Figure 3.
Egr4, but not Egr1, strongly induces the activity of KCC2 reporter constructs containing the Egr4KCC2 site. a, Basal activity of KCC2 promoter constructs in N2a cells. Activity of the KCC2(−1398/+42) construct, containing a 1.4 kb KCC2 promoter sequence upstream of the transcription start site, is ∼12 times stronger than the activity of the promoterless pGL3-Basic vector. Activities of KCC2(−1398/+42) and KCC2(−309/+42) constructs are similar, implying that the proximal promoter region is mostly sufficient to provide a basal level of KCC2 promoter activity. Note the significantly lower activity of the KCC2(−1398/+5445) construct, which may be explained by the presence of unknown repressor elements in the proximal part of intron-1 (Uvarov et al., 2005). b, Egr4 overexpressed in N2a cells strongly (from 15- to 22-fold) induces luciferase activity of previously described KCC2 reporter constructs (gray bars). Egr4 does not induce activity of the KCC2(−180/+42) construct lacking the Egr4KCC2 site. Egr1 expression (open bars) moderately upregulates activity of the KCC2(−1398/+5445) construct but has no significant effect on the activity of other KCC2 reporters. To take into account different basal levels of activity for KCC2 reporter constructs, we present the results as fold inductions of Egr4 or Egr1 overexpression in N2a cells in comparison with empty vector transfection. c, Simultaneous coexpression of Egr1 and Egr4 does not prevent induction of the KCC2(−1398/+42) construct. However, activity of the KCC2(−1398/+42) construct is upregulated slightly by increasing amounts of Egr1 plasmid transfected into N2a cells (*p < 0.05; Student's t test; n = 3). Error bars indicate SEM.