Although, LEDGF is present in the nucleus of most unstressed cells, oxidative- and heat-stress elevate LEDGF levels. Unstressed cells transfected to over-expressing LEDGF also up-regulated Hsp27, alphaB-crystallin, and AOP2, and these cells acquired a strong resistance against environmental stresses. Stress-induced elevation of LEDGF may be a basic mechanism by which cells increase their tolerance to environmental stress. In lens epithelial cells (LECs) and cos7 cells, stress elevates not only LEDGF but also chaperones and other stress-related proteins (Hsp27, alphaB-crystallin and AOP2). We hypothesized that LEDGF bound to and up-regulated stress-related genes. We have found considerable evidence in support of this hypothesis that LEDGF bound to the stress response elements (STRE; consensus sequence A/TGGGGA/T) and the heat shock element (HSE; consensus sequence nGAAn) to up-regulate stress-related genes. (See Appendices 8, 9). Among the up-regulated stress-related genes, Hsp27, alphaB-crystallin, and AOP2 are most relevant to cataractogenesis. Hsp27 and alphaB-crystallin, both chaperone proteins, are essential for refolding denatured proteins. AOP2, a relatively new antioxidant protein, protects cells from oxidative stress. To increase our understanding of how LECs resist stress, we have focused our study on the up-regulation of stress-related genes by LEDGF. I have selected the genes for Hsp27, alphaB-crystallin and AOP2 and will study the interaction between their promoter elements and LEDGF and heat shock transcriptional factor1 (HSF1). In addition, I will study the functional role of AOP2 and its relationship to LEDGF in the context of age related cataract. This application contains three Specific Aims; 1) To characterize and define the DNA-binding domains of LEDGF, their binding affinity, functional interaction(s) and transactivation potential to two distinct regulatory elements (HSE and STRE) of the Hsp27 and AOP2 gene promoter. 2) To understand the functional significance of the interactions between HSE-HSF1 and HSE-LEDGF in the normal (unstressed) cells and in the stress-activation of the Hsp27, alphaB-crystallin and AOP2 genes. 3) To determine transcriptional regulation of AOP2 by LEDGF and the molecular basis of antioxidant potency of AOP2 under oxidative stress. This application will provide novel insights into the mechanisms by which stress-response genes are regulated by LEDGF. It will also expand our understanding of roles of these genes and LEDGF in survival of cells under stress. In particular, our studies in LECs may increase our understanding of age-related cataractogenesis and means of slowing this blinding degenerative process.
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