Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3
Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3
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DOI：
10.1074/jbc.m401273200
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发表时间：
2004-07-02
影响因子：
4.8
通讯作者：
Toguchida, J
中科院分区：
文献类型：
--
作者：
Aoyama, T;Okamoto, T;Toguchida, J
细胞和阶段特异性基因的转录调控是间充质组织发育的关键过程。在这里，我们已经调查了软骨细胞特异性基因之一的软骨调节素-I（ChM-I）基因的表达的调控机制，在成骨细胞使用骨肉瘤（OS）细胞作为模型。甲基化特异性序列分析显示，在核心启动子区的ChM-I基因的甲基化程度与在OS原发性肿瘤和细胞系中的ChM-I基因的表达呈负相关。5-氮杂脱氧胞苷处理诱导ChM-I阴性OS细胞系中ChM-I基因的表达，并且表达的诱导与Sp1/3结合位点中间的-52（C（-52））处的胞嘧啶的去甲基化紧密相关，Sp3而不是Sp1与该结合位点结合。用甲基胞嘧啶或胸腺嘧啶取代C（-52）可消除Sp3结合，也可消除包括C（-52）的基因组片段的转录活性。小干扰RNA对Sp3表达的抑制降低了ChM-I阳性正常软骨细胞中ChM-I基因的表达，表明Sp3是ChM-I基因的生理转录激活因子。这些结果表明，核心启动子区域的甲基化状态是通过调节Sp3的结合来决定ChM-1基因的细胞特异性表达的机制之一。
Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma ( OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.