Identification of a cis-acting sequence in the collagen II enhancer required for chondrocyte expression and the binding of a chondrocyte nuclear factor.

Collagen II is synthesized at high levels by differentiated chondrocytes. A 620-base pair DNA sequence in the first intron of the rat collagen II gene was previously determined to have chondrocyte-specific enhancer activity. Using mobility shift assays, we have defined a decamer sequence, 5'-CACAATGCAT-3', in the middle of the enhancer that binds a protein or protein complex expressed by chondrocytes but not by NIH3T3 cells or L2 rat fibroblasts. This protein was also induced during the differentiation of limb bud mesenchymal cells into chondrocytes. Mutational analyses, coupled with both in vitro binding studies and direct assays of enhancer activity following transfection into cells, demonstrated that this sequence is involved in the binding of the chondrocyte nuclear protein(s) and is necessary for the enhancer activity. This sequence has homology to the consensus binding sequence for helix-loop-helix transcription factors.