Regulatory region in choline acetyltransferase gene directs developmental and tissue-specific expression in transgenic mice.

Abstract

Acetylcholine, one of the main neurotransmitters in the nervous system, is synthesized by the enzyme choline acetyltransferase (ChAT; acetyl-CoA:choline 0-acetyltransferase, EC 2.3.1.6). The molecular mechanisms controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo are largely unknown. A previous report showed that a 3800-bp, but not a 1450-bp, 5' flanking segment from the rat ChAT gene promoter directed cell type-specific expression of a reporter gene in cholinergic cells in vitro. Now we have characterized a distal regulatory region of the ChAT gene that confers cholinergic specificity on a heterologous downstream promoter in a cholinergic cell line and in transgenic mice. A 2342-bp segment from the 5' flanking region of the ChAT gene behaved as an enhancer in cholinergic cells but as a repressor in noncholinergic cells in an orientation-independent manner. Combined with a heterologous basal promoter, this fragment targeted transgene expression to several cholinergic regions of the central nervous system of transgenic mice, including basal forebrain, cortex, pons, and spinal cord. In eight independent transgenic lines, the pattern of transgene expression paralleled qualitatively and quantitatively that displayed by endogenous ChAT mRNA in various regions of the rat central nervous system. In the lumbar enlargement of the spinal cord, 85-90% of the transgene expression was targeted to the ventral part of the cord, where cholinergic av-motor neurons are located. Transgene expression in the spinal cord was developmentally regulated and responded to nerve injury in a similar way as the endogenous ChAT gene, indicating that the 2342-bp regulatory sequence contains elements controlling the plasticity of the cholinergic phenotype in developing and injured neurons.

Specific Vesicular Acetylcholine Transporter Promoters Lie within the First Intron of the Rat Choline Acetyltransferase Gene

“…Indeed, a rat genomic sequence upstream from the R-type exon has been shown to confer cholinergic specificity to the R-type promoter in vitro (12). Moreover, this region directs the in vivo expression of a heterologous downstream promoter in the cholinergic neurons (13). These results raise the question of whether this region controls the tissue-specific expression of both the ChAT and the VAChT genes.…”

Expression of the Rat m4 Muscarinic Acetylcholine Receptor Gene Is Regulated by the Neuron-restrictive Silencer Element/Repressor Element 1

“…The m4 mAChR gene is the first example of a NRSE/RE1-regulated gene among genes whose products are involved in neurotransmission, such as neurotransmitter-synthesizing enzymes, neuropeptide, and receptors. The choline acetyltransferase gene is a candidate for another such gene, since silencers have been implicated in its cholinergic neuron-specific expression, and a sequence highly homologous to the NRSE/RE1 is present in the 5Ј-flanking region of the rat gene (21)(22)(23)(24). Although the NRSE/RE1 regulates the neuron-specific expression of m4 mAChR gene, an additional mechanism is necessary to explain the restricted expression of this gene to specific subsets of neurons.…”

The Sympathetic Neurotransmitter Switch Depends on the Nuclear Matrix Protein Satb2

“…Our overexpression results, showing that Satb2 is capable of activating Vacht expression in the absence of CNTF, indicate a more direct role in augmenting cholinergic gene expression, not simply ensuring favorable transcriptional environment. One possible mechanism is that Satb2 may cooperate with REST4 to counteract the repressor activity of REST (RE-1 silencing transcription factor), a transcriptional repressor that has previously been shown to bind Chat promoter and downregulate cholinergic gene transcription in both cholinergic and noncholinergic cells (Lonnerberg et al, 1995;Hahm et al, 1997;Hersh and Shimojo, 2003).…”