メタロチオネイン1発現制御に関わるCpGサイトの同定

抄録

Metallothionein (MT) is a small, cysteine-rich protein active in zinc homeostasis, cadmium detoxification, and protection against reactive oxygen species. Mouse MT-1 gene transcription is regulated by metal response element (MRE)-binding transcription factor-1 (MTF-1), which is strongly recruited to the promoter in response to zinc. MTF-1 shows the highest affinity to MREd among 5 MREs (MREa-MREe) in MT1 promoter. Several studies showed that epigenetics, such as methylation of CpG, is also involved in the MT1 gene expression. Here, we examined whether the specific CpG sites involved in regulation of MT1 expression using repoter vector, pCpGfree-basic-Lucia, which is devoid of CpG. Twenty-nine CpG sites are in the MT1 promoter spanning bases －264 to +10 (relative to the transcription start site). Complete CpG methylation by M.SssI (CG → ^5mCG) inhibited MT1 promoter-mediated Lucia expression. The inhibitory effect of M.SssI-methylation was not observed in MREd/MREe-deleted reporter vector. The inhibitory effect was remained in MREa/MREb/MREc-deleted reporter vector. Similar results were observed in point mutated reporter vectors. To clarify the effect of partial CpG methylation, we used HhaI (GCGC → G^5mCGC)-methylated reporter vectors. Point mutation analysis revealed that methylation of CpG site near MREd/MREe is strongly inhibit MT1 gene expression. Our results suggest that methylation/demethylation of the CpG near MREd/MREe is important for regulation of the MT-1 gene expression.