Regulation of human myocilin/TIGR gene transcription in trabecular meshwork cells and astrocytes: role of upstream stimulatory factor.
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. We 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.
Kirstein
L
L
University of Erlangen-Nürnberg, Department of Anatomy, D-91054 Erlangen, Germany.
Cvekl
A
A
Chauhan
B K
BK
Tamm
E R
ER
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001483:Base Sequence; D003598:Cytoskeletal Proteins; D018983:DNA Footprinting; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I / Q000378:metabolism; D005136:Eye Proteins / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005902:Glaucoma, Open-Angle / Q000235:genetics*; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid; D014129:Trabecular Meshwork / Q000378:metabolism*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D051720:Upstream Stimulatory Factors; D000099179:Myocilin
D003598:Cytoskeletal Proteins; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D005136:Eye Proteins; D006023:Glycoproteins; D016328:NF-kappa B; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D051720:Upstream Stimulatory Factors; D000099179:Myocilin; C497181:USF1 protein, human; C497183:Usf1 protein, mouse