Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells.
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+1GG. 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 GC-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 (GFP) exhibited expression of GFP 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.
Yanagihara
I
I
Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Japan.
Yamagata
M
M
Sakai
N
N
Shukunami
C
C
Kurahashi
H
H
Yamazaki
M
M
Michigami
T
T
Hiraki
Y
Y
Ozono
K
K
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002356:Cartilage / Q000166:cytology / Q000378:metabolism*; D002460:Cell Line; D002522:Chlorocebus aethiops; D002882:Chromosomes, Human, Pair 13 / Q000235:genetics; D003360:Cosmids; D018076:DNA, Complementary / Q000235:genetics; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D015723:Gene Library; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006133:Growth Substances / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D036341:Intercellular Signaling Peptides and Proteins; D007438:Introns / Q000235:genetics; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D008565:Membrane Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009928:Organ Specificity; D012150:Polymorphism, Restriction Fragment Length; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic; D014162:Transfection
C489408:Cnmd protein, mouse; D018076:DNA, Complementary; D006133:Growth Substances; D036341:Intercellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C068675:CNMD protein, human; D049452:Green Fluorescent Proteins; D008156:Luciferases