Abstract
Acetylcholine Receptor (AChR)-inducing activity (ARIA) is believed to be the trophic factor utilized by motoneurons to stimulate AChR synthesis in the subsynaptic area. Among the four AChR subunit genes, the epsilon subunit gene is strictly expressed in nuclei localized to the synaptic region of the muscle. To understand mechanisms of the regulation of synapse-specific transcription, we studied the promoter activity of the 5'-flanking region of the AChR epsilon subunit gene in response to ARIA. Transgenes containing the wild type or mutant 5'-flanking regions upstream of a luciferase gene were transfected in C2C12 muscle cells. The promoter activity of these transgenes was determined by assaying activity of expressed luciferase. Analyzing a combination of 5' deletion and site-directed mutants, we identified a 10-nucleotide element (position -55/-46), which was crucial for ARIA-induced expression from the epsilon subunit promoter. This element was named ARE for ARIA-responsive element. Mutation of ARE greatly diminished ARIA-induced transgene expression and deletion of ARE abolished completely the ARIA response. Electrophoretic mobility shift analyses revealed a DNA binding activity in muscle nuclear extract that interacted with ARE. Such interaction was enhanced by ARIA stimulation of muscle cells and appeared to be dependent on nuclear protein phosphorylation.
Highlights
The development and maintenance of a functional neuromuscular junction require that expression of all the molecular components be temporally and spatially regulated at the nervemuscle contact
Localization of the ARIA-responsive Promoter Activity in 34 Nucleotides between Ϫ78 and Ϫ45 in the 5Ј-Flanking Region of the ⑀ Subunit Gene—To determine the minimum length of the 5Ј regulatory region required for promoter activity in response to ARIA, we generated transgenes containing a series of deletion mutants in the 5Ј-flanking region of the ⑀ subunit gene
Transgenes were transfected in C2C12 muscle cells, and promoter activity was characterized by luciferase assay in ARIAstimulated C2C12 myotubes
Summary
The development and maintenance of a functional neuromuscular junction require that expression of all the molecular components be temporally and spatially regulated at the nervemuscle contact. We provide evidence that a 10-nucleotide element in the sequence upstream of the transcription initiation site, termed ARE, is required for ARIAinduced ⑀ subunit expression in muscle cells. Expression of the Ϫ416-Luc transgene, which contains 416 nucleotides upstream from the transcription initiation site, was increased by ARIA stimulation in a concentration-dependent manner (Fig. 1).
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