Cancer Research Institute, Seoul National University College of Medicine, Seoul, Korea.
Int J Oncol. 2010 Jun;36(6):1563-72. doi: 10.3892/ijo_00000643.
DNA methyltransferase (DNMT) 1 and 3 are primarily responsible for abnormal methylation in cancer. Unlike these DNMTs, DNA methyltransferase 3-like (DNMT3L) harbors no conserved catalytic domain, and has been shown to function as a regulatory cofactor for DNA methylation. However, it is unclear whether DNMT3L directly regulates DNA methylation in cancer cells. To address this, we investigated the methylation targets of DNMT3L by conducting methylation microarray trials after the siRNA-induced knockdown. We determined that methylation of 242 out of 1,505 CpG sites was significantly altered by DNMT3L knockdown. Among these 242 CpG sites, 204, 12, and 11 CpG sites were identified as common targets of DNMT 1/3B/3L, 1/3L, and 3B/3L, respectively; this indicates that DNMT3L participates in DNA methylation via cooperation with other DNMTs. However, we also determined that the methylation of 15 CpG sites was significantly altered by DNMT3L knockdown only. As a validation, we confirmed that thymine DNA glycosylase (TDG), an enzyme involved in the base excision repair of mismatched-DNA, was up-regulated in DNMT3L knockdown cells, but neither in DNMT1 nor 3B knockdown cells. Methylation-specific PCR (MSP) also showed that promoter methylation of TDG was decreased in DNMT3L knockdown cells. Interestingly, 5-aza-2'-deoxycitidine (5-aza-dC) re-expressed DNMT3L, leading to down-regulation of TDG. This study is the first to show that DNMT3L exerts a major effect on the transcriptional regulation of a specific target gene, such as TDG, despite the absence of enzymatic activity.
DNA 甲基转移酶（DNMT）1 和 3 主要负责癌症中的异常甲基化。与这些 DNMT 不同，DNA 甲基转移酶 3 样（DNMT3L）没有保守的催化结构域，已被证明作为 DNA 甲基化的调节辅因子发挥作用。然而，DNMT3L 是否直接调节癌细胞中的 DNA 甲基化尚不清楚。为了解决这个问题，我们通过 siRNA 诱导敲低后进行甲基化微阵列试验，研究了 DNMT3L 的甲基化靶标。我们确定，DNMT3L 敲低后，1505 个 CpG 位点中有 242 个的甲基化明显改变。在这 242 个 CpG 位点中，204、12 和 11 个 CpG 位点分别被鉴定为 DNMT1/3B/3L、1/3L 和 3B/3L 的共同靶标；这表明 DNMT3L 通过与其他 DNMT 合作参与 DNA 甲基化。然而，我们还确定，DNMT3L 敲低仅使 15 个 CpG 位点的甲基化明显改变。作为验证，我们证实胸腺嘧啶 DNA 糖基化酶（TDG）在 DNMT3L 敲低细胞中上调，而在 DNMT1 或 3B 敲低细胞中均未上调，该酶参与错配 DNA 的碱基切除修复。甲基化特异性 PCR（MSP）也显示，DNMT3L 敲低细胞中 TDG 的启动子甲基化减少。有趣的是，5-氮杂-2'-脱氧胞苷（5-aza-dC）重新表达了 DNMT3L，导致 TDG 下调。这项研究首次表明，尽管缺乏酶活性，DNMT3L 对特定靶基因（如 TDG）的转录调节仍有重大影响。
