COLLAGEN-II PROMOTER AND ENHANCER INTERACT SYNERGISTICALLY THROUGH SP1 AND DISTINCT NUCLEAR FACTORS

The collagen II gene is expressed primarily in chondrocytes. Its transcription is activated through the interaction of cell type-specific regulatory elements located in the promoter region and in the first intron. In this study, we found that a short promoter sequence including two GC boxes was required for efficient enhancer-mediated transcription, Gel-shift analysis, site mutations, and footprint analysis showed that one of the GC boxes bound functionally to an Sp1-related factor and that an oligonucleotide containing this GC box did interact with an enhancer-nuclear factor complex. Additionally, an enhancer-derived oligonucleotide was found to complex CIIZFP, a zinc-finger protein that binds to the enhancer within the first intron and Sp1, but only in presence of CIIZFP. Antibodies against Sp1 specifically inhibited the formation of this complex. Western/Southwestern analysis also showed that a protein complex including Sp1 was able to bind the enhancer and the promoter regions in non-denaturing conditions. This complex was dissociated by denaturation. These results suggest that the formation of a nuclear protein-mediated loop structure between the promoter and enhancer may regulate transcription of the collagen II gene transcription.