title: Adjacent DNA sequences modulate Sox9 transcriptional activation at paired Sox sites in three chondrocyte-specific enhancer elements.
abstract: Expression of the type XI collagen gene Col11a2 is directed to cartilage by at least three chondrocyte-specific enhancer elements, two in the 5' region and one in the first intron of the gene. The three enhancers each contain two heptameric sites with homology to the Sox protein-binding consensus sequence. The two sites are separated by 3 or 4 bp and arranged in opposite orientation to each other. Targeted mutational analyses of these three enhancers showed that in the intronic enhancer, as in the other two enhancers, both Sox sites in a pair are essential for enhancer activity. The transcription factor Sox9 binds as a dimer at the paired sites, and the introduction of insertion mutations between the sites demonstrated that physical interactions between the adjacently bound proteins are essential for enhancer activity. Additional mutational analyses demonstrated that although Sox9 binding at the paired Sox sites is necessary for enhancer activity, it alone is not sufficient. Adjacent DNA sequences in each enhancer are also required, and mutation of those sequences can eliminate enhancer activity without preventing Sox9 binding. The data suggest a new model in which adjacently bound proteins affect the DNA bend angle produced by Sox9, which in turn determines whether an active transcriptional enhancer complex is assembled.
authors.affiliation: Department of Microbiology and Molecular Biology, Brigham Young University, 591 WIDB, Provo, UT 84602, USA. laura_bridgewater@byu.edu
authors.forename: Laura C
authors.initials: LC
authors.lastname: Bridgewater
authors.forename: Marlan D
authors.initials: MD
authors.lastname: Walker
authors.forename: Gwen C
authors.initials: GC
authors.lastname: Miller
authors.forename: Trevor A
authors.initials: TA
authors.lastname: Ellison
authors.forename: L Daniel
authors.initials: LD
authors.lastname: Holsinger
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Potter
authors.forename: Todd L
authors.initials: TL
authors.lastname: Jackson
authors.forename: Reuben K
authors.initials: RK
authors.lastname: Chen
authors.forename: Vicki L
authors.initials: VL
authors.lastname: Winkel
authors.forename: Zhaoping
authors.initials: Z
authors.lastname: Zhang
authors.forename: Sandra
authors.initials: S
authors.lastname: McKinney
authors.forename: Benoit
authors.initials: B
authors.lastname: de Crombrugghe
mesh_terms: D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism*; D024161:Collagen Type XI / Q000235:genetics; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay; D004742:Enhancer Elements, Genetic / Q000235:genetics*; D006609:High Mobility Group Proteins / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009841:Oligonucleotides / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics; D055758:SOX9 Transcription Factor; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
chemical_list: D024161:Collagen Type XI; D006609:High Mobility Group Proteins; D009841:Oligonucleotides; D011993:Recombinant Fusion Proteins; D055758:SOX9 Transcription Factor; C527933:Sox9 protein, mouse; D014157:Transcription Factors; D004247:DNA; D008156:Luciferases