Transcriptional control of glaucoma pathogenesis.

Abstract

Purpose : Glaucoma is recognized as a complex disease in which multiple genetic and environmental factors interact. Recently, we used a mouse model system to study the effects of Six6-His risk variant in glaucoma pathogenesis. We observed that upon experimentally increased IOP, expression of Six6 increases to directly regulate the expression of p16Ink4a, leading to enhanced senescence in RGCs and most likely causing RGC death. Our study also indicated that SIX6-His variant, despite binding the p16Ink4a regulatory regions with the same efficiency as SIX6-Asn variant, is a more potent activator of p16Ink4a expression, raising an intriguing question about the molecular difference between those two SIX6 variants. We therefore hypothesized that Six6-HIS and ASN variants interact with different transcriptional co-activators repertoire in the cell nucleus.

Methods : Full length cDNAs encoding SIX6-His (risk) and SIX6-Asn (non-risk) variants, were overexpressed in HEK293T cell line. Both proteins were tagged with HA to allow robust and comparable immunoprecipitation using known and validated HA antibodies. Washed immunoprecipitates were subjected to mass spectrometry to investigate the composition of each complex. Only the peptides with a confidence level greater than 95% were considered. The mass-spectrometry analysis for several proteins was confirmed by co-immunoprecipitation followed by Western blotting of both SIX6 variants from HEK293T extracts.

Results : Mass spectrometry revealed that several proteins involved in nucleus biology differentially interact with Six6 variants. Of particular interest are the interactions with proteins involved in nuclear organization, transcription regulation, and splicing. Co-immunoprecpitation experiments followed by Western blotting confirmed specific interactions detected by mass-spectrometry.

Conclusions : The results presented here are consistent with our hypothesis that Six6 glaucoma risk variant SIX6-HIS interacts with co-factors that are most likely important for its increased transcriptional potency. Further molecular. biochemical and in vivo analyses are focused on understanding the causative role of the newly identified Six6 co-factors in the etiology of glaucoma. This study elucidates the mechanism of how multiple pathways converge on a single key effector, the p16ink4A, to cause RGC death in glaucoma.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.