Figure 3

NURR1 activates the Top IIβ promoter through NURR1 binding response elements. A. NURR1 transactivates the Top IIβ promoter in a dose-dependent manner in SH-SY5Y cells. The effect plasmid pCI-Nurr1 was co-transfected with the reporter plasmid pGL3-Top IIβ pro into SH-SY5Y cells. The luciferase activity in the presence of empty vector (pCI) was set to 1. B. Structural requirement for activation of the Top IIβ promoter. SH-SY5Y cells were transiently transfected with Nurr1 mutation construct, which contains a DNA binding deficient mutant (Nurr1^R334A). Data is shown as fold induction relative to cells transfected with empty vector. C. Point mutations that disrupt the two putative NBREs clearly reduced the ability of NURR1 to activate the promoter. Data is shown as percentage of the luciferase activity relative to cells transfected with the original promoter construct. D. Responsiveness to NURR1 is increased when either NBRE1 or NBRE2 site is converted to a consensus NBRE motif. Data is shown as fold induction relative to cells transfected with the original promoter construct. E. Down-regulation of the basal activity of the Top IIβ promoter by dnNURR1. SH-SY5Y cells were transiently transfected with the promoter construct together with increasing amounts of dnNURR1 expression plasmids. Data is shown as percentage of the basal activity of the promoter constructs. For the experiments above, the means of relative luciferase activity (induction fold) ± SEM are presented as the average values from three independent experiments. Samples are triplicate. *p < 0.05 and **p < 0.01 vs. control. F. ChIP analysis of N2a cells shows recruitment of NURR1 to the endogenous Top IIβ promoter. RT-PCR was performed with primers directed towards NBRE1, NBRE2 or a control region located 3700 base pairs downstream of Top IIβ transcriptional start site. Only the fragment contains NBRE2 was precipitated by NURR1 antibody.