The Ets transcription factor GABP is required for postsynaptic differentiation in vivo

Abstract

At chemical synapses, neurotransmitter receptors are concentrated in the postsynaptic membrane. During the development of the neuromuscular junction, motor neurons induce aggregation of acetylcholine receptors (AChRs) underneath the nerve terminal by the redistribution of existing AChRs and preferential transcription of the AChR subunit genes in subsynaptic myonuclei. Neural agrin, when expressed in nonsynaptic regions of muscle fibers in vivo, activates both mechanisms resulting in the assembly of a fully functional postsynaptic apparatus. Several lines of evidence indicate that synaptic transcription of AChR genes is primarily dependent on a promoter element called N-box. The Ets-related transcription factor growth-associated binding protein (GABP) binds to this motif and has thus been suggested to regulate synaptic gene expression. Here, we assessed the role of GABP in synaptic gene expression and in the formation of postsynaptic specializations in vivo by perturbing its function during postsynaptic differentiation induced by neural agrin. We find that neural agrin-mediated activation of the AChR epsilon subunit promoter is abolished by the inhibition of GABP function. Importantly, the number of AChR aggregates formed in response to neural agrin was strongly reduced. Moreover, aggregates of acetylcholine esterase and utrophin, two additional components of the postsynaptic apparatus, were also reduced. Together, these results are the first direct in vivo evidence that GABP regulates synapse-specific gene expression at the neuromuscular junction and that GABP is required for the formation of a functional postsynaptic apparatus.

Fig. 1.

Structure of GABP α and β subunits and expression in COS-7 cells. A, Scheme of the α and β subunits of GABP and the dominant-negative construct. The black region (aa 318–399) indicates the Ets-related DNA binding domain of GABPα. The region between aa 318–454 is necessary for the binding to GABPβ. In GABPβ, the gray region (aa 1–130) represents four tandem repeats of a Notch/ankyrin motif required for heterodimerization with GABPα. The black bar indicates the approximate location of a nuclear localization sequence. The hatched region (aa 341–370) represents the transactivation domain. In the dominant-negative mutant (GABPβ^DN) used in this study, the last 52 amino acids of GABPβ were deleted. B, Intracellular anti-myc staining of COS cells transfected with myc-tagged GABPα constructs. GABPα is localized in the cytoplasm (left). Upon cotransfection with GABPβ^DN, GABPα accumulates in the cell nucleus (right). This indicates that the truncated GABPβ^DN protein dimerizes with the α subunit and translocates into the cell nucleus. Scale bar, 20 μm.

Fig. 2.

Agrin induces the expression of an AChRε subunit reporter construct via a GABP-dependent mechanism in vivo. A, Diagram of the AChRε subunit reporter construct used in this study. A fragment of the ε subunit gene (gray) extending 216 bp upstream from the transcription start site drives the expression of a luciferase gene. This fragment contains two E-boxes (E), putative binding sites for basic helix-loop-helix myogenic factors, and one N-box (N). B, In each individual experiment, luciferase activity of the AChRε reporter construct injected alone was set as 100% (first column). Coinjection of expression constructs encoding full-length neural agrin (cAgrin_7A4B8) increases luciferase activity more than threefold (second column; p < 0.01). This increase is abolished by expression of the dominant-negative mutant of GABPβ (third column;p < 0.01). Data represent mean ± SDs of three independent injections (3 injected rats). Note that, in each individual injection, luciferase activity was normalized to the β-galactosidase activity derived from the coinjected NLS-LacF construct. The effect of neural agrin and GABPβ^DN is specific for the AChRε promoter because a muscle creatine kinase promoter construct was not affected by either condition (reporter alone, 100%; reporter plus cAgrin_7A4B8, 88.3%; reporter plus cAgrin_7A4B8 plus GABPβ^DN, 87.2%).

Fig. 3.

Whole-mount confocal views of cDNA-injected muscle fibers. A, Two representative examples of control muscle fibers that were injected with a construct encoding NLS-LacZ, full-length neural agrin and NLS-GFP. The injected fibers are identified by the presence of GFP-positive nuclei (GFP). AChR clusters are stained with rhodamine_α-bungarotoxin (Rh_α-bgt). Many injected fibers form AChR clusters in response to neural agrin and aggregates of GFP-positive nuclei are found underneath the AChR clusters (arrowheads). B, Two representative examples of muscle fibers expressing the dominant-negative mutant of GABPβ, neural agrin, and NLS-GFP. GFP-positive muscle fibers are devoid of AChR clusters and aggregates of myonuclei. Note that, in neighboring noninjected muscle fibers, AChR aggregates were formed (asterisks). The fluorescence spots that appear in both channels are unspecific staining. Scale bar, 50 μm.

Fig. 4.

Quantification of the influence of GABPβ^DN on AChR clustering. A,B, Quantitative assessment of the effect of GABPβ^DN in one individual cDNA injection experiment. Histograms of the number of GFP-positive fibers that formed AChR clusters (C/GFP; yellow bars), GFP-positive muscle fibers (GFP; green line), and the total number of fibers that formed AChR aggregates (Ctot; red line) counted on 14-μm-thick consecutive cross-sections through the entire injection sites. For illustration, a camera lucida drawing of section 37 (#37) is shown in A. In this particular section, 16 GFP-positive muscle fibers (GFP) were counted, and seven fibers formed AChR clusters (Ctot). Five of these fibers were also GFP-positive (C/GFP). Scale bar, 50 μm. C, Confocal views of cross-sections of a control-injected muscle fiber (left) and a GABPβ^DN-injected muscle fiber (right). Injected fibers are identified by the expression of GFP (green). In the control, AChR clusters (red) are associated with myofibers containing GFP-positive nuclei. In contrast, in the GABPβ^DN-injected muscle, AChR clusters are found in adjacent, GFP-negative myofibers. Scale bar, 20 μm.

Fig. 5.

Distribution of utrophin and AChE in GABPβ^DN-injected muscles. A,B, The distribution of utrophin was examined by immunostaining and compared with that of AChRs on cross-sections made through the injected muscle fibers. The evaluation was made as described in Figure 4. Utrophin and AChR clusters colocalized on each cross-section, throughout the injection site, in the control as well as in the GABPβ^DN situation. C/GFP, GFP-positive fibers that have AChR clusters; U/GFP, GFP-positive fibers that have utrophin clusters; GFP, GFP-positive (injected) fibers; Ctot, total number of fibers having AChR clusters. C, The distribution of AChE was examined by choline esterase staining and compared with that of AChRs stained with alexa α-bungarotoxin (green). Both proteins colocalized on each analyzed cross-section. Scale bar, 20 μm.