Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo

To understand the molecular mechanisms by which mesenchymal cells differentiate into chondrocytes, we have used the gene for an early and abundant marker of chondrocytes, the mouse pro-alpha 1(II) collagen gene (Col2a1), to delineate a minimal sequence needed for chondrocyte-specific expression and to identify the DNA-binding proteins that mediate its activity, We show here that a 48-base pair (bp) Col2a1 intron 1 sequence specifically targets the activity of a heterologous promoter to chondrocytes in transgenic mice. Mutagenesis studies of this 48-bp element identified three separate sites (sites 1-3) that were essential for its chondrocyte-specific enhancer activity in both transgenic mice and transient transfections. Mutations in sites 1 and 2 also severely inhibited the chondrocyte-specific enhancer activity of a 468-bp Col2a1 intron 1 sequence in vivo, SOX9, an SRY-related high mobility group (HMG) domain transcription factor, was previously shown to bind site 3, to bend the 48-bp DNA at this site, and to strongly activate this 48-bp enhancer as well as larger Col2a1 enhancer elements. All three sites correspond to imperfect binding sites for HMG domain proteins and appear to be involved in the formation of a large chondrocyte-specific complex between the 48-bp element, Sox9, and other protein(s), Indeed, mutations in each of the three HMG-like sites of the 48-bp element, which abolished chondrocyte-specific expression of reporter genes in transgenic mice and in transiently transfected cells, inhibited formation of this complex, Overall our results suggest a model whereby both Sox9 and these other proteins bind to several HMG-like sites in the Col2a1 gene to cooperatively control its expression in cartilage.