Glaucoma is the leading cause of irreversible blindness worldwide, yet its etiology is poorly understood. Elevated intraocular pressure (IOP), its major risk factor, is caused by impaired aqueous humour drainage through the trabecular meshwork (TM). Primary Open Angle Glaucoma (POAG) is the most prevalent type of glaucoma. However, mutations in five POAG causing genes account for <10% of POAG cases. The combined effect of several genes and gene-environment interactions are expected to be important in the etiology of POAG in patients without mutations in said genes. FOXC1 (forkhead box c1) is a transcription factor with a crucial role in the differentiation of ocular tissues. Additionally, mutations in FOXC1 causes Axenfeld-Rieger Syndrome (ARS), a disorder characterized by anterior eye segment defects and systemic anomalies. Approximately 50% of ARS patients secondarily develop glaucoma, and patients with FOXC1 mutations are yet more likely to develop glaucoma. We earlier identified 849 genes whose mRNA levels were affected by FOXC1 knock-down in whole genome microarray gene expression analysis in primary human trabecular cell lines. Among FOXC1 direct targets, there is MEIS2, an important transcription factor in ocular development. Here, we attempted to expand the genetic network relevant to trabecular meshwork functions that include FOXC1 by identification of a potentially glaucoma relevant gene that is indirectly affected by FOXC1 and directly affected by MEIS2. The target gene studied was OLFM3 that encodes Olfactomedin 3 which interacts with Myocillin, both secreted proteins, and mutations in MYOC lead to glaucoma.

Initially, OLFM3 was identified as an appropriate possible target gene for MEIS2 by bioinformatics tools. Subsequently, using dual luciferase assay, direct targeting of the promoter of OLFM3 by MEIS2 was tested. The experiment was performed by cloning a promoter region of OLFM3 containing MEIS2 binding sites upstream of a firefly luciferase gene and comparison of expression of the luciferase in the presence and absence of MEIS2 expression vectors in the HEK293T cell line.

Dual luciferase assays in HEK293T cells showed that MEIS2 directly affects the OLFM3 promoter and increases the expression of downstream sequences.

MEIS2 can indeed directly affect the transcription of OLFM3