The basic helix-loop-helix differentiation factor Nex1/MATH-2 functions as a key activator of the GAP-43 gene

Abstract

Nex1/MATH-2 is a neurogenic basic Helix-Loop-Helix (bHLH) transcription factor that belongs to the NeuroD subfamily. Its expression parallels that of the GAP-43 gene and peaks during brain development, when neurite outgrowth and synaptogenesis are highly active. We previously observed a direct correlation between the levels of expression of Nex1 and GAP-43 proteins, which resulted in extensive neurite outgrowth and neuronal differentiation of PC12 cells in the absence of nerve growth factor. Since the GAP-43 gene is a target for bHLH regulation, we investigated whether Nex1 could regulate the activity of the GAP-43 promoter. We found that among the members of the NeuroD subfamily, Nex1 promoted maximal activity of the GAP-43 promoter. The Nex1-mediated activity is restricted to the conserved E1-E2 cluster located near the major transcription start sites. By electrophoretic mobility shift assay and site-directed mutagenesis, we showed that Nex1 binds as homodimers and that the E1 E-box is a high affinity binding site. We further found that Nex1 released the ME1 E-protein-mediated repression in a concentration dependent manner. Thus, the E1-E2 cluster has a dual function: it can mediate activation or repression depending on the interacting bHLH proteins. Finally, a series of N-terminal and C-terminal deletions revealed that Nex1 transcriptional activity is linked to two distinct transactivation domains, TAD1 and TAD2, with TAD1 being unique to Nex1. Together, our results suggest that Nex1 may engage in selective interactions with components of the core transcriptional machinery whose assembly is dictated by the architecture of the GAP-43 promoter and cellular environment.

Fig. 1

Transcriptional properties of the members of the NeuroD subfamily on the GAP-43 promoter. N18 cells were cotransfected with the reporter-promoter plasmid GHC (1 μg) and eukaryotic expression plasmid containing either NeuroD, NeuroD2, or Nex1 (8 μg). CAT activities are corrected for transfection efficiency and expressed as fold activation relative to the value obtained by transfection of the GHC plasmid alone. Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate. The reporter-promoter GAP-43/CAT construct (GHC) is illustrated below the graph.

Fig. 2

Nex1 activates the GAP-43 promoter through the E1–E2 cluster. (a) The proximal E1–E2 cluster of the GAP-43 promoter is sufficient for maximal Nex1-mediated regulation. N18 cells were cotransfected with the reporter-promoter plasmids (1 μg) GXC or GHC alone (open bars) or with the Nex1 expression plasmid (4 μg) (filled bars). Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate. A map of the GXC and GHC plasmids is shown on the left of the graph. (b) Nex1 also regulates the activity of the E1–E2 cluster in the context of the chimeric thymidine kinase promoter. N18 cells were cotransfected with the reporter-promoter plasmids (1 μg) G2minCAT or G4minCAT alone (open bars) or with the Nex1 expression plasmid (4 μg) (filled bars). Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate. A map of the G2minCAT and G4minCAT plasmids is shown on the left of the graph. (c) Mutation of the E1 E box alters the Nex1-mediated regulation of the GAP-43 promoter. N18 cells were cotransfected with the reporter-promoter plasmids (1 μg) GHC, GHC/E1m, or GHC/E2m alone (open bars) or with the Nex1 expression plasmid (4 μg) (filled bars). Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate. A map of the GHC/E1m and GHC/E2m plasmids is shown on the left of the graph.

Fig. 3

Nex1 binds to the E1 and E2 E-boxes as homodimers. (a) Nex1 binds to the E1 E-box with high affinity. EMSA was performed with 60 ng of purified recombinant Nex1 protein in the absence (lane 2) or presence of increasing amounts of unlabeled specific competitor (lanes 3–6) or unlabeled non-specific competitor in which the E1 E-box was mutated (lanes 7–10). Lane 1 shows the probe E1 alone. The DNA-protein complexes were resolved by native polyacrylamide gel electrophoresis. The free probe E1 is indicated by an arrow. (b) Nex1 binds poorly to the E2 E-box. EMSA was performed as described in (a) in the absence (lane 2) or presence of increasing amounts of unlabeled specific competitor (lanes 3–5) or unlabeled non-specific competitor in which the E1 E-box was mutated (lanes 6–8). Lane 1 shows the probe E2 alone. The free probe E2 is indicated by an arrow.

Fig. 4

Nex1-mediated regulation of the GAP-43 promoter is insensitive to the dominant negative regulator Id2. N18 cells were transfected with the reporter-promoter plasmid GHC alone (1 μg) or in the presence of eukaryotic expression plasmid containing either Id2 (6 μg) (gray bar) or Nex1 (4 μg) (filled bar) or a combination of both plasmids (hatched bar). CAT activities are corrected for transfection efficiency and expressed as fold activation relative to the value obtained by transfection of the GHC plasmid alone. Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate. The reporter-promoter GAP-43/CAT construct (GHC) is illustrated below the graph.

Fig. 5

Nex1 releases the ME1-mediated repression of the GAP-43 promoter in a concentration-dependent manner. N18 cells were transfected with the reporter-promoter plasmid GHC alone (open bar) or in the presence of Nex1 expression plasmid (4 μg) (filled bar) or in the presence of a constant amount of ME1 expression plasmid (8 μg) and various amounts of Nex1 expression plasmid (0–12 μg) (hatched bars). CAT activities are corrected for transfection efficiency and expressed as fold activation relative to the value obtained by transfection of the GHC plasmid alone. Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate. The reporter-promoter GAP-43/CAT construct (GHC) is illustrated below the graph.

Fig. 6

Nex1 activates the GAP-43 promoter through two distinct transactivation domains. The Nex1 mutants used for this analysis are schematically represented at the top of the figure. Both TAD1 and TAD2 domains are indicated by a shaded box, the nuclear localization domain (NL) is indicated by a hatched box and the bHLH motif as well as the stretch of 12 glutamic acid residues are indicated by open boxes. The activity of the wild-type Nex1 protein and each Nex1 mutant was challenged on the two reporter-promoter plasmids, GHC on the left and G2minCAT on the right. Each plasmid is illustrated at the bottom of the figure. N18 cells were transfected with either the reporter-promoter plasmid alone (1 μg) (open bars) or in the presence of wild-type or mutant Nex1 (4 μg) (filled bars). CAT activities are corrected for transfection efficiency and expressed as fold activation relative to the value obtained by transfection of the GHC plasmid alone. Relative CAT activities were presented as means ± SD and represented results of at least four independent experiments performed in duplicate.