Abstract
This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pair-long region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. A 21-base pair-long neuron-restrictive silencer element (NRSE) was identified in the proximal part of this region. This element was recognized by the neuron-restrictive silencer factor (NRSF), previously shown to regulate expression of other neuron-specific genes. The ChAT NRSE was inactive in both cholinergic and non-cholinergic neuronal cells, but repressed expression from a heterologous promoter in non-neuronal cells. Specific deletion of this element allowed ChAT gene promoter activity in non-neuronal cells, and overexpression of NRSF repressed ChAT gene promoter activity in cholinergic cells. The distal part of the ChAT gene promoter showed cholinergic-specific enhancing activity, which stimulated promoter activity in cholinergic cells, but was inactive in non-cholinergic neuronal and non-neuronal cells. This enhancer region suppressed the activity of the ChAT NRSE in cholinergic cells, even after NRSF overexpression. Thus, at least two kinds of regulatory elements cooperate to direct ChAT gene expression to cholinergic neurons, namely a neuron-restrictive silencer element and a cholinergic-specific enhancer.
Highlights
This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pairlong region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells
In support of this notion, we have recently demonstrated that this segment from the ChAT gene promoter is able to confer cholinergic specificity to a non-cholinergic promoter in an orientation-independent manner and to direct correct developmental and tissue-specific expression to cholinergic cells in transgenic mice [15]
We report on the identification and functional analysis of positive and negative regulatory elements in a 2336bp-long upstream region of the rat ChAT gene promoter capable of directing cell type-specific gene expression
Summary
This study demonstrates the presence of positive and negative regulatory elements within a 2336-base pairlong region of the rat choline acetyltransferase (ChAT) gene promoter that cooperate to direct cell type-specific expression in cholinergic cells. Cholinergic neurons in the central nervous system include cells in the basal forebrain, cortex, motor nuclei, and spinal cord These neurons acquire their cholinergic phenotype progressively as the expression of the ChAT gene increases during development. Developing neurons may switch from a non-cholinergic to a cholinergic phenotype only after reaching the target of innervation [2] and may become cholinergic in response to injury [3] In most of these circumstances, a major point of control of the cholinergic phenotype has been demonstrated at the transcriptional level [4, 5], suggesting the presence of regulatory elements in the ChAT gene controlling accurate timeand cell-specific expression. We report the functional dissection of this region and the identification of a neuron-restrictive silencer element and cholinergic-specific enhancer sequences that cooperate to direct cell type-specific expression in cholinergic neurons
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