Methylation in the core-promoter region of the chondromodulin-I gene determines the cell-specific expression by regulating the binding of transcriptional activator Sp3.
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.
Aoyama
Tomoki
T
Institute for Frontier Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan.
Okamoto
Takeshi
T
Nagayama
Satoshi
S
Nishijo
Koichi
K
Ishibe
Tatsuya
T
Yasura
Ko
K
Nakayama
Tomitaka
T
Nakamura
Takashi
T
Toguchida
Junya
J
D044503:5-Methylcytosine / Q000737:chemistry; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001665:Binding Sites; D015153:Blotting, Western; D001842:Bone and Bones / Q000378:metabolism; D002356:Cartilage / Q000378:metabolism; D045744:Cell Line, Tumor; D019902:Chondrocytes / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D018899:CpG Islands; D003596:Cytosine / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000378:metabolism*; D000077209:Decitabine; D017930:Genes, Reporter; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000235:genetics*; D008156:Luciferases / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics*; D011233:Precipitin Tests; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016329:Sp1 Transcription Factor / Q000378:metabolism; D051705:Sp3 Transcription Factor; D013447:Sulfites / Q000494:pharmacology; D013941:Thymine / Q000737:chemistry; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D002843:Chromatin; D004268:DNA-Binding Proteins; D036341:Intercellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D013447:Sulfites; D014157:Transcription Factors; C068675:CNMD protein, human; D051705:Sp3 Transcription Factor; D044503:5-Methylcytosine; D000077209:Decitabine; D003596:Cytosine; D008156:Luciferases; D001374:Azacitidine; D013941:Thymine; C009279:sodium bisulfite