Nishita Y, Sado T, Yoshida I, Takagi N
Research Center for Molecular Genetics Hokkaido University, Sapporo,
Gene. 1999 Jan 21;226(2):199-209. doi: 10.1016/s0378-1119(98)00576-9.
We previously reported isolation of the mouse gene, Mest (mesoderm-specific transcripts), which is mapped to the proximal part of chromosome 6 and predominantly expressed in the mesoderm and its derivatives during development. Peg1, a paternally expressed gene isolated by a systematic screening of imprinted genes, was recently demonstrated to be identical to Mest. We and others have shown that the human homolog (MEST) of Mest is also imprinted so as to be expressed from the paternal copy and maps to 7q32. To study transcriptional regulation of Mest/Peg1, we examined the effect of DNA methylation on its expression. In the embryonal carcinoma (EC) cell line, MC12, from which Mest was originally isolated, the 5'-region harboring presumptive promoter of the gene was undermethylated. On the other hand, C4XX, a subclone of MC12 which had lost expression of Mest, was characterized by extremely high levels of methylation in the 5'-region, demethylation of which resulted in activation of Mest. Furthermore, a methylated reporter construct with the luciferase gene under the control of the putative promoter region of Mest was not competent to produce luciferase activity in MC12 cells. These results suggest a suppressive role for DNA methylation in Mest expression. However, neither methylated nor unmethylated reporter constructs showed luciferase activity in a primary culture from the adult kidney, in which Mest is down-regulated despite apparent unmethylation of the paternal allele. Taken together, the data suggest that there are probably two modes of regulation for the Mest gene; one being a methylation-dependent mechanism that regulates imprinted expression of Mest during development, and the other being a methylation-independent mechanism that is involved in down-regulation of Mest in adult tissues.
我们先前报道了小鼠基因Mest（中胚层特异性转录本）的分离，该基因定位于6号染色体近端，在发育过程中主要在中胚层及其衍生物中表达。Peg1是通过对印记基因进行系统筛选分离出的一个父本表达基因，最近被证明与Mest相同。我们和其他人已经表明，Mest的人类同源基因（MEST）也被印记，从而从父本拷贝表达，并定位于7q32。为了研究Mest/Peg1的转录调控，我们检测了DNA甲基化对其表达的影响。在最初分离出Mest的胚胎癌细胞系MC12中，该基因假定启动子所在的5'-区域未发生甲基化。另一方面，MC12的一个亚克隆C4XX失去了Mest的表达，其特征是5'-区域甲基化水平极高，该区域去甲基化导致Mest激活。此外，一个带有荧光素酶基因的甲基化报告构建体，在Mest假定启动子区域的控制下，在MC12细胞中无法产生荧光素酶活性。这些结果表明DNA甲基化在Mest表达中起抑制作用。然而，甲基化和未甲基化的报告构建体在成年肾脏原代培养物中均未显示荧光素酶活性，在该培养物中，尽管父本等位基因明显未甲基化，但Mest仍被下调。综上所述，数据表明Mest基因可能存在两种调控模式；一种是甲基化依赖机制，在发育过程中调节Mest的印记表达，另一种是甲基化非依赖机制，参与成年组织中Mest的下调。
