FMNL2 with Functions Related to the Cytoskeleton is Partially Regulated by PAX6

PURPOSE We aimed to assess whether the transcription factor PAX6 affects transcription of FMNL2. PAX6 is a transcription factor with significant roles in development of the eye and eye-related functions. FMNL2 encodes a member of the formin family of proteins and has roles in polymerization of actin and features of the cytoskeleton. The state of the cytoskeleton affects the flow of aqueous humor, disruption of which is a cornerstone of glaucoma pathology. METHODS Initially, bioinformatics were used extensively to identify FMNL2 as an appropriate candidate gene for possible targeting by PAX6. Subsequently, direct targeting of the promoter of FMNL2 by PAX6 was tested using the dual luciferase assay. The experiment was performed by cloning a promoter region of FMNL2 that contains PAX6 binding sitesupstream of a firefly luciferase gene and comparison of expression of luciferase in the presence and absence of PAX6 expression vectors in the HEK293T cell line. The effect of PAX6 on endogenous expression of FMNL2 in primary trabecular meshwork (TM) cells was assessed by real-time polymerase chain reaction. RESULTS Dual luciferase assays in HEK293T cells clearly demonstrated that PAX6 directly affects the FMNL2 promoter to increase expression of downstream sequences. However, overexpression of PAX6 in TM cells caused mild but statistically significant downregulation of endogenous FMNL2 as assessed by real-time polymerase chain reaction. CONCLUSION It is concluded that PAX6 can indeed directly affect transcription of FMNL2. However, regulation of FMNL2 expression in TM cells is complicated and not limited to the direct effects of PAX6.