The objective of the research proposed here is to understand transcriptional mechanisms that control cholinergic neurotransmitter system development in vertebrates. A conserved cluster of vertebrate genes ordered beta4, alpha3, and alpha5 encode subunits that are assembled into several neuronal nicotinic acetylcholine receptor (nAchR) subtypes in peripheral and central neurons. One subtype composed of all three subunits is essential for excitatory post-synaptic transmission in adrenal chromaffin cells and between pre- and post-ganglionic neurons of the sympathetic and parasympathetic nervous systems. Subtypes containing beta4 and alpha3 subunits are important for cholinergic synaptic transmission in retina. Before these subtypes can be assembled the genes encoding beta4, alpha3, and alpha5 must be transcribed in appropriate neurons at the correct time. However, the transcriptional mechanisms that control subunit expression are poorly understood. We have identified an enhancer (beta43') within the cluster that is likely to be important for transcription of the subunit genes in neurons. We also showed that ETS domain factor interactions are important for neuronal activity of beta43'. In this proposal we wish to determine the biological relevance of beta43' and its interactions with ETS factors for the assembly of different receptor subtypes made from beta4, alpha3, and alpha5. Our hypothesis is that beta43' controls neuron specific transcription of the clustered genes through ETS interactions. We have prepared transgenic mice expressing each of the clustered genes from a Pl-bacterial artificial chromosome (PAC). We will prepare additional lines of transgenic mice that carry mutations in the enhancer ETS binding sites. Then transcription of each clustered gene will be investigated in various neuronal cell types that express the cluster. We will also investigate whether the cluster is a transcriptional target of the Pet-1 ETS factor by investigating subunit RNA and protein levels in our Pet-1 knock out mice. We will also investigate ETS factor expression in neuronal cell types that express the cluster. This research will further our understanding of the mechanisms governing the development of neuronal cholinergic systems and assembly of specific nAchR subtypes. It will also help to reveal the poorly defined functions of ETS factors in vertebrate neurons.
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