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Z. Jing, S. P. Bhat; Sequences Immediately Upstream of the Heat Shock Element in B-Crystallin Gene Are Critical for Expression in ARPE Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):481.
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© ARVO (1962-2015); The Authors (2016-present)
The small heat shock protein B-crystallin (B) is expressed in retinal pigment epithelium in a developmentally controlled fashion. The expression of this gene has been associated with a number of neurodegenerative disorders including Alzheimer’s, Alexander’s, Parkinson’s diseases, multiple sclerosis, and age-related macular degeneration. We have previously characterized the heat shock promoter of this gene in the ocular lens. The rat B promoter contains a canonical heat shock element (-40/-54) that interacts with heat shock factor4 (HSF4) (J. Biol. Chem. 279:44497-503, 2004). In this investigation we have analyzed the import of the heat shock element (HSE) and its surrounding sequences in the expression of this gene in ARPE cells.
We have used gel-shift experiments to characterize binding activities in the nuclear extracts made from the ocular lens and ARPE cells. Specific antibodies to HSF4 were used for the characterization of the promoter-HSF4 complexes. B promoter-GFP recombinant molecules transfected into ARPE cells were used to assess the impact of various sequence manipulations on the expression of the reporter GFP; GFP expression was quantified by immunoblotting. We used both human and rat promoter sequences (about 900 bp upstream of ATG) with GFP reporters for these studies.
The rat promoter sequences showed appreciably higher activity in ARPE cells than the promoter sequences from the human B gene. In vitro, gel-shift experiments, performed with a B promoter probe (30 bp), containing the canonical HSE, demonstrated that manipulations of either the HSE or the sequences surrounding HSE result in the loss of interaction between HSF4 and the promoter probe. In vivo, using the ARPE cells in culture, as expected, manipulations of the canonical trimeric 15 bp HSE element impact expression of GFP negatively. Importantly, changes in the sequences (not part of the HSE) immediately upstream of the HSE, drastically inhibit expression of GFP.
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