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 express ion of MYOC/TIGR in the trabecular meshwork (TM) of the eye is considerably higher than in normals. We performed transfection, DNAse I footprinting, m utagenesis and electrophoretic mobility shift assays (EMSA) to identify ele ments responsible for the basal transcription of MYOC/TIGR in TM cells and astrocytes. Results: DNAse I footprinting experiments of the human MYOC/TIGR promoter s howed a major protected area between nt -106 to -77, which was not conserve d in the homologous region of the mouse myoc/tigr promoter. In addition, th e TATA-box was protected, as well as at least three downstream sites, inclu ding an AP-1-like sequence. Deletion of the -106 to -77 region caused a sub stantial loss of functional promotor activity in all cell types. Site-direc ted mutagenesis and EMSA experiments revealed the presence of two regulator y elements in the -106 to -77 region. Each of these cis-elements is essenti al for minimal promoter activity. The 5'-half of the region contains a sequ ence with similarities to NF-kappa B-related sites, however, binding of NF- kappa B 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 supershi fted by specific antibodies. Conclusions: Several DNA-protein binding elements contribute to a transcrip tion of MYOC/TIGR, and USF is critically required for its basal transcripti on in trabecular meshwork cells and astrocytes.