Figure 5.

MEF2 cooperates with CREB to mediate BDNF-induced NT-3 transcription. A, The NT-3 promoter contains a CREB-binding sequence. Extracts from cerebellar granule neurons (CGN; P6 + 3DIV) were incubated in the presence or absence a 30 bp oligonucleotide containing the putative NT-3 CRE, with or without a 10-fold excess of the wild-type oligonucleotide (10x WT CRE), an oligonucleotide containing a mutant CRE (10x Mut CRE), or a polyclonal antibody against either CREB or MEF2A. FP, Free oligonucleotide probe. B, The CRE is required for BDNF-induction of NT-3 transcription in response to BDNF. An NT-3-luciferase reporter gene containing nucleotides -1087 to +91 of the NT-3 promoter with either the wild-type CRE (WT) or a point mutation of the CRE (Mut CRE) that abolished CREB binding in vitro was introduced to P6 + 3DIV granule neurons and analyzed as in Figure 4C. BDNF induced the wild-type -1087 NT-3-luciferase reporter significantly (ANOVA; p < 0.005; n = 6) but not the Mut CRE -1087 NT-3-luciferase reporter gene. C, Dominant-negative CREB prevents BDNF induction of NT-3. Granule neurons were transfected with the -1087 NT-3-luciferase reporter gene together with an expression plasmid encoding CREBM1 or a control plasmid. Transfected cultures were analyzed as in Figure 4C. BDNF induced the NT-3 promoter significantly in control-transfected cultures (ANOVA; p < 0.0005; n = 3) but not in CREBM1-expressing granule neurons. D, The ERK1/2 pathway is necessary for BDNF-induced NT-3 transcription. Newly generated granule neurons (P6 + 3DIV) were transfected as in Figure 4C with the -1087 NT-3-luciferase reporter gene and an expression vector encoding dominant-negative MEK1 (MEK1KA97) or its control vector and analyzed as in Figure 4C. BDNF significantly induced the NT-3 reporter in vector-transfected cultures (ANOVA; p < 0.05; n = 3) but not in MEK1KA97-expressing granule neurons.