG dinucleotides therefore comprise a novel paternal germline imprint mark and are located in a binding motif for the insulator protein CTCF. Methylation of the CpG dinucleotides is positively correlated with IGF2 transcription, indicating that increased methylation represses insulator function. These combined results suggest that methylation and CTCF binding at this region play important roles in regulating the level of IGF2 transcription. Our data have revealed a novel epigenetic regulatory element within the IGF2/H19 imprinted domain that is highly relevant to aberrant IGF2 expression in ovarian malignancies.
胰岛素样生长因子-II（IGF2）的过度表达是许多上皮性卵巢恶性肿瘤的一个显著特征。IGF2 的印迹和转录部分受 DNA 甲基化调控，而 DNA 甲基化又调节着 IGF2/H19 印迹中心内绝缘子蛋白 CTCF 的结合。我们已经表明，卵巢癌中 IGF2 的过度表达与 IGF2/H19 印迹中心内 CTCF 结合位点的高甲基化有关。本研究的目的是研究位于 IGF2 基因内的一个新的假定 CTCF 结合基序的甲基化和结合能力，并确定其与 IGF2 表达的关系。在 35 例原发性浆液性上皮性卵巢癌标本中，18 例 IGF2 表达升高的癌症中两个 CpG （包括核心结合基序内的一个 CpG 和该基序附近的另一个 CpG）的甲基化程度高于 10 例 IGF2 表达较低的癌症（平均 68.2%对 38.5%；p<0.0001）。我们还发现，CTCF 结合基序内的 CpG 位点在精子中高度甲基化（>92%；平均 93.2%；N=16）。我们在卵巢癌细胞中证实了 CTCF 与该区域的结合，以及 CTCF 的同源物，即印迹调节因子的兄弟（BORIS）的结合，BORIS 在癌症中经常过度表达。在表达 CTCF 或 BORIS 的细胞中，未甲基化的 CTCF 结合基序具有绝缘子活性，但在同时表达 CTCF 和 BORIS 的细胞中没有。因此，这些内含子 CpG 二核苷酸构成了一个新的父系生殖系印记标记，位于绝缘子蛋白 CTCF 的结合基序中。CpG 二核苷酸的甲基化与 IGF2 转录呈正相关，表明甲基化增加抑制了绝缘子功能。这些综合结果表明，该区域的甲基化和 CTCF 结合在调节 IGF2 转录水平方面发挥着重要作用。我们的数据揭示了 IGF2/H19 印迹域内的一个新的表观遗传调节元件，与卵巢恶性肿瘤中 IGF2 表达异常高度相关。
