Regulation of the Primary Open-Angle Glaucoma Gene Myocilin by PITX2

Abstract

Abstract: : Purpose: PITX2 mutations are associated with a wide spectrum of anterior segment dysgeneses (ASD) that have a high risk for developing glaucoma. In silico promoter analyses searching for PITX2 DNA-binding sites identified the primary open-angle glaucoma gene Myocilin (MYOC) promoter as a possible downstream target of PITX2 in the eye. We investigated whether PITX2 can regulate MYOC expression, and the impact of PITX2 mutation upon MYOC regulation. Methods: Co-expression of MYOC and PITX2 in the adult anterior segment was examined by RT-PCR and Northern analysis. The MYOC promoters of human, mouse, and rat were tested in luciferase reporter gene assays to assess transactivation potential by PITX2 through co-transfection into cultured and transformed non-pigmented ciliary epithelium (NPCE) cells of the anterior segment. Serial deletions and mutations of the human MYOC promoter were further tested to identify regions within the MYOC promoter necessary for transactivation by PITX2. The PITX2 Thr30Pro homeodomain mutation was also tested to examine its ability to transactivate the MYOC promoter. Electrophoretic mobility shift assays (EMSAs) with PITX2 protein and fragments of the human MYOC promoter as probes examined direct DNA-binding of the promoter by PITX2. Results: MYOC and PITX2 are co-expressed in tissues of the adult anterior ocular segment. Reporter gene assays revealed that PITX2 can transactivate the human, mouse, and rat MYOC promoters. Serial deletion and mutation of the human MYOC promoter has identified regions that are required for transactivation by PITX2, in addition to regions that are required for basal transcriptional initiation from the MYOC promoter. In addition, the Thr30Pro PITX2 mutant was unable to transactivate the MYOC promoter. EMSAs indicate that PITX2 protein was able to bind fragments of the MYOC promoter that contain putative PITX2 DNA-binding sites and regions that are important for transactivation. Conclusions: MYOC is the first putative PITX2 target gene to be described in the eye. Aberration of MYOC regulation by PITX2 mutant proteins may contribute to the development of glaucoma in ASD patients with PITX2 mutations.

Keywords: transcription factors • gene/expression • anterior segment