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Potential for Transcriptional Upregulation of Cochlin in Glaucomatous Trabecular Meshwork: a Combinatorial Bioinformatic and Biochemical Analytical Approach

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Potential for Transcriptional Upregulation of Cochlin in Glaucomatous Trabecular Meshwork: a Combinatorial Bioinformatic and Biochemical Analytical Approach Potential for Transcriptional Upregulation of Cochlin in Glaucomatous Trabecular Meshwork: A Combinatorial Bioinformatic and Biochemical Analytical Approach Renata G. Picciani, Anthony Diaz, Richard K. Lee, and Sanjoy K. Bhattacharya PURPOSE. To determine the existence of a relatively higher Cochlin, a secretory extracellular matrix (ECM) protein of abundance of potential TFs in glaucomatous trabecular mesh- unknown function, was identified by proteomic analyses to be work (TM) that may bind putative promoter regions and affect differentially expressed in glaucomatous compared with nor- cochlin protein expression in glaucomatous compared to nor- mal TM.2 Cochlin is the product of the COCH gene3 located on mal TM. human chromosome 14, region q12-13.3,4 The cochlin protein METHODS. Combinatorial bioinformatics and biochemical anal- sequence is highly conserved, with 94% and 79% amino acid ϭ identity with human to mouse and chicken sequences, respec- yses, using human glaucomatous and normal donor tissue (n 5 4 each). Biochemical analysis included electrophoretic mobil- tively. Cochlin contains a short signal peptide, an N-terminal factor C homology, and two von Willebrand factor A-like do- ity shift assays (EMSAs), filter binding assays (FBAs), coupled in 2,4 vitro transcription–translation (TNT) assays and promoter mu- mains. In situ hybridization has shown that cochlin mRNA is tation analysis. expressed in the TM, suggesting that the protein is likely expressed and deposited locally.2 RESULTS. Combinatorial bioinformatics and biochemical analy- Elevated IOP is a significant risk factor for optic nerve ses revealed the existence of a higher abundance of TFs in damage. Changes in fluid dynamics and incremental fluctua- glaucomatous than in normal TM nuclear extracts. The evi- tions in IOP results in stress and stretch on TM cells and are dence of a relatively high abundance of TFs, leading to in- thought to trigger early biochemical responses.6 Stress- and creased expression of cochlin predicted by bioinformatic and stretch-induced modulation of protein expression are mediated biochemical analyses (EMSA and FBA), was further supported by transcription factors (TFs).6,7 by TNT and promoter mutation TNT assays. Increased cochlin expression has been reported in the TM8 CONCLUSIONS. These results support the finding that the ob- and in the inner ear4; however, the promoter region of cochlin served increased cochlin expression in glaucomatous TM is and the details of cochlin gene expression remain to be char- due to relative elevated abundance of TFs. The results also acterized. Deciphering mechanisms that lead to transcriptional demonstrate the utility of combinatorial bioinformatic and bio- regulation of cochlin expression in the TM is critical for un- chemical analyses for genes with uncharacterized promoter derstanding the role cochlin may play in glaucoma’s pathogen- regions. (Invest Ophthalmol Vis Sci. 2009;50:3106–3111) DOI: esis. We used a combinatorial approach of bioinformatics and 10.1167/iovs.08-3106 molecular and biochemical analyses to determine whether an increased abundance of transcription factors with the potential laucoma is a group of irreversible blinding eye diseases to bind and enhance transcription in the promoter region of Gassociated with optic neuropathy. Primary open-angle cochlin was present in nuclear extracts of glaucomatous TM glaucoma (POAG) is often associated with elevated intraocular compared with the control. pressure (IOP), which is due to an imbalance between aqueous humor production and outflow in the anterior chamber of the eye.1 Aqueous humor is a clear liquid produced by the ciliary MATERIAL AND METHODS epithelium that exits through the trabecular meshwork (TM) after bathing the anterior segment structures, such as the Tissue Procurement and Preparation of cornea and lens, with nutrients. Aqueous outflow is believed to Nuclear Extracts encounter increased resistance at the level of the TM in glau- Glaucomatous and normal control eyes were obtained from the Na- coma. The mechanisms that impede aqueous outflow elevate tional Disease Research Institute (Philadelphia, PA) and the Lions Eye IOP are poorly understood. Bank (Miami, FL), respectively. The eyes had been enucleated within 10 hours of death and placed in a moisture chamber at 4°C and transported. They were dissected within 48 hours, and the TM was From the Bascom Palmer Eye Institute, University of Miami Miller carefully excised for study. The available details of the donor were School of Medicine, Miami, Florida. recorded. According to available information, all glaucomatous donor Supported by National Institutes of Health Grants R01 EY16112, eyes had POAG (see Supplementary Table S1; all Supplementary Tables K08 EY016775, and P30 EY014801; a career development award (SKB) are online at http://www.iovs.org/cgi/content/full/50/7/3106/DC1). and an unrestricted grant to the University of Miami from Research to Prevent Blindness; and the Howard Hughes Medical Institute’s scholar Bioinformatic Analyses program (AD). Submitted for publication November 3, 2008; revised November The human cochlin upstream promoter gene region was analyzed up 30, 2008; accepted March 12, 2009. to 5000 bp upstream of the translational start site (ATG; accession Disclosure: R.G. Picciani, None; A. Diaz, None; R.K. Lee, None; number BC007230; National Center for Biotechnology Information S.K. Bhattacharya, None [NCBI], Bethesda, MD). The cochlin DNA sequence was obtained from The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertise- the UCSC genome browser (http://genome.ucsc.edu/ provided in the ment” in accordance with 18 U.S.C. §1734 solely to indicate this fact. public domain by the University of California at Santa Cruz). Putative Corresponding author: Sanjoy K. Bhattacharya, Bascom Palmer TF binding sites were identified with commercial software (MatInspec- Eye Institute, University of Miami, Miller School of Medicine, 1638 NW tor; Matrix Family Library Version 6.3; Genomatix, Munich, Germany). 10th Avenue, Miami, FL 33136; [email protected]. The analysis parameters used have been provided in respective tables. Investigative Ophthalmology & Visual Science, July 2009, Vol. 50, No. 7 3106 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 09/27/2021 IOVS, July 2009, Vol. 50, No. 7 Cochlin Promoter Region TF-Binding Analyses 3107 TABLE 1. Relative Abundance of Select Transcription Factors According to the Filter Binding Assay for Consensus Binding Sites in the Cochlin Upstream Region Reported Association with Oligonucleotide Filter Binding Assay Sequences (Consensus Ant. Transcription Factor Binding Sites) Glaucoma TM Chamber Eye Normal Glaucomatous Fork head–related activator-3 (FoxC1) ataaaGTAAaaaaagac ϩϩϩϩ25.4 Ϯ 0.7 17.6 Ϯ 2.1 aaaaaGTAAaaaatgag ϩϩϩϩ22.0 Ϯ 1.0 18.3 Ϯ 0.5 gccatGAAAataaacat ϩϩϩϩ23.7 Ϯ 0.6 17.3 Ϯ 0.5 Paired domain and homeodomain (Pax6)* cggcgacttCCAGctccgc ϩϩϩϩ29.2 Ϯ 2.8 29.5 Ϯ 2.3 cccgctgctCCAGgccagc ϩϩϩϩ25.8 Ϯ 0.7 24.7 Ϯ 0.6 POU-IV protein (Brn3) ctatgatagATTAtagagc ϩϩ26.8 Ϯ 1.7 27.8 Ϯ 2.0 ataatagTAATtaataaca ϩϩ25.4 Ϯ 0.7 27.1 Ϯ 1.0 ttgttatTAATtactatta ϩϩ25.4 Ϯ 0.7 32.2 Ϯ 2.9 ttcatctTAATtattttgt ϩϩ24.7 Ϯ 0.6 30.9 Ϯ 1.8 HNF-3/Fkh homolog 1 (FoxQ1) cctataTAAActaagag ϩϩ 22.7 Ϯ 2.1 28.8 Ϯ 1.7 acaataTAAActttttc ϩϩ 26.8 Ϯ 1.7 31.9 Ϯ 1.8 Nuclear factor (erythroid-derived 2)-like 2, gcatagttTTGActctgccaaatca ϩϩ23.4 Ϯ 1.5 27.8 Ϯ 0.7 (Nrf2) ttcagtgaGTGAtttggcagagtca ϩϩ26.8 Ϯ 1.7 30.2 Ϯ 1.3 Homeobox TAAT motif-binding transcription aataTAATtggtctggg ϩϩ25.8 Ϯ 1.7 31.2 Ϯ 2.9 factor (Barx2) ttatTAATtactattat ϩϩ25.8 Ϯ 0.7 28.8 Ϯ 0.7 caaaTAATgaggccggg ϩϩ24.8 Ϯ 1.6 30.5 Ϯ 2.3 atctTAATtattttgtt ϩϩ24.1 Ϯ 1.1 29.2 Ϯ 1.2 aaaaTAATtaagatgaa ϩϩ26.1 Ϯ 1.2 29.1 Ϯ 1.0 ttttTAATggttacaga ϩϩ24.7 Ϯ 0.6 29.5 Ϯ 0.8 tataTAATtaggaagag ϩϩ28.5 Ϯ 2.3 30.9 Ϯ 3.4 Association of a TF in the literature with glaucoma. TM, anterior chamber or eye is represented by plus sign. * Several binding sites were identified as being present in the cochlin promoter region (Supplementary Table S3); however, the two oligonucleotide sequences used were arbitrarily selected for filter binding or gel mobility shift assays. TFs related to the eye (see Supplementary Table S2) were further antibodies against Nrf2, FoxC1, FoxQ1 (cat. nos. ART38754_T100, investigated for their correlation with glaucoma, the TM, the anterior ARP32300_T100, ARP39754_T100; Aviva Systems Biology, San Diego, chamber, or the eye, as reported in the literature and for its expression CA, respectively), and Brn3a (cat. no. AB5945; Chemicon Inc., Te- in the eye per the UniGene database (http://www.ncbi. mecula, CA), as well as a goat polyclonal antibody against Brn3b (cat. nlm.nih.gov/UniGene; provided in the public domain by NCBI; see no. sc-6026; Santa Cruz Biotechnology Inc., Santa Cruz, CA) were used. Supplementary Table S3). Tissue-specific associations of TFs in putative The Pax6 protein was detected using previously published rabbit cochlin promoter regions for ear, brain, central nervous system (CNS), antisera against a 17-residue C-terminal mouse Pax6 peptide.9,10 All embryonic tissue and liver were also analyzed (MatInspector; Geno- primary antibodies were detected with the appropriate horseradish matix), and a list of tissue-specific TFs was generated (not shown). peroxidase–conjugated secondary antibodies and ECL (cat. no. 32106; Only those TFs present in at least two search terms were investigated Pierce Biotechnology). further. For the selected TFs, 5Ј biotin end-labeled oligonucleotide sequences were generated for the relevant consensus binding sites as Gel Mobility Shift Experiments well as for their respective complementary sequences. These results were confirmed and/or cross-validated with other bioinformatic Web- The nonradioactive light shift chemiluminescent electrophoretic mo- based programs including the International HapMap Project (www.
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