Identification of a DNA element determining synaptic expression of the mouse acetylcholine receptor delta-subunit gene.
mRNAs for acetylcholine receptor genes are highly concentrated in the endplate region of adult skeletal muscle largely as a result of a transcription restricted to the subneural nuclei. To identify the regulatory elements involved, we employed a DNA injection of a plasmid containing a fragment of the acetylcholine receptor delta-subunit gene promoter (positions -839 to +45) linked to the reporter gene lacZ with a nuclear localization signal. Injection of the wild-type construct into mouse leg muscles yielded preferential expression of the reporter gene in the synaptic region. Analysis of various mutant promoters resulted in the identification of a DNA element (positions -60 to -49), referred to as the N box, that plays a critical role in subneural expression. Disruption of this 12-bp element in the context of a mouse delta-subunit promoter from positions -839 to +45 gives widespread expression of the reporter gene throughout the entire muscle fiber, indicating that this element is a silencer that represses delta-subunit gene transcription in extrajunctional areas. On the other hand, this element inserted upstream of a heterologous basal promoter preferentially enhances expression in the endplate region. This element therefore regulates the restricted expression of the delta-subunit gene both as an enhancer at the endplate level and as a silencer in extrajunctional areas. Furthermore, gel-shift experiments with mouse muscle extracts reveal an activity that specifically binds the 6-bp sequence TTCCGG of this element, suggesting that a transcription factor(s) controls the expression of the delta-subunit gene via this element.
D000818:Animals; D002451:Cell Compartmentation; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D007273:Injections, Intramuscular; D051379:Mice; D009132:Muscles / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011485:Protein Binding; D011950:Receptors, Cholinergic / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid; D017384:Sequence Deletion; D013569:Synapses / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011950:Receptors, Cholinergic