Identification of an element crucial for the sub-synaptic expression of the acetylcholine receptor epsilon-subunit gene.
The adult neuromuscular junction displays an accumulation of both the acetylcholine receptor (AChR) protein in the subneural domain of the post-synaptic membrane and the mRNAs coding for all its subunits at the level of the subjunctional "fundamental nuclei." In the course of end plate development, the epsilon-subunit, at variance with other subunits, becomes exclusively expressed at the level of the fundamental nuclei, yet at a rather late stage (around birth). To analyze the promoter region of the epsilon-subunit gene which directs its specific expression at the synapse, we used a quantitative transient in vivo expression assay in intact muscle tissue using constructs of the epsilon-subunit promoter placed upstream of the beta-galactosidase reporter gene. One crucial element for synapse-specific expression was detected between the -11 and -6 positions. Disruption of this element, either by a scanning mutation or single base mutations, greatly diminishes, or even completely inhibits, preferential expression of the transgene at the end plate. Gel shift experiments reveal the presence of a complex in nuclear muscle extracts that bind the core sequence of this element. The identification of such a site opens the possibility to identify regulatory factors responsible for compartmentalized expression at the neuromuscular junction.
Duclert
A
A
URA CNRS 0210 "Neurobiologie Moléculaire," Département des Biotechnologies, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France.
Savatier
N
N
Schaeffer
L
L
Changeux
J P
JP
D000375:Aging / Q000502:physiology*; D000818:Animals; D001483:Base Sequence; D018507:Gene Expression Regulation, Developmental; D046911:Macromolecular Substances; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D015345:Oligonucleotide Probes; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D011950:Receptors, Cholinergic / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000096:biosynthesis
D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011950:Receptors, Cholinergic; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase