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C. Colitz, V.J. Kuonen; Use of a Protein Array to Identify Transcription Factor Interactions with TERT in Cataractous and Normal Lens Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):4500.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Telomerase activity is associated with rapidly proliferating cell types including tumors, germ-line cells and some stem cells. Lens epithelial cells (LEC) are not highly proliferative but still have a significant amount of telomerase activity and cataractous LEC have almost three times more than normal LEC. The promoter of the catalytic subunit of telomerase (TERT) has numerous transcription factor (TF) sites, therefore we were interested in determining if there were differences in TF interactions with TERT between cataractous and normal LEC. Methods: The TranSignal TM TF-TF Interaction Array I from Panomics was used to screen for 54 TF-protein interactions. Briefly, nuclear protein was extracted from a normal and a cataractous canine anterior lens capsule and quantified by Bradford assay. Each sample was then incubated with the biotin-labeled, double-stranded oligonucleotide probes provided, allowing the TF cis-elements to bind the TERT protein in the sample extracts. An immunoprecipitation was then performed using an antibody to TERT (Calbiochem) in order to pull out the TF cis-elements interacting with TERT. Non-specific binding was then washed away and then cis-elements bound to TERT and anti-TERT antibody were then eluted and hybridized to the TranSignal Array membrane (1 membrane per sample and a negative control). The membranes are spotted with 54 different TF consensus sequences and direct comparisons of spot intensity were made between the two sample blots to identify differences in TF interactions. Results: Twelve TFs that interacted with TERT were up-regulated and three were down-regulated in the cataractous LEC compared to normal LEC. The up-regulated TFs were AP-1, AP-2, ERE (estrogen response element), IRF-1 (interferon regulatory factor), NFkB, p53, GAS/ISRE, NF-1, CREB, Pbx-1, Smad3/4, and TFIID. The down-regulated TFs were TR (thyroid hormone receptor), PPAR (peroxisome proliferator-activated receptor), and MEF2. Conclusions: Previous studies have found that AP-1, IRF-1, p53, and CREB play varying roles in lens physiology. AP-1 is up-regulated in lens wound repair, p53 is strongly expressed in the LEC nuclei, and CREB plays a role in crystallin regulation. The other TFs may play strong roles in lens physiology and biochemistry and need to be investigated. The results of this study will be confirmed using super gel shift assays.
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