Epigenetic repression of RARRES1 is mediated by methylation of a proximal promoter and a loss of CTCF binding.
Epigenetic repression of RARRES1 is mediated by methylation of a proximal promoter and a loss of CTCF binding.
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DOI：
10.1371/journal.pone.0036891
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Lin HJ
中科院分区：
文献类型：
--
作者：
Peng Z;Shen R;Li YW;Teng KY;Shapiro CL;Lin HJ
通过甲基化导致视黄酸受体应答器 1 (RARRES1) 表观遗传沉默的顺式作用启动子元件尚不清楚。同样，异常甲基化如何与效应器相互作用并从而影响乳腺肿瘤特征仍然很大程度上未知。我们首先比较了原发性乳腺癌和邻近良性组织中 RARRES1 启动子侧翼序列 (-664∼+420) 发生的甲基化。令人惊讶的是，肿瘤核心显示仅在上游区域（-664∼-86）显着升高的甲基化，而靠近转录起始位点（+1）的下游元件（-85∼+420）基本保持不变。然而，前者的高甲基化并没有导致明显的沉默效应。相反，近端序列显示出完整的启动子活性和甲基化，其显着沉默了 RARRES1 转录。这种现象在乳腺癌细胞系中得到了重现，其中近端区域的甲基化与下调惊人地一致。我们还发现 CTCF 占据在与转录活性组蛋白标记结合的非甲基化启动子处富集。此外，敲低 CTCF 表达阻碍了 RARRES1 表达，表明 CTCF 可能通过与处于转录就绪状态的未甲基化启动子结合而正向调节 RARRES1 转录。此外，RARRES1的恢复不仅阻碍细胞侵袭，还促进化疗药物诱导的细胞死亡，表明其具有肿瘤抑制作用。其减弱侵袭的作用与临床标本产生的数据一致，表明与肿瘤核心相比，RARRES1 在转移淋巴结中普遍下调。该报告描述了近端启动子元件上发生的超甲基化导致的 RARRES1 沉默，并与与 CTCF（RARRES1 表达激活剂）结合的丧失相关。我们还揭示了 RARRES1 的肿瘤抑制作用，部分是通过促进乳腺上皮细胞死亡和阻止细胞侵袭而发挥的，而细胞侵袭是转移扩散的重要特性。
The cis-acting promoter element responsible for epigenetic silencing of retinoic acid receptor responder 1 (RARRES1) by methylation is unclear. Likewise, how aberrant methylation interplays effectors and thus affects breast neoplastic features remains largely unknown. We first compared methylation occurring at the sequences (−664∼+420) flanking the RARRES1 promoter in primary breast carcinomas to that in adjacent benign tissues. Surprisingly, tumor cores displayed significantly elevated methylation occurring solely at the upstream region (−664∼−86), while the downstream element (−85∼+420) proximal to the transcriptional start site (+1) remained largely unchanged. Yet, hypermethylation at the former did not result in appreciable silencing effect. In contrast, the proximal sequence displayed full promoter activity and methylation of which remarkably silenced RARRES1 transcription. This phenomenon was recapitulated in breast cancer cell lines, in which methylation at the proximal region strikingly coincided with downregulation. We also discovered that CTCF occupancy was enriched at the unmethylayed promoter bound with transcription-active histone markings. Furthermore, knocking-down CTCF expression hampered RARRES1 expression, suggesting CTCF positively regulated RARRES1 transcription presumably by binding to unmethylated promoter poised at transcription-ready state. Moreover, RARRES1 restoration not only impeded cell invasion but also promoted death induced by chemotherapeutic agents, denoting its tumor suppressive effect. Its role of attenuating invasion agreed with data generated from clinical specimens revealing that RARRES1 was generally downregulated in metastatic lymph nodes compared to the tumor cores. This report delineated silencing of RARRES1 by hypermethylation is occurring at a proximal promoter element and is associated with a loss of binding to CTCF, an activator for RARRES1 expression. We also revealed the tumor suppressive roles exerted by RARRES1 in part by promoting breast epithelial cell death and by impeding cell invasion that is an important property for metastatic spread.
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