title: Three high mobility group-like sequences within a 48-base pair enhancer of the Col2a1 gene are required for cartilage-specific expression in vivo.
abstract: 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-alpha1(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.
authors.affiliation: Department of Molecular Genetics, the University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, USA.
authors.forename: G
authors.initials: G
authors.lastname: Zhou
authors.forename: V
authors.initials: V
authors.lastname: Lefebvre
authors.forename: Z
authors.initials: Z
authors.lastname: Zhang
authors.forename: H
authors.initials: H
authors.lastname: Eberspaecher
authors.forename: B
authors.initials: B
authors.lastname: de Crombrugghe
mesh_terms: D000818:Animals; D001483:Base Sequence; D002356:Cartilage / Q000254:growth & development*; D002454:Cell Differentiation / Q000235:genetics; D019902:Chondrocytes / Q000378:metabolism; D003094:Collagen / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000032:analysis; D004622:Embryo, Mammalian / Q000166:cytology; D005314:Embryonic and Fetal Development; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D018507:Gene Expression Regulation, Developmental / Q000235:genetics*; D017930:Genes, Reporter / Q000235:genetics; D006609:High Mobility Group Proteins / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009687:Nuclear Proteins / Q000032:analysis; D011401:Promoter Regions, Genetic / Q000235:genetics; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection / Q000235:genetics
chemical_list: D004268:DNA-Binding Proteins; D006609:High Mobility Group Proteins; D009687:Nuclear Proteins; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D003094:Collagen