(A) Schematic overview of the in vitro DNA pulldown assay to determine transcription factors binding to the +3 kb enhancer region. The illustration was created with BioRender.com. (B) Gene names of the transcription factors identified in the in vitro DNA pulldown assay in two biological replicates of postnatal day 8 (P8) rat cortices. Semicolon between gene names indicates uncertainty in the peptide to protein assignment between the genes separated by the semicolons. (C) Previously published ChIP-seq experiments showing binding of different transcription factors to the +3 kb enhancer region. (D) ChIP-qPCR assay in cultured cortical neurons at 8 DIV with anti-CREB or anti-TCF4 antibody. Enrichment is shown relative to the enrichment of unrelated region (URR) with the respective antibody. The +21 kb region (downstream of the Bdnf exon VII) was used as a negative control. pIV indicates Bdnf promoter IV region. (E–H) Rat cortical neurons were transfected at 5 DIV (F) or 6 DIV (E–H) with reporter constructs where the +3 kb enhancer region was cloned in front of the luciferase coding sequence (E–G, see also Figure 2A), or with reporter constructs where the +3 kb enhancer region was cloned downstream of the Bdnf promoter I-controlled firefly luciferase expression cassette (H, see Figure 3A). Schematic representations of the used reporter constructs are shown above the graphs, with Luc designating luciferase coding sequence and pA polyadenylation sequence. At 8 DIV, neurons were left untreated (CTRL) or treated with 25 mM KCl (with 5 µM D-APV) or 50 ng/ml brain-derived neurotrophic factor (BDNF) for the indicated time, after which luciferase activity was measured. Luciferase activity is depicted relative to the luciferase activity in respectively treated cells transfected with enhanced green fluorescent protein (EGFP)-encoding plasmid and the respective +3 kb enhancer construct (E), relative to the luciferase activity in untreated cells co-transfected with control shRNA (scr) and the respective +3 kb enhancer construct (F), relative to the luciferase activity in untreated cells transfected with the respective wild-type (wt) +3 kb enhancer construct (G), or relative to the luciferase activity in untreated cells transfected with rat Bdnf promoter I construct containing no enhancer region (H). Eboxmut indicates mutation of a putative E-box element in the +3 kb enhancer region. Numbers above the columns indicate average, error bars represent SEM (n = 4 [D, CREB antibody], n = 3 [D, TCF4 antibody], n = 5–6 [E], n = 4–5 [F], n = 4 [G], and n = 7 [H] independent experiments). Statistical significance was calculated compared to the ChIP enrichment of DNA at the URR region using respective antibody (D), compared to the luciferase activity in respectively treated cells transfected with the respective +3 kb enhancer construct and EGFP (E), scr shRNA (F), or the respective wt +3 kb enhancer construct (G, H). NS: not significant. *p<0.05, **p<0.01, ***p<0.001 (paired two-tailed t-test).