Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells
Short hairpin RNA-directed cytosine (CpG) methylation of the RASSF1A gene promoter in HeLa cells
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DOI：
10.1016/j.ymthe.2005.03.003
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发表时间：
2005-07-01
影响因子：
12.4
通讯作者：
Rossi, JJ
中科院分区：
文献类型：
--
作者：
Castanotto, D;Tommasi, S;Rossi, JJ
CpG基序中胞嘧啶的甲基化是哺乳动物细胞基因表达表观遗传调控的重要机制。哺乳动物细胞，尤其是癌症细胞中的从头甲基化的启动事件(S)尚不清楚。在植物中，已知与DNA序列同源的短RNA启动从头开始甲基化。为了研究短发夹状RNAs(ShRNAs)是否也可以作为人类细胞从头甲基化的启动子，我们表达了与CpG岛互补的短发夹状RNAs，包括人RASSF1A基因的启动子和早期转录区域。RASSF1A编码一种可能的肿瘤抑制因子，在各种人类癌症中高度甲基化，而在一些人类细胞系中，如HeLa，RASSF1A是非甲基化的，并且在转录上是活性的。我们证明，与RASSF1A启动子或早期转录区域互补的shRNAs可以在HeLa细胞中引导低水平的从头DNA甲基化和部分基因沉默。相反，与靶标存在四个中心错配的shRNA不能指导这种甲基化。这一结果提示了癌细胞中通过DNA甲基化而导致转录基因沉默的潜在的激发机制。
Methylation of cytosines in CpG motifs is an important mechanism for epigenetic regulation of gene expression in mammalian cells. The initiating event(s) for de novo methylation in mammalian cells, particularly in cancer, is unknown. In plants, short RNAs homologous to DNA sequences are known to initiate de novo methylation. To investigate whether short hairpin RNAs (shRNAs) may also serve as initiators for de novo methylation in human cells we have expressed short hairpin RNAs complementary to the CpG island including the promoter and early transcribed regions of the human RASSF1A gene. RASSF1A encodes a putative tumor suppressor that is hypermethylated in a variety of human cancers, whereas in some human cell lines, such as HeLa, RASSF1A is unmethylated and transcriptionally active. We demonstrate that shRNAs complementary to the RASSF1A promoter or early transcribed regions can direct low levels of de novo DNA methylation and partial gene silencing in HeLa cells. In contrast, an shRNA harboring four central mismatches with the target cannot direct such methylation. The results presented suggest provocative potential mechanisms for transcriptional gene silencing via DNA methylation in cancer cells.