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. 2010 Nov 26;18(5):769–782. doi: 10.1038/cdd.2010.142

Figure 3.

Figure 3

Transcriptional upregulation of parkin under ER stress is mediated by ATF4. (a) Schematic representation of the consensus ATF4-binding site, the putative ATF4-binding site within the parkin promoter and the luciferase reporter constructs cloned for the analysis described in the following: park-luc contains the putative ATF4-binding site of the parkin promoter in triplicate, mutant park-luc habors two point mutations within the putative ATF4-binding site, and ATF4RE-luc contains the confirmed ATF4-binding site of the insulin growth factor binding protein 1 (IGFBP1) promoter in triplicate. Of note, the putative binding site for ATF4 within the parkin promoter is located on the complementary strand in 5′ → 3′ direction. (b) The park-luc reporter construct is induced after ER stress. HEK293 T cells or SH-SY5Y cells were transfected with either the control luciferase reporter construct pGL3-luc (vector), the ATF4RE-luc construct containing the confirmed ATF4-binding site, the park-luc construct or the park-luc construct with a mutated ATF4 binding site (mut. park). At 8 h after transfection, cells were incubated with 1 μM thapsigargin (TG) and collected after 14 h of treatment. Shown is the fold induction of luciferase activity in stressed cells compared with the non-stressed control based on triplicates of at least three independent experiments. (c) Increased expression of ATF4 or upstream PERK induces transcription from the park-luc reporter construct. HEK293T cells were co-transfected with the ATF4RE-luc reporter plasmid or the park-luc reporter plasmid and ATF4, PERK or GFP (as a control). As a positive control, one set of cells was treated with TG as described under (b) Shown is the fold induction of luciferase activity compared with GFP-expressing control cells based on triplicates of at least three independent experiments (left panel). Expression levels of ATF4 and PERK were analyzed by immunoblotting using the anti-ATF4 pAb C-20 or the anti-myc mAb 9E10 (right panels). Notably, TG treatment (1 μM, 14 h) induced the increased expression of endogenous ATF4. Loading was controlled by re-probing the blots for β-actin. (d) A dominant-negative mutant of ATF4 (ATF4ΔN) interferes with the activation of the park-luc reporter construct in response to ER stress. HEK293T cells were co-transfected with the park-luc reporter plasmid and ATF4, ATF4ΔN, or GFP (as a control). At 8 h after transfection, cells were incubated with 1 μM TG for 14 h. Shown is the fold induction of luciferase activity in comparison with GFP-expressing control cells based on triplicates of at least three independent experiments (left panel). Expression levels of ATF4 and ATF4ΔN were analyzed by immunoblotting using the anti-ATF4 pAb C-20 (right panel). Loading was controlled by re-probing the blots for β-actin. ***P<0.001, **P<0.01, n.s.=not significant