title: Analysis of the zebrafish sox9b promoter: Identification of elements that recapitulate organ-specific expression of sox9b.
abstract: The SRY-related high-mobility box 9 (SOX9) gene is expressed in many different tissues. To better understand the DNA elements that control tissue-specific expression, we cloned and sequenced a 2.5 kb fragment lying 5' to the zebrafish sox9b gene transcriptional start site. Three regions of this clone contained stable secondary structures that hindered cloning, sequencing, and amplification. This segment and smaller fragmentswere inserted 5' of an EGFP reporter and transgenic fish were raised with the different reporters. Reporter expression was also observed in embryos directly injected with the constructs to transiently express the reporter. Heart expression required only a very short 5' sequence, as a 0.6 kb sox9b fragment produced reporter expression in heart in transgenic zebrafish, and transient experiments showed heart expression from a minimal sox9b promoter region containing a conserved TATA box and an EGR2 element (-74/+29 bp). Reporter expression in transgenic skeletal muscle was consistently lower than in other tissues. Jaw, brain, and notochord expression was strong with the full-length clone, but was dramatically reduced as the size of the fragment driving the reporter decreased from approximately 1.8 to 0.9 kb. The 2.5 kb region 5' of the sox9b contained 7 conserved non-coding elements (CNEs) that included putative hypoxia inducible factor 1α (HIF1α), CAAT box (CCAAT), early growth response protein 2 (EGR2), and core promoter elements. While a synthetic fragment containing all 7 CNEs produced some degree of reporter expression in muscle, jaw, heart and brain, the degree of reporter expression was considerably lower than that produced by the full length clone. These results can account for the tissue-specific expression of sox9b in the developing zebrafish.
authors.affiliation: Molecular and Environmental Toxicology Center, School of Medicine and Public Health, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA. Electronic address: flpburns@gmail.com.
authors.forename: Felipe R
authors.initials: FR
authors.lastname: Burns
authors.affiliation: Molecular and Environmental Toxicology Center, School of Medicine and Public Health, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Kevin A
authors.initials: KA
authors.lastname: Lanham
authors.affiliation: Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA; Department of Biomolecular Chemistry, School of Medicine and Public Health, University of Wisconsin, 420 Henry Mall, Madison, WI 53705, USA; Immunoassay Research and Development, Beckman Coulter Inc., 1000 Lake Hazeltine Dr., Chaska, MN 55318, USA.
authors.forename: Kong M
authors.initials: KM
authors.lastname: Xiong
authors.affiliation: Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Alex J
authors.initials: AJ
authors.lastname: Gooding
authors.affiliation: Molecular and Environmental Toxicology Center, School of Medicine and Public Health, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Richard E
authors.initials: RE
authors.lastname: Peterson
authors.affiliation: Molecular and Environmental Toxicology Center, School of Medicine and Public Health, University of Wisconsin, 1300 University Avenue, Madison, WI 53706, USA; Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin, 777 Highland Avenue, Madison, WI 53705, USA.
authors.forename: Warren
authors.initials: W
authors.lastname: Heideman
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001921:Brain / Q000254:growth & development / Q000378:metabolism; D004625:Embryo, Nonmammalian; D018507:Gene Expression Regulation, Developmental; D006321:Heart / Q000254:growth & development; D007568:Jaw / Q000378:metabolism; D018482:Muscle, Skeletal / Q000254:growth & development / Q000378:metabolism; D009672:Notochord / Q000254:growth & development / Q000378:metabolism; D009928:Organ Specificity / Q000235:genetics*; D011401:Promoter Regions, Genetic; D055758:SOX9 Transcription Factor / Q000096:biosynthesis / Q000235:genetics*; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development; D029961:Zebrafish Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D055758:SOX9 Transcription Factor; C504919:Sox9b protein, zebrafish; D029961:Zebrafish Proteins
keywords: CAAT box; CCAAT; CNE, conserved non-coding elements; CPE, core promoter element; EGFP, enhanced green fluorescent protein; EGR2, early growth response protein 2; HIF1α, hypoxia inducible factor 1α; SOX9, SRYrelated high-mobility box 9; TSS, transcriptional start site