Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells

被引:12
|
作者
Yanagihara, I
Yamagata, M
Sakai, N
Shukunami, C
Kurahashi, H
Yamazaki, M
Michigami, T
Hiraki, Y
Ozono, K
机构
[1] Osaka Med Ctr, Dept Environm Med, Osaka 5941101, Japan
[2] Res Inst Maternal & Child Hlth, Osaka 5941101, Japan
[3] Kyoto Univ, Inst Frontier Med Sci, Dept Mol Interact & Tissue Engn, Kyoto, Japan
[4] Osaka Univ, Biomed Res Ctr, Dept Med Genet, Osaka, Japan
关键词
chondromodulin-1; chondrocyte; gene structure; gene expression; initiator;
D O I
10.1359/jbmr.2000.15.3.421
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Chondromodulin-1 (ChM-1) is a cartilage-specific glycoprotein that stimulates the growth of chondrocytes and inhibits the tube formation of endothelial cells. To clarify the tissue-specific expression and the role of ChM-1 in pathophysiological conditions, we analyzed the structure of the human ChM-1 gene and its promoter. On the screening of a human genomic cosmid library using the human ChM-1 complimentary DNA (cDNA) as a probe, two clones were obtained that contained ChM-1 cDNA, The restriction enzyme map and nucleotide sequence revealed the human ChM-1 gene consisting: of seven exons and exon-intron boundaries. The human ChM-1 gene was assigned to chromosome 13q14-21 by fluorescence in situ hybridization (FISH) using the clone as a probe. A primer extension analysis using total RNA extracted from human cartilage revealed a major transcription start site with the sequence CGCT(+1)GG. The region approximately 3-kilobase (kb) nucleotides upstream of the translation start site was then sequenced and analyzed in terms of promoter activity. We found that a region 446 base pairs (bp) upstream of the start site had promoter activity in COS7, HeLa, and ATDC5 cells. In structure the promoter is a TATA-less type without a CC-rich region.-The transcription factors Sox9, Og12, and Cart-1 did not affect the promoter activity,The transcription factor Ying-Yang1 suppressed the promoter activity but GABP protein did not change the promoter activity. The construct containing -446/+87 fused to the SV40 enhancer and green fluorescent protein (CFP) exhibited expression of CFP corresponding to the differentiation of ATDC5 cells to mature chondrocytes, These results suggest that the element -446/+87 confers the cartilage-specific expression of this gene by some factor(s) other than Sox9, Og12, and Cart-1.
引用
收藏
页码:421 / 429
页数:9
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