Article Figures & Data
Figures
- Figure 1.
CaRE1 is required for membrane depolarization-induced activation of Bdnf promoter IV in CaRF knock-out neurons. A, Schematic of the Bdnf pIV-luciferase reporter plasmids used. The numbers indicate the position of the promoter 5′ end relative to the initiation site of transcription. pIV-Luc (Tao et al., 2002) contains the proximal 170 bp of Bdnf pIV; pIV(mCaRE1)-Luc has the CaRE1 site mutated to a sequence that does not bind CaRF. B, Mutation of the CaRE1 element leads to a reduction in membrane depolarization-induced Bdnf pIV activity in both Carf+/+ and Carf−/− cortical neurons. Single pup cultures were independently transfected and treated with KCl. n = 2 KO and 3 WT pups. *p < 0.05, pIV(mCaRE1) compared with pIV.
- 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.
- Figure 3.
Region-specific expression and calcium-dependent regulation of MEF2s. A, B, Relative expression of MEF2 family members was determined by quantitative PCR from cultured embryonic rat hippocampal (A) or cortical (B) neurons. n = 4. C, MEF2C splice variants are differentially responsive to membrane depolarization. Cultured embryonic rat cortical neurons were cotransfected with a pUAS–Luc reporter vector along with plasmids expressing Gal4 fusions of the indicated MEF2C splice variants. Δγ, A γ-containing MEF2C variant with an S396A point mutation. The bars show the mean values from four independent experiments scaled to the untreated empty vector control from each day. In all graphs, error bars represent SEM. *p < 0.05 6 h KCl versus none.
- Figure 4.
Differential regulation of endogenous Bdnf transcription by MEF2 family members in cortical neurons. A, Cultured embryonic rat cortical neurons were infected with lentiviruses containing shRNAs targeting individual MEF2 family members. Knockdown (KD) of endogenous mRNA levels was validated by quantitative PCR. MEF2 expression is normalized to levels in cells infected with the empty vector control viruses. n = 4. B, C, Cortical neurons were infected with the indicated lentiviruses then left untreated or stimulated with 55 mm KCl for 3 h. Endogenous Fos (B) or Bdnf exon IV (C) mRNA levels were determined by quantitative PCR. D, Luciferase expression from Bdnf pIV-Luc cotransfected with the indicated plasmids. Control 1, Transfected with empty pLKO.1 vector; Control 2, transfected with pTRIPZ dox-inducible MEF2C shRNAmir in the absence of doxycycline. E, Luciferase expression from the Bdnf pIV(ΔCaRE1–2xUAS)-Luc plasmid cotransfected with the indicated Gal4 expression plasmids. F, Quantitative PCR for Bdnf exon I in cortical neurons infected with the indicated lentiviruses. G, Luciferase expression from a Bdnf pI-Luc reporter plasmid (−6143) or a truncated reporter lacking the −4.8 kb upstream MEF2 binding site (−4495) contransfected with the indicated plasmids. n = 3–4. *p < 0.05 versus Control (Ctrl).
