May 2003
Volume 44, Issue 13
ARVO Annual Meeting Abstract  |   May 2003
The UV-Responsive Elements in the Human Mimecan Promoter - Functional Characterization
Author Affiliations & Notes
  • E.S. Tasheva
    Biology Department, Kansas State University, Manhattan, KS, United States
  • G.W. Conrad
    Biology Department, Kansas State University, Manhattan, KS, United States
  • Footnotes
    Commercial Relationships  E.S. Tasheva, None; G.W. Conrad, None.
  • Footnotes
    Support  NIH Grant EY13395 to GWC and EST and NIH Grant EY00952 to GWC.
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 870. doi:
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      E.S. Tasheva, G.W. Conrad; The UV-Responsive Elements in the Human Mimecan Promoter - Functional Characterization . Invest. Ophthalmol. Vis. Sci. 2003;44(13):870.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract: : Purpose: Mammalian defense mechanisms in response to adverse effects of UV light result in induction of a number of genes. Studies on the transcriptional regulation of genes that are expressed in the eye will increase understanding of both the physiological functions of these genes in the mammalian UV response, and the molecular bases for abnormalities associated with diseases caused by exposure to UV. The purpose of this study was to determine and characterize the UV-responsive regulatory elements of the human mimecan promoter. Methods:Transcriptional activity of the promoter was evaluated, before and after UV irradiation, using transient transfections of human mimecan promoter/luciferase reporter constructs into corneal keratocytes and non-corneal cells. Site-directed mutagenesis and corresponding functional assays were used to determine the contribution of UV-responsive regions to human mimecan transcription. Co-transfection experiments were used to investigate the role of transcription factors that bind these elements in the promoter and mediate the UV response. mRNA expression was analyzed by reverse transcription- polymerase chain reaction (RT-PCR).Results:The shortest promoter construct that was strongly activated following UV-irradiation contained three initiator elements, an E-box element that is conserved between species, and the entire first intron of the human mimecan gene. Deletion of the intronic p53-binding site from this construct considerably diminished transcription and the UV response of the promoter. Surprisingly, deletion of the E-box sequence from this construct completely abolished both transcription and UV response of the promoter. The role of the E-box, and the E-box-binding protein, USF-1, in transcription and UV responses of the human mimecan promoter were confirmed by co-transfection experiments using dominant negative transcription factor, A-USF. In addition to these positive regulators, we demonstrate that the region between nucleotides -1314 and -1907 contains a transcriptional repressor site that is active in a time-dependent manner following UV-irradiation. Finally, we show that UV-irradiation results in changes in mimecan mRNA levels in bovine corneal keratocytes in a time-dependent manner. Conclusions:The human mimecan promoter contains several UV-responsive regulatory elements that are conserved between human and bovine species and include the intronic p53 DNA-binding site, the E-box in the proximal promoter, and the region between nucleotides -1314 and -1907. The E-box plays an important role in transcription and UV response of the human mimecan promoter.

Keywords: cornea: stroma and keratocytes • gene/expression • molecular biology 

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