1. Proximal E-box sequence in Bdnf promoter 4 functions as a transcriptional suppressor.
Proximal E-box sequence in Bdnf promoter 4 functions as a transcriptional suppressor. A pDsRed Bdnf promoter 4 reporter plasmid having a mutated proximal E-box element was constructed by site-directed mutagenesis (see Materials and Methods). Positions of the mutated bases are shown in lowercase letters (A). Differences in pDsRed expression by transfected hippocampal neurons with Bdnf reporter promoter 4 constructs deleted for either the IS region or having a mutated E-box element. Expression was relative to the intact promoter (WT). The deletion mutant and the dinucleotide mutant had threefold higher activity than the construct with the intact Bdnf promoter. Experiments were performed five times in triplicate wells and fluorescence is expressed as mean ± SEM (n = 15). **p < 0.01 versus wild-type promoter (A). In a second series of experiments, double-stranded decoy DNAs composed of the IS region (−110 to −87), a consensus E-box, or a mutated E-box were used to transfect hippocampal neurons in culture for 24 h followed by determination of Bdnf exon 4-specific mRNA levels. Expression was determined by relative fluorescence relative to untreated control neurons, normalized for transfection efficiency. Compared with untreated neurons, the IS or consensus E-box decoy DNAs increased basal expression sevenfold and fourfold, respectively. The mutated E-box decoy showed no significant change relative to the no addition control neurons (mean ± SEM; n = 12). **p < 0.01 IS decoy versus no addition; *p < 0.05 consensus E-box versus no addition (B).
Xueying Jiang et al. J. Neurosci. 2008;28:
©2008 by Society for Neuroscience