title: Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene.
abstract: The type II collagen gene (Col2a1) is expressed primarily in chondrocytes. Transcription of Col2a1 is mediated by cell-specific regulatory elements located within the promoter and first intron. Here, we map a minimal enhancer and identify elements that determine cartilage-specific Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated to the chloramphenicol acetyltransferase reporter gene. We show that a 100-base pair (bp) segment within the first intron is the minimum size necessary for high level, cell type-specific expression of Col2a1. Sequence analysis of this 100-bp Col2a1 enhancer revealed several sequence motifs similar to motifs present within the regulatory region of the link protein gene, another cartilage gene. These motifs include an AT-rich element, a C1 motif and a C3 motif. Deletion of any of these elements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer-mediated activity in CFK2 cells which differentiate to a chondrogenic phenotype and begin to express type II collagen mRNA after extended culture. In stably transfected CFK2 cells, constructs containing the 100-bp enhancer were activated during the transition from prechondrogenic to chondrogenic cell populations and deletions within the enhancer strongly down-regulated activity. Chondrocyte-specific DNA-protein complexes were identified using nuclear extracts prepared from chick embryo chondrocytes and 32P-labeled oligonucleotides from these regions of the first intron. These results suggest that interaction of chondrocyte specific nuclear factors with multiple core elements from a small region within the first intron are important for cell-type specific Col2a1 enhancer activity.
authors.affiliation: Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
authors.forename: P H
authors.initials: PH
authors.lastname: Krebsbach
authors.forename: K
authors.initials: K
authors.lastname: Nakata
authors.forename: S M
authors.initials: SM
authors.lastname: Bernier
authors.forename: O
authors.initials: O
authors.lastname: Hatano
authors.forename: T
authors.initials: T
authors.lastname: Miyashita
authors.forename: C S
authors.initials: CS
authors.lastname: Rhodes
authors.forename: Y
authors.initials: Y
authors.lastname: Yamada
mesh_terms: D000818:Animals; D001483:Base Sequence; D002356:Cartilage / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002642:Chick Embryo; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D003094:Collagen / Q000096:biosynthesis / Q000235:genetics*; D004742:Enhancer Elements, Genetic; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D007438:Introns; D008969:Molecular Sequence Data; D016296:Mutagenesis; D015345:Oligonucleotide Probes; D014176:Protein Biosynthesis; D011506:Proteins / Q000235:genetics*; D011509:Proteoglycans; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D017384:Sequence Deletion; D012689:Sequence Homology, Nucleic Acid; D014162:Transfection; D000099249:Proteoglycan Link Protein
chemical_list: D015500:Chloramphenicol O-Acetyltransferase; D003094:Collagen; D016326:Extracellular Matrix Proteins; D015345:Oligonucleotide Probes; D011506:Proteins; D011509:Proteoglycans; D011993:Recombinant Fusion Proteins; D000099249:Proteoglycan Link Protein