Figure 2. MEF2 binds to CaRE1 and drives CaRE1-dependent gene transcription. A, Alignment of the consensus CaRF binding site (Pfenning et al., 2010), the Bdnf pIV CaRE1 element (Tao et al., 2002), and a canonical MRE (Gossett et al., 1989). Vertical lines indicate nucleotide sequences shared with CaRE1. B, MEF2 binds CaRE1. The binding of MEF2A/D heterodimers to a radiolabeled MRE probe was either not competed (−) or competed with the addition of a 10-, 100-, or 1000-fold molar excess of unlabeled MRE, CaRE1, or cCaRE. The right triangles indicate increasing concentrations of unlabeled competitor (Comp), and the arrow indicates the transcription factor–radiolabeled probe complexes. C, MEF2 activates Bdnf exon IV transcription in a CaRE1-dependent manner. A wild-type Bdnf pIV reporter plasmid (pIV-Luc) or a version bearing mutations at CaRE1 [pIV(mCaRE1)-Luc] were transfected into HEK 293T cells with mammalian expression vectors containing CaRF, MEF2A and MEF2D, CREB, or an empty vector control. The bars show the mean values from three independent experiments scaled to the vector control. *p < 0.05, pIV(mCaRE1)-Luc vs pIV-Luc. D, Endogenous MEF2 contributes to CaRE1-dependent transcription in neurons. Luciferase reporter plasmids enhanced by MRE, CaRE1, or cCaRE were transfected into cultured mouse cortical neurons along with shRNA vectors that knock down (KD) MEF2A and MEF2D. An empty shRNA vector (pSuper) was used as a control (Ctrl). The bars show the mean values from three independent experiments scaled to the vector control from each day. *p < 0.05 KD versus Ctrl.