Identification of a neuregulin and protein-tyrosine phosphatase response element in the nicotinic acetylcholine receptor « subunit gene: Regulatory role of an Ets transcription factor

At the neuromuscular synapse, innervation induces endplate-specific expression of adult-type nicotinic acetylcholine receptors by selective expression of their subunit-encoding genes (a2b«d) in endplate-associated myonuclei. These genes are specifically regulated by protein-tyrosine phosphatase (PTPase) activity. In addition, neureguliny acetylcholine-receptor-inducing activity, a nerve-derived factor that stimulates nicotinic acetylcholine receptor synthesis, induces adult-type specific « subunit gene expression via activation of a Rasymitogen-activated protein kinase pathway. However, the DNA regulatory elements and the binding proteins that mediate PTPase and neuregulin-dependent gene expression remain unknown. Herein we report that PTPase, neuregulin, and Ras-dependent regulation of the « subunit gene map to a 15-bp promoter sequence. Interestingly, this same 15-bp sequence appears to be necessary for low « subunit gene expression in extrajunctional regions of the muscle fiber. Site-directed mutagenesis of a putative Ets binding site located within this 15-bp sequence, reduced PTPase, neuregulin, and Ras-dependent regulation. Overexpression of the rat muscle Ets-2 transcription factor resulted in a sequencespecific induction of « subunit promoter activity. Further, a dominant negative mutant of Ets-2 abolished neuregulindependent induction of « subunit gene expression. Thus, these results indicate a crucial role for the 15-bp element in determining synapse-specific and neuregulin-mediated motor neuron control of « subunit gene expression and suggest the participation of Ets transcription factor(s) in this control. Synapse formation and subsequent maturation involves complex interactions between the presynaptic cell and its postsynaptic target. As a model synapse, the neuromuscular junction provides a relatively simple system to study molecular mechanisms mediating some of these interactions. At this synapse, synaptic transmission between the motor neuron and its target muscle fiber is mediated by the muscle nicotinic acetylcholine receptor (nAChR), a multisubunit ligand-gated ion channel (1). During the development of the neuromuscular synapse, nerve-evoked muscle activity suppresses expression of embryonic-type nAChRs (a2bgd) throughout the muscle fiber (1, 2), whereas muscle innervation induces expression of adult-type nAChRs (a2b«d) at the endplate. This spatially restricted expression of adult-type nAChRs is largely a result of selective induction of their subunit encoding genes in endplateassociated myonuclei (1). The transcriptional mechanisms by which the motor neuron regulates gene expression in these subsynaptic nuclei are not well understood. Motor-neuron-derived trophic factors named neuregulins and their receptors have been colocalized at the neuromuscular endplate (3–5). Neuregulins include neu differentiation factor, heregulin, glial growth factor, and acetylcholinereceptor-inducing activity, which are alternatively spliced products of a single gene (6). Several lines of evidence point to the crucial role of neuregulins in directing synapse-specific expression of the nAChRs at the neuromuscular junction (3–11). However, the underlying mechanism of this regulation is unclear. Previously, we showed that protein-tyrosine phosphatase (PTPase) activity selectively suppresses muscle adult-type nAChR genes and proposed that changes in protein-tyrosine phosphorylation contributed to synapse-specific gene expression (12). Interestingly, neuregulins function as ligands for epidermal growth factor receptor-related (erbB) tyrosine kinases and stimulate adult-type nAChR synthesis (3–10). Whether the neuregulin signaling pathway differs from that perturbed by PTPase overexpression is not known. Further, synaptic induction of nAChR genes by neuregulin has been shown to require activation of a Rasymitogen-activated protein (MAP) kinase pathway (13, 14). This raises the possibility that MAP-kinase-responsive transcription factors such as the Ets family of proteins (15–18) may be involved, at least in part, in mediating this regulation. Because the « subunit gene is unique to adult-type nAChRs and is locally expressed at the endplate, it serves as a marker for regulatory mechanisms involved in synapse-specific expression. In addition, this gene is most sensitive to neuregulin induction (9). However, there is no clear consensus regarding the element that mediates this induction. For example, one study identified a 6-bp element, referred to as the N-box (CCGGAA), that is crucial for synapse-specific expression of this gene (19), and a subsequent study concluded that this element is not required for acetylcholine-receptor-inducing activity (neuregulin)-dependent induction (20). Further, the transcription factors mediating this neuregulin-dependent and synapse-specific expression have not been identified. In the present study, we show that a 15-bp « promoter sequence plays a crucial role in PTPase, neuregulin, and Ras-dependent regulation of the rat nAChR « subunit gene. In vivo expression suggest that this same DNA sequence participates in suppressing « gene expression in extrajunctional regions of the muscle fiber. Further, overexpression of Ets-2 and a dominant negative Ets-2 mutant suggest the participation of Ets transcription factor(s) in neuregulin-mediated motor neuron control of « subunit gene expression. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact. © 1998 by The National Academy of Sciences 0027-8424y98y951289-6$2.00y0 PNAS is available online at http:yywww.pnas.org. Abbreviations: nAChR, nicotinic acetylcholine receptor; PTPase, protein tyrosine phosphatase; CAT, chloramphenicol acetyltransferase; MAP kinase, mitogen-activated protein kinase; CMV, cytomegalovirus. ‡To whom reprint requests should be addressed at: Mental Health Research Institute, University of Michigan, 205 Zina Pitcher Place, Ann Arbor, MI 48109.