Identification of a minimum enhancer sequence for the type II collagen gene reveals several core sequence motifs in common with the link protein gene.
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.
Krebsbach
P H
PH
Laboratory of Developmental Biology, NIDR, National Institutes of Health, Bethesda, Maryland 20892, USA.
Nakata
K
K
Bernier
S M
SM
Hatano
O
O
Miyashita
T
T
Rhodes
C S
CS
Yamada
Y
Y
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
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