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 chondrocy tes. Transcription of Col2a1 is mediated by cell-specific regulatory e lements located within the promoter and first intron. Here, we map a m inimal enhancer and identify elements that determine cartilage-specifi c Col2a1 expression by analyzing the activity of a series of chimeric genes consisting of rat Col2a1 first intron deletion mutants ligated t o 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. Se quence analysis of this 100-bp Col2a1 enhancer revealed several sequen ce motifs similar to motifs present within the regulatory region of th e link protein gene, another cartilage gene. These motifs include an A T-rich element, a C1 motif and a C3 motif. Deletion of any of these el ements reduced Col2a1 enhancer activity in chick embryo chondrocytes. We also tested enhancer-mediated activity in CFK2 cells which differen tiate to a chondrogenic phenotype and begin to express type II collage n mRNA after extended culture. In stably transfected CFK2 cells, const ructs containing the 100-bp enhancer were activated during the transit ion from prechondrogenic to chondrogenic cell populations and deletion s within the enhancer strongly down-regulated activity. Chondrocyte-sp ecific DNA-protein complexes were identified using nuclear extracts pr epared from chick embryo chondrocytes and P-32-labeled oligonucleotide s from these regions of the first intron. These results suggest that i nteraction 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.