AN 18-BASE-PAIR SEQUENCE IN THE MOUSE PRO-ALPHA-1(II) COLLAGEN GENE IS SUFFICIENT FOR EXPRESSION IN CARTILAGE AND BINDS NUCLEAR PROTEINS THAT ARE SELECTIVELY EXPRESSED IN CHONDROCYTES

The molecular mechanisms by which mesenchymal cells differentiate into chondrocytes are still poorly understood, We have used the gene for a chondrocyte marker, the pro alpha 1(II) collagen gene (Col2 alpha 1), as a model to delineate a minimal sequence needed for chondrocyte exp ression and identify chondrocyte-specific proteins binding to this seq uence. We previously localized a cartilage-specific enhancer to 156 bp of the mouse Col2 alpha 1 intron 1. We show here that four copies of a 48-bp subsegment strongly increased promoter activity in transiently transfected rat chondrosarcoma (RCS) cells and mouse primary chondroc ytes but not in 10T1/2 fibroblasts. They also directed cartilage speci ficity in transgenic mouse embryos. These 48 bp include two 11-bp inve rted repeats with only one mismatch. Tandem copies of an 18-bp element containing the 3' repeat strongly enhanced promoter activity in RCS c ells and chondrocytes but not in fibroblasts. Transgenic mice harborin g 12 copies of this 18-mer expressed luciferase in ribs and vertebrae and in isolated chondrocytes but not in noncartilaginous tissues excep t skin and brain, In gel retardation assays, an RCS cell-specific prot ein and another closely related protein expressed only in RCS cells an d primary chondrocytes bound to a 10-bp sequence within the 18-mer. Mu tations in these 10 bp abolished activity of the multimerized 18-bp en hancer, and deletion of these 10 bp abolished enhancer activity of 465 - and 231-bp intron 1 segments. This sequence contains a low-affinity binding site for POU domain proteins, and competition experiments with a high-affinity POU domain binding site strongly suggested that the c hondrocyte proteins belong to this family. Together, our results indic ate that an 18-bp sequence in Col2 alpha 1 intron 1 controls chondrocy te expression and suggest that RCS cells and chondrocytes contain spec ific POU domain proteins involved in enhancer activity.