The cholinergic locus: ChAT and VAChT genes.

The gene encoding the vesicular acetylcholine transporter has been localized within the first intron of the gene encoding choline acetyltransferase and is in the same transcriptional orientation. These two genes, whose products are required for the expression of the cholinergic phenotype, could therefore be coregulated. The promoters of both genes have been identified. The mechanisms that account for the regulation of the expression of both genes are now being investigated.

Specific vesicular acetylcholine transporter promoters lie within the first intron of the rat choline acetyltransferase gene.

The sequence encoding the vesicular acetylcholine transporter (VAChT) has recently been localized within the first intron of the choline acetyltransferase (ChAT) gene in various species. In rat, we previously identified a class of VAChT mRNAs that may originate from the same promoter region as two ChAT mRNAs. Here, we demonstrate by a detailed analysis of the 5'-noncoding region of the VAChT gene, that two specific VAChT promoters lie within the first intron of the ChAT gene. Two VAChT mRNAs are generated from these promoters. These results demonstrate that the promoter regions of these two genes are intermingled, which highlight the unique organization of the ChAT/VAChT gene locus.

A unique gene organization for two cholinergic markers, choline acetyltransferase and a putative vesicular transporter of acetylcholine.

Choline acetyltransferase (ChAT) is the biosynthetic enzyme of acetylcholine. In mammalian tissues, it is encoded by multiple mRNAs with different 5'-ends. This diversity results from the alternative usage of three promoters and from differential splicing events. Here, we show that the first intron of the rat ChAT gene contains an open reading frame that encodes a potential vesicular acetylcholine transporter based on the following criteria. (i) The encoded protein is structurally similar to transporter proteins, the highest identity being found with the vesicular acetylcholine transporters from Torpedo and Caenorhabditis elegans (77 and 56%, respectively, in 352 amino acids). (ii) The corresponding mRNAs exhibit a cholinergic expression profile. Amplification experiments with spinal cord cDNA revealed that at least three mRNAs encode this transporter. Two contain the same 5' non-coding region as two ChAT mRNAs and, therefore, are derived from the ChAT transcription unit by alternative splicing. The third mRNA may be transcribed from an additional internal promoter.

Regulation by CDF/LIF and retinoic acid of multiple ChAT mRNAs produced from distinct promoters.

The cholinergic differentiation factor/leukaemia inhibitory factor (CDF/LIF) and retinoic acid (RA) induce in sympathetic neurones, a switch from the noradrenergic to the cholinergic neurotransmitter phenotype. In particular, these molecules alter the activities of the biosynthetic enzymes choline acetyltransferase (ChAT), tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). Recently, five rat ChAT mRNA species have been identified although no data have yet been reported concerning their production and regulation in sympathetic neurones. By use of the reverse transcription polymerase chain reaction technique we analysed the effects of CDF/LIF and RA on the levels of ChAT, TH and DBH mRNAs. Each ChAT mRNA was produced in sympathetic neurones and was induced by both molecules, whereas the mRNAs encoding TH and DBH enzymes were down-regulated.

Negative regulatory elements upstream of a novel exon of the neuronal nicotinic acetylcholine receptor alpha 2 subunit gene.

The expression of the nicotinic acetylcholine receptor alpha 2 subunit gene is highly restricted to the Spiriform lateralis nucleus of the Chick diencephalon. As a first step toward understanding the molecular mechanism underlying this regulation, we have investigated the structural and regulatory properties of the 5' sequence of this gene. A strategy based on the ligation of an oligonucleotide to the first strand of the cDNA (SLIC) followed by PCR amplification was used. A new exon was found approximately 3kb upstream from the first coding exon, and multiple transcription start sites of the gene were mapped. Analysis of the flanking region shows many consensus sequences for the binding of nuclear proteins, suggesting that the 1 kb flanking region contains at least a portion of the promoter of the gene. We have analysed the negative regulatory elements present within this region and found that a silencer region located between nucleotide -144 and +76 is active in fibroblasts as well as in neurons. This silencer is composed of six tandem repeat Oct-like motifs (CCCCATGCAAT), but does not bind any member of the Oct family. Moreover these motifs were found to act as a silencer only when they were tandemly repeated. When two, four or five motifs were deleted, the silencer activity of the motifs unexpectedly became an enhancer activity in all cells we have tested.

Promoter elements of the rat choline acetyltransferase gene allowing nerve growth factor inducibility in transfected primary cultured cells.

Choline acetyltransferase, the enzyme responsible for the synthesis of acetylcholine, provides a convenient index for cholinergic neurons. Using a previously identified rat cDNA clone, we have isolated several corresponding genomic clones and have characterized a 1,902-bp fragment that contains part of the first noncoding exon as well as promoter sequences. The promoter activity of this fragment was tested, taking advantage of the recently developed lipopolyamine-mediated DNA transfer method, which allows transfection of primary neurons. The 1,902-bp sequence drives the expression of the bacterial chloramphenicol acetyltransferase (CAT) reporter gene in a culture of dissociated cells prepared from the septal area of fetal (embryonic day 17) rats, a structure rich in cholinergic neurons. Moreover, addition of nerve growth factor to the culture increases CAT expression by approximately 56-fold, indicating that our DNA fragment contains sequences required for NGF induction. In addition, it contains consensus sequences for various transcription factors, including those of the basic helix-loop-helix family. Finally, experiments to characterize the transcription start site are presented.