Transcriptional control of the neuronal nicotinic acetylcholine receptor gene cluster by the β43′ enhancer, Sp1, SCIP and ETS transcription factors

Abstract

Receptors assembled from the products of a neuronal β4α3α5 NAChR gene cluster depend on these genes being coordinately regulated in particular populations of neurons. Little is known, however, about the transcriptional mechanisms that are likely to underlie their co-expression in correct neuronal cell types. We have identified several regulatory elements and transcription factors that influence transcription of the α3 and β4 genes. The promoters of these genes appear to contain a common cis element that binds Sp1 transcription factors. They can be activated by the POU-domain factor SCIP and activation does not require SCIP binding sites. Between these two promoters is a cell type specific enhancer called β43′. This enhancer has little activity in non-neuronal cells and is preferentially active in particular populations of central neurons. The clustered genes are potential targets of ETS factors as the ETS domain factor, Pet-1 can activate β43′-dependent transcription. The neuron-selective properties of β43′ and its location suggest that it is a component of the cis regulatory information required to control expression of the β4 and α3 genes in specific populations of neurons.

Introduction

The vertebrate genome provides a tremendous potential for generating nicotinic acetylcholine receptor diversity by encoding at least sixteen different subunits that can be used to assemble receptors. An important question currently under investigation is what are the transcriptional mechanisms that control the distribution of subunit mRNA within various populations of vertebrate neurons? As most nicotinic receptor subtypes expressed in neurons are heteromeric assemblies of two, three, or even four different subunits, the genes encoding them must be coordinately regulated to allow the co-expression of appropriate subunit mRNA in the correct neuronal cell types. Thus, as the protein-coding regions of the neuronal nicotinic receptor subunit genes diversified over time, the genetic regulatory information required to restrict and coordinate neuronal co-expression of individual subunit mRNAs must have evolved in parallel.

Three of the neuronal nicotinic receptor subunit genes are clustered in the order β4, α3, and α5 over about 50 kb in the vertebrate genome Boulter et al., 1990, Couturier et al., 1990, Raimondi et al., 1992. Clustering of the β4, α3, and α5 genes suggests that this organization has been evolutionarily conserved in order to preserve regulatory information needed to control cell-type specific transcription of these genes. It is straightforward to imagine what might be the functional significance of this organization as the β4, α3, and α5 subunits are assembled together into at least one major ganglionic nicotinic receptor subtype Conroy and Berg, 1995, Vernallis et al., 1993. β4 and α3 but not α5 are also likely to be assembled together into at least one retinal subtype well as other brain subtypes Vailati et al., 1999, Zoli et al., 1998. It seems reasonable to expect that the clustered organization of their respective genes is a particularly efficient solution to coordinating peripheral and central neuronal co-expression of one or more of them. Experimentally, the cluster offers a relatively compact genetic system with which to identify transcriptional mechanisms that coordinate expression of subunits that are to be assembled together into receptors in particular neuronal populations.

One way in which coordinate expression might be achieved is through the action of neuron selective enhancers that activate transcription of the clustered genes in specific populations of neurons. Perhaps clustering permits the sharing of these cell-type specific cis elements in order to coordinate subunit expression. We have searched for neuron-selective cis elements upstream of the rat β4 and α3 genes using a combination of reporter assays in cell lines, central and peripheral primary neurons, and transgenic animals. This investigation has resulted in the identification of a non-cell type-specific α3 and β4 promoters and neuron selective enhancer within the β4 3′-untranslated region.