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. 1998 Sep 1;18(17):6723–6739. doi: 10.1523/JNEUROSCI.18-17-06723.1998

Fig. 5.

Fig. 5.

Mutation sensitive scan of the proximal GluR2 promoter identifies both positive and negative regulatory regions.A, The promoter activity of the base construct R2(−302/+320)luc in cultured neurons (N;n = 63) and glia (G;n = 46), relative to SV40lucactivity, is shown. Data are the mean ± SEM combined from all experiments. The average raw luciferase activity recovered from neurons and glia for the unmutated R2(−302/+320)luc control was 1320 ± 120 and 31 ± 14 attomoles per μg of DNA per well, respectively. B, A series of short internal deletions (ΔA through ΔN, typically 24–31 bp long, except ΔK, ΔL, and ΔM) were generated by replacement with unique 6 bp restriction sites in the R2(−302/+320)luc GluR2 context. All deletions were generated by PCR and are shown schematically at thetop. Dashed lines indicate the sequence deleted for the indicated construct (uppercase letters).Gray and solid circles and open triangles on the line indicate the locations of primer extension, RNase protection, and RACE 5′ ends, respectively.Open boxes identify the locations of Sp1/Krox-24 and NRF-1 consensus sequences; the black box identifies the GluR2 silencer that is homologous with the RE1/NRSE silencers. The effect of each internal mutation on promoter activity in cultured cortical neurons and glia is shown. Luciferase activity was normalized to that of the unmutated R2(−302/+320)luc control construct. Note the log scale on the y-axis. The R2(−302/+320)luc-ΔB construct resulted in a significant twofold increase in promoter activity in glia. Likewise the R2(−302/+320)luc-ΔD deletion resulted in a significant increase in promoter activity in neurons. Three other deletions, R2(−302/+320)luc-ΔG, -ΔH, and -ΔI, resulted in significant decreases in promoter activity in both glia and neurons. This internal deletion scan identified both a silencer region that negatively regulates GluR2 promoter activity in glia and a larger 78 bp region that positively regulates promoter activity in both neurons and glia. Data shown are the mean ± SEM for 6–18 plasmid transfections in 21 cultured neuronal preparations and for 6–8 transfections in 18 cultured glial preparations. lucif, Luciferase. *p < 0.05 and **p< 0.01 indicate significance from the control construct by ANOVA andpost hoc Dunnett’s tests.