Suppression of metallothionein gene expression in a rat hepatoma because of promoter-specific DNA methylation
Suppression of metallothionein gene expression in a rat hepatoma because of promoter-specific DNA methylation
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DOI：
10.1074/jbc.275.1.539
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发表时间：
2000-01-07
影响因子：
4.8
通讯作者：
Jacob, ST
中科院分区：
文献类型：
--
作者：
Ghoshal, K;Majumder, S;Jacob, ST
金属硫蛋白 I 可以响应多种物质（包括重金属和氧化应激）而被诱导，相反，它的诱导在一些淋巴源性癌细胞中受到抑制。这种镇压的机制尚未阐明。在这里，我们展示了大鼠实体移植肿瘤中 MT I 基因的沉默，这是由于跨越 -225 bp 至 +1 bp 区域的所有 21 个 CpG 二核苷酸的启动子甲基化所致。相比之下，这些 CpG 二核苷酸在带有肿瘤的大鼠的肝脏中没有被甲基化，这与硫酸锌在该组织中有效诱导该基因一致。基因组足迹揭示了肝癌中甲基化 MT-I 启动子的相应顺式作用元件缺乏转录激活因子的通路。足迹的缺失并不是由于金属调节转录因子MTF-1失活，因为它在肝癌中高度活跃。用5-氮杂胞苷（一种去甲基化剂）治疗携带肝癌的大鼠，诱导了肝癌中基础的以及重金属激活的MT-I基因表达，这意味着甲基化确实导致了该基因的沉默。亚硫酸氢盐基因组测序显示，用 5-AzaC 处理后，肝癌中跨越 MT-I 启动子的 CPG 二核苷酸显着（>90%）去甲基化。MT-I 启动子的高甲基化可能是由于相对于宿主肝脏而言，肝癌中 DNA 甲基转移酶活性水平显着升高（高达 7 倍）以及其基因表达增强所致。这些数据首次阐明了与相应亲代组织中的强诱导相比，在动物移植的实体瘤中高度诱导基因沉默的分子机制，并讨论了在某些肿瘤中抑制该基因的可能原因。
Metallothionein I can be induced in response to a variety of agents that include heavy metals and oxidative stress, On the contrary, its induction was suppressed in some lymphoid-derived cancer cells. The mechanism of this repression has not been elucidated. Here, we show silencing of MT I gene in a solid transplanted rat tumor as a result of promoter methylation at all the 21 CpG dinucleotides that span the region from -225 bp to +1 bp. By contrast, none of these CpG dinucleotides were methylated in the livers from the rats bearing the tumor, which was consistent with the efficient induction of the gene in this tissue by zinc sulfate. Genomic footprinting revealed lack of access of the transcriptional activators to the respective cis-acting elements of the methylated MT-I promoter in the hepatoma. The absence of footprinting was not due to inactivation of the metal regulatory transcription factor MTF-1, because it was highly active in the hepatoma. Treatment of the hepatoma bearing rats with 5-azacytidine, a demethylating agent, induced basal as well as heavy metal-activated MT-I gene expression in the hepatoma, implying that methylation was indeed responsible for silencing the gene. Bisulfite genomic sequencing showed significant (>90%) demethylation of CPG dinucleotides spanning MT-I promoter in the hepatoma following treatment with 5-AzaC, The hypermethylation of MT-I promoter was probably caused by significantly higher (as much as 7-fold) level of DNA methyl transferase activity as well as enhanced expression of its gene in the hepatoma relative to the host liver. These data elucidated for the first time the molecular mechanism for the silencing of a highly inducible gene in a solid tumor transplanted in an animal, as compared with the robust induction in the corresponding parental tissue and have discussed the probable reasons for the suppression of this gene in some tumors.