Identification and Transgenic Analysis of a Murine Promoter that Targets Cholinergic Neuron Expression

Abstract : Choline acetyltransferase (ChAT) is a specific phenotypic marker of cholinergic neurons. Previous reports showed that different upstream regions of the ChAT gene are necessary for cell type-specific expression of reporter genes in cholinergic cell lines. The identity of the mouse ChAT promoter region controlling the establishment, maintenance, and plasticity of the cholinergic phenotype in vivo is not known. We characterized a promoter region of the mouse ChAT gene in transgenic mice, using β-galactosidase ( LacZ ) as a reporter gene. A 3,402-bp segment from the 5'-untranslated region of the mouse ChAT gene (from -3,356 to +46, +1 being the translation initiation site) was sufficient to direct the expression of LacZ to selected neurons of the nervous system ; however, it did not provide complete cholinergic specificity. A larger fragment (6,417 bp, from -6,371 to +46) of this region contains the requisite regulatory elements that restrict expression of the LacZ reporter gene only in cholinergic neurons of transgenic mice. This 6.4-kb DNA fragment encompasses 633 bp of the 5'-flanking region of the mouse vesicular acetylcholine transporter (VAChT), the entire open reading frame of the VAChT gene, contained within the first intron of the ChAT gene, and sequences upstream of the start coding sequences of the ChAT gene. This promoter will allow targeting of specific gene products to cholinergic neurons to evaluate the mechanisms of diseases characterized by dysfunction of cholinergic neurons and will be valuable in design strategies to correct those disorders.     

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.     

Regulation of choline acetyltransferase/iacZ fusion gene expression in putative cholinergic neurons of drosophila melanogaster

We have analyzed the distribution of putative cholinergic neurons in whole-mount preparations of adult Drosophila melanogaster. Putative cholinergic neurons were visualized by X-gal staining of P-element transformed flies carrying a fusion gene consisting of 5′ flanking DNA from the choline acetyltransferase (ChAT) gene and a lacZ reporter gene. We have previously demonstrated that cryostat sections of transgenic flies carrying 7.4 kb of ChAT 5′ flanking DNA show reporter gene expression in a pattern essentially similar to the known distribution of ChAT protein. Whole-mount staining of these same flies by X-gal should thus represent the overall distribution of ChAT-positive neurons. Extensive staining was observed in the cephalic, thoracic, and stomodeal ganglia, primary sensory neurons in antenna, maxillary palps, labial palps, leg, wing, and male genitalia. Primary sensory neurons associated with photoreceptors and tactile receptors were not stained. We also examined the effects of partial deletions of the 7.4 kb fragment on reporter gene expression. Deletion of the 7.4 kb fragment to 1.2 kb resulted in a dramatic reduction of X-gal staining in the peripheral nervous system (PNS). This indicates that important regulatory elements for ChAT expression in the PNS exist in the distal region of the 7.4 kb fragment. The distal parts of the 7.4 kb fragment, when fused to a basal heterologous promoter, can independently confer gene expression in subsets of putative cholinergic neurons. With these constructs, however, strong ectopic expression was also observed in several non-neuronal tissues. © 1995 John Wiley & Sons, Inc.     

Promotor and enhancer like activity at the 5'-end of normal and T24 Ha-ras1 genes

We have used a short-term transfection technique, in which we monitor the ability of DNA fragments to induce the expression of the chloramphenicol acetyltransferase (CAT) gene in rat 208F fibroblast cells. Using appropriate vectors we have assayed for promoter or enhancer activity of the 0.8 kb SstI fragment located within the 5'-flanking sequences of the first coding exon of the human T24 and normal Ha-ras1 genes. We find that this fragment contains promoter and enhancer activities in both the normal and the T24 Ha-ras1 gene.     

Nerve growth factor regulates the expression of the cholinergic locus and the high-affinity choline transporter via the Akt/PKB signaling pathway

Nerve growth factor (NGF) is a trophic and survival factor for cholinergic neurons, and it induces the expression of several genes that are essential for synthesis and storage of acetylcholine (ACh), specifically choline acetyltransferase, vesicular ACh transporter (VAChT), and choline transporter. We have found previously that the phosphatidylinositol 3'-kinase pathway, but not the MEK/MAPK pathway, is the mediator of NGF-induced cholinergic differentiation. Here we demonstrate, in the rat pheochromocytoma cell line PC12 and in primary mouse neuronal cultures, that NGF-evoked up-regulation of these three cholinergic-specific genes is mediated by the anti-apoptotic signaling molecule Akt/protein kinase B. Inhibition of Akt activation by the pharmacological inhibitor 1L-6-hydroxymethyl-chiro-inositol 2(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), or by a peptide fragment derived from the proto-oncogene TLC1, eliminated NGF-stimulated increases in cholinergic gene expression, as demonstrated by RT-PCR and reporter gene assays. Moreover, treatment with HIMO reversed NGF-evoked increases in choline acetyltransferase activity and ACh production. In co-transfection assays with the reporter construct, a dominant-negative Akt plasmid and Akt1-specific small interfering RNA also attenuated NGF-induced cholinergic promoter activity. Our data indicate that, in addition to its well-described role in promoting neuronal survival, Akt can also mediate signals necessary for neurochemical differentiation.