A dual function activity-dependent, muscle-specific enhancer from rat nicotinic acetylcholine receptor delta-subunit gene.

Nicotinic acetylcholine receptors (nAChR) mediate communication between nerve and muscle. The expression of these receptors increases dramatically during muscle development when myoblasts are fusing into multinucleated myotubes. The molecular mechanisms mediating this muscle developmental stage specific expression are not well understood. We report here the identification of nAChR delta-subunit promoter DNA sequences that differentially interact with nuclear proteins isolated from myoblasts, myotubes, and nonmuscle cells. The functional role these sequences play in mediating muscle-specific expression was explored using mutagenesis and enhancer assays. These studies resulted in the identification of a 47-bp muscle-specific enhancer that mediates increased expression of the nAChR delta-subunit gene during myotube formation. This enhancer contains an E-box and an element with similarity to the SV40 core enhancer (SVCE). Point mutations throughout this 47-bp enhancer showed that the E-box and the SVCE sequence are both necessary for conferring muscle-specific expression onto a heterologous promoter. Interestingly, this same DNA sequence also functions as an activity-dependent enhancer.

Identification and characterization of a 47 base pair activity-dependent enhancer of the rat nicotinic acetylcholine receptor delta-subunit promoter.

Nicotinic acetylcholine receptor (nAChR) genes are regulated by muscle electrical activity. E-box sequences found in their promoters are necessary for this regulation. However, many muscle genes contain E-boxes, yet are not regulated by muscle depolarization. This suggests that other elements are necessary, perhaps working in conjunction with E-boxes, to confer depolarization-dependent control onto promoter activity. We have used direct DNA injection into muscle as an in vivo assay to identify and characterize these additional elements. Mutagenesis and expression assays identified multiple elements within the first 81 base pairs (bp) of the nAChR delta-subunit promoter that contribute to its regulation by muscle electrical activity. Within this 81 bp sequence, two regions of DNA were identified that were capable of conferring activity-dependent regulation onto a heterologous promoter. The stronger of these two putative enhancers was characterized further. It is a 47 bp sequence that contains an E-box along with sequences similar to the SV40 core enhancer and an SP1 site. Site-directed mutagenesis identified residues within each of these sequences that were necessary for enhancer activity. Furthermore, methylation interference DNA footprinting assays showed increased nuclear protein binding to sequences within both these enhancers after muscle denervation, and this pattern of binding was very similar to that observed with nuclear protein isolated from myotube extracts. These latter results suggest that similar mechanisms may mediate increased nAChR expression during muscle development and after muscle denervation.

Cloning and characterization of a novel transcriptional repressor of the nicotinic acetylcholine receptor delta-subunit gene.

We have identified a negative cis-acting regulatory element in the nicotinic acetylcholine receptor delta-subunit gene's promoter. This element resides within a previously identified 47-base pair activity-dependent enhancer. Proteins that bind this region of DNA were cloned from a lambdagt11 innervated muscle expression library. Two cDNAs (MY1 and MY1a) were isolated that encode members of the Y-box family of transcription factors. MY1/1a RNAs are expressed at relatively high levels in heart, skeletal muscle, testis, glia, and specific regions of the central nervous system. MY1/1a are nuclear proteins that bind specifically to the coding strand of the 47-base pair enhancer and suppress delta-promoter activity in a sequence-specific manner. These results suggest a novel mechanism of repression by MY1/1a, which may contribute to the low level expression of the delta-subunit gene in innervated muscle. Finally, the gene encoding MY1/1a, Yb2, maps to the mid-distal region of mouse chromosome 6.

Calcium-dependent regulation of rat and chick muscle nicotinic acetylcholine receptor (nAChR) gene expression.

Muscle depolarization leads to decreased expression of nicotinic acetylcholine receptor (nAChR) genes in extrajunctional regions of the muscle fiber with little effect on their expression at the neuromuscular junction (NMJ). Depolarization-dependent decreases in nAChR gene expression have been linked to a cAMP-dependent signaling system in rat (Chahine, K. G., Baracchini, E., and Goldman, D. (1993) J. Biol. Chem. 2893-2898), and a calcium-dependent protein kinase C (PKC) signaling system in chick (Klarsfeld, A., Laufer, R., Fontaine, B., Devillers-Thiery, A., Bubreuil, C., and Changeux, J. P. (1989) Neuron 2, 1229-1236). We report here on experiments investigating the role of calcium and PKC in regulating rat muscle nAChR gene expression. These studies indicate that depolarization-dependent regulation of rat muscle nAChR gene expression is independent of PKC activity. However, these genes are regulated by a calcium-dependent signal transduction system. Calcium influx across the plasma membrane decreases nAChR gene expression in inactive rat myotubes. Surprisingly, this influx of extracellular calcium is most effective at reducing nAChR epsilon-subunit gene expression. We also provide evidence that a similar signal transduction system is capable of regulating nAChR gene expression in chick muscle. Based on these data we propose that calcium, in addition to mediating depolarization-dependent regulation of nAChR expression, may also participate in restricting their expression to the neuromuscular junctions of adult muscle fibers.

A 102 base pair sequence of the nicotinic acetylcholine receptor delta-subunit gene confers regulation by muscle electrical activity.

Muscle electrical activity suppresses expression of the embryonic-type (alpha, beta, gamma, and delta) nicotinic acetylcholine receptor (nAChR) genes. The molecular mechanism by which electrical activity regulates these genes is not known. One approach to this problem is to identify regions of the nAChR genes that mediate electrical regulation. Here we report results from such a study of the nAChR delta-subunit gene. We cloned the rat delta-subunit promoter region and created an expression vector in which this DNA controlled the expression of a down-stream luciferase structural gene. The effect that muscle electrical activity had on the expression from this promoter was assayed by introducing this expression vector into electrically stimulated and tetrodotoxin (TTX)-treated rat primary myotubes, and assaying for luciferase activity. These myotubes, when stimulated with extracellular electrodes, suppressed endogenous embryonic-type nAChR gene expression compared to those treated with TTX. Transfection of these cells with delta-promoter-luciferase expression vectors resulted in the delta-promoter conferring electrical regulation on luciferase expression. Additional experiments using deletions from the 5' end of the delta-promoter region have identified an element between -677 and -550 bp that suppressed delta-promoter activity and a minimal 102 bp sequence that promotes and regulates luciferase expression in response to muscle electrical activity. This latter sequence also contains all the necessary elements to confer tissue and developmental stage-specific expression.