PURPOSE: To determine the existence of a relatively higher abundance of potential TFs in glaucomatous trabecular meshwork (TM) that may bind putative promoter regions and affect cochlin protein expression in glaucomatous compared to normal TM. METHODS: Combinatorial bioinformatics and biochemical analyses, using human glaucomatous and normal donor tissue (n = 4 each). Biochemical analysis included electrophoretic mobility shift assays (EMSAs), filter binding assays (FBAs), coupled in vitro transcription-translation (TNT) assays and promoter mutation analysis. RESULTS: Combinatorial bioinformatics and biochemical analyses revealed the existence of a higher abundance of TFs in glaucomatous than in normal TM nuclear extracts. The evidence of a relatively high abundance of TFs, leading to increased expression of cochlin predicted by bioinformatic and biochemical analyses (EMSA and FBA), was further supported by TNT and promoter mutation TNT assays. CONCLUSIONS: These results support the finding that the observed increased cochlin expression in glaucomatous TM is due to relative elevated abundance of TFs. The results also demonstrate the utility of combinatorial bioinformatic and biochemical analyses for genes with uncharacterized promoter regions.
PURPOSE: To determine the existence of a relatively higher abundance of potential TFs in glaucomatous trabecular meshwork (TM) that may bind putative promoter regions and affect cochlin protein expression in glaucomatous compared to normal TM. METHODS: Combinatorial bioinformatics and biochemical analyses, using humanglaucomatous and normal donor tissue (n = 4 each). Biochemical analysis included electrophoretic mobility shift assays (EMSAs), filter binding assays (FBAs), coupled in vitro transcription-translation (TNT) assays and promoter mutation analysis. RESULTS: Combinatorial bioinformatics and biochemical analyses revealed the existence of a higher abundance of TFs in glaucomatous than in normal TM nuclear extracts. The evidence of a relatively high abundance of TFs, leading to increased expression of cochlin predicted by bioinformatic and biochemical analyses (EMSA and FBA), was further supported by TNT and promoter mutation TNT assays. CONCLUSIONS: These results support the finding that the observed increased cochlin expression in glaucomatous TM is due to relative elevated abundance of TFs. The results also demonstrate the utility of combinatorial bioinformatic and biochemical analyses for genes with uncharacterized promoter regions.
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