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

Itaru Yanagihara, Masayo Yamagata, Norio Sakai, Chisa Shukunami, Hiroki Kurahashi, Miwa Yamazaki, Toshimi Michigami, Yuji Hiraki, Keiichi Ozono

Research output: Contribution to journalArticle

11 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)421-429
Number of pages9
JournalJournal of Bone and Mineral Research
Volume15
Issue number3
Publication statusPublished - 16-03-2000
Externally publishedYes

Fingerprint

Reporter Genes
Genetic Promoter Regions
Cartilage
Chondrocytes
Green Fluorescent Proteins
Genes
Exons
Transcription Factors
Clone Cells
GC Rich Sequence
Chromosomes, Human, Pair 21
Cosmids
Genomic Library
Transcription Initiation Site
DNA
Fluorescence In Situ Hybridization
HeLa Cells
Base Pairing
Introns
Cell Differentiation

All Science Journal Classification (ASJC) codes

  • Endocrinology, Diabetes and Metabolism
  • Orthopedics and Sports Medicine

Cite this

Yanagihara, Itaru ; Yamagata, Masayo ; Sakai, Norio ; Shukunami, Chisa ; Kurahashi, Hiroki ; Yamazaki, Miwa ; Michigami, Toshimi ; Hiraki, Yuji ; Ozono, Keiichi. / Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells. In: Journal of Bone and Mineral Research. 2000 ; Vol. 15, No. 3. pp. 421-429.
@article{b8460af767a944a2a10bc816651e3ad5,
title = "Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells",
abstract = "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.",
author = "Itaru Yanagihara and Masayo Yamagata and Norio Sakai and Chisa Shukunami and Hiroki Kurahashi and Miwa Yamazaki and Toshimi Michigami and Yuji Hiraki and Keiichi Ozono",
year = "2000",
month = "3",
day = "16",
language = "English",
volume = "15",
pages = "421--429",
journal = "Journal of Bone and Mineral Research",
issn = "0884-0431",
publisher = "Wiley-Blackwell",
number = "3",

}

Yanagihara, I, Yamagata, M, Sakai, N, Shukunami, C, Kurahashi, H, Yamazaki, M, Michigami, T, Hiraki, Y & Ozono, K 2000, 'Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells', Journal of Bone and Mineral Research, vol. 15, no. 3, pp. 421-429.

Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells. / Yanagihara, Itaru; Yamagata, Masayo; Sakai, Norio; Shukunami, Chisa; Kurahashi, Hiroki; Yamazaki, Miwa; Michigami, Toshimi; Hiraki, Yuji; Ozono, Keiichi.

In: Journal of Bone and Mineral Research, Vol. 15, No. 3, 16.03.2000, p. 421-429.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Genomic organization of the human chondromodulin-1 gene containing a promoter region that confers the expression of reporter gene in chondrogenic ATDC5 cells

AU - Yanagihara, Itaru

AU - Yamagata, Masayo

AU - Sakai, Norio

AU - Shukunami, Chisa

AU - Kurahashi, Hiroki

AU - Yamazaki, Miwa

AU - Michigami, Toshimi

AU - Hiraki, Yuji

AU - Ozono, Keiichi

PY - 2000/3/16

Y1 - 2000/3/16

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=0034007870&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0034007870&partnerID=8YFLogxK

M3 - Article

C2 - 10750556

AN - SCOPUS:0034007870

VL - 15

SP - 421

EP - 429

JO - Journal of Bone and Mineral Research

JF - Journal of Bone and Mineral Research

SN - 0884-0431

IS - 3

ER -