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Abstract #15924 Published in IGR 2-3
Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor
Kirstein L; Cvekl A; Chauhan BK; Tamm ERGenes to Cells 2000; 5: 661-676
BACKGROUND: Mutations in the myocilin (MYOC)/TIGR gene are responsible for autosomal-dominant juvenile primary open-angle glaucoma (POAG). In patients with non-autosomal-dominant POAG, such mutations are rare, but the expression of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. The authors performed transfection, DNAse I footprinting, mutagenesis and electrophoretic mobility shift assays (EMSA) to identify elements responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes. RESULTS: DNAse I footprinting experiments of the human MYOC/TIGR promoter showed a major protected area between nt -106 to -77, which was not conserved in the homologous region of the mouse myoc/tigr promoter. In addition, the TATA-box was protected, as well as at least three downstream sites, including an AP-1-like sequence. Deletion of the -106 to -77 region caused a substantial loss of functional promotor activity in all cell types. Site-directed mutagenesis and EMSA experiments revealed the presence of two regulatory elements in the -106 to -77 region. Each of these cis-elements is essential for minimal promoter activity. The 5'-half of the region contains a sequence with similarities to NF-kappaB-related sites, however, binding of NF-kappaB could not be confirmed by EMSA. The 3'-half contains a canonical E-box sequence. EMSA experiments showed that the upstream regulatory factor (USF) was binding to the E-box sequence and that the binding can be supershifted by specific antibodies. CONCLUSIONS: Several DNA-protein binding elements contribute to a transcription of MYOC/TIGR, and USF is critically required for its basal transcription in trabecular meshwork cells and astrocytes.
Dr. L. Kirstein, University of Erlangen-Nürnberg, Department of Anatomy, D-91054 Erlangen, Germany
Classification:
1.2 Population genetics (Part of: 1 General aspects)
