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. 2011 Mar 2;31(9):3295–3308. doi: 10.1523/JNEUROSCI.4540-10.2011

Figure 5.

Figure 5.

PasRE and CRE-like elements regulate hBDNF pIX induction. A, Rat primary neurons transfected with hBDNF pIX-dependent FFluc construct or indicated derivative thereof and hEF1α promoter-dependent hRluc construct, were treated with 25 mm KCl and luciferase activities were measured. Data are represented as fold induced promoter activities over activities measured from untreated control neurons. Numbers in the schematic representations of the promoters indicate nucleotide positions relative to the 3′ TSS (TSS2) of hBDNF exon IX. TSS1, 5′ transcription start site in hBDNF exon IX. 5′, 5′ part of hBDNF pIX; 3′, 3′ part of hBDNF pIX; NdeI, NdeI restriction site used to generate the pIX 3′ NdeI construct. 3′ 112bp, hBDNF pIX-luciferase construct with minimal KCl inducible sequence. mut, mutations in the PasRE and CRE-like elements. B, Alignment of the region responsible for neuronal activity-dependent induction of hBDNF pIX with the respective region in the rat promoter. Numbers indicate nucleotide positions relative to the TSS2 of hBDNF exon IX. C, EMSA showing CREB1-, USF-, and ARNT2-NPAS4-containing complexes binding to the PasRE and CRE-like element containing oligo pIX PasRE/CRE-like. Addition of unlabeled oligo or ab to the reaction and the lysates used are designated as in Figure 2D. Representative image of three independent experiments is shown. D, Rat primary neurons transfected with 3′ 112bp hBDNF pIX-dependent FFluc construct and hEF1α promoter-dependent hRluc construct together with pRC or with a construct coding the indicated protein, were left untreated or were treated for 8 h with 25 mm KCl and luciferase activities were measured. Promoter activities are presented in RLUs relative to the promoter activity measured from untreated pRC-transfected neurons that was arbitrarily set as 1. Results of at least three independent experiments with duplicate measurements are shown. Error bars represent SD. Statistical significance denoted by asterisks is between the bars indicated by lines in A and relative to promoter activity in pRC-transfected untreated control or pRC-transfected KCl-treated neurons, respectively, in D (***p < 0.001; **p < 0.01; *p < 0.05; t test).