NUCLEIC ACIDS, PROTEIN SYNTHESIS, AND MOLECULAR GENETICS
Three High Mobility Group-like Sequences within a 48-Base Pair Enhancer of the Col2a1 Gene Are Required for Cartilage-specific Expression in Vivo *

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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-α1(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-bpCol2a1 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 largerCol2a1 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.

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This work was supported in part by National Institutes of Health Grants R01 AR42909 and P01 AR42919-02 (to B. d. C.). The University of Texas M. D. Anderson Cancer Center Core Sequencing Facility, in which DNA sequencing was performed, is supported by National Institutes of Health Grant CA16672 (NCI).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement ” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Current address: Dept. of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030.

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Recipient of an Arthritis Investigator Award from the Arthritis Foundation.