Upstream sequencing and functional characterization of the human cholinergic gene locus (original) (raw)

Is RE1/NRSE a Common cis-Regulatory Sequence for ChAT and VAChT Genes?

Journal of Biological Chemistry, 2000

Choline acetyltransferase (ChAT), the biosynthetic enzyme of acetylcholine, and the vesicular acetylcholine transporter (VAChT) are both required for cholinergic neurotransmission. These proteins are encoded by two embedded genes, the VAChT gene lying within the first intron of the ChAT gene. In the nervous system, both ChAT and VAChT are synthesized only in cholinergic neurons, and it is therefore likely that the cell typespecific expression of their genes is coordinately regulated. It has been suggested that a 2336-base pair genomic region upstream from the ChAT and VAChT coding sequences drives ChAT gene expression in cholinergic structures. We investigated whether this region also regulates VAChT gene transcription. Transfection assays showed that this region strongly represses the activity of the native VAChT promoters in non-neuronal cells, but has no major effect in neuronal cells whether or not they express the endogenous ChAT and VAChT genes. The silencer activity of this region is mediated solely by a repressor element 1 or neuron-restrictive silencer element (RE1/NRSE). Moreover, several proteins, including RE1-silencing transcription factor or neuron-restrictive silencer factor, are recruited by this regulatory sequence. These data suggest that this upstream region and RE1/NRSE co-regulate the expression of the ChAT and VAChT genes.

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

Journal of Neurochemistry, 2002

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 ␤-galactosidase (LacZ) as a reporter gene. A 3,402-bp segment from the 5Ј-untranslated region of the mouse ChAT gene (from Ϫ3,356 to ϩ46, ϩ1 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 Ϫ6,371 to ϩ46) 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 5Ј-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. Key Words: Choline acetyltransferase promoter-Choline acetyltransferase-Vesicular acetylcholine transporter-Cholinergic neurons.

Independent patterns of transcription for the products of the rat cholinergic gene locus

Neuroscience, 2001

öThe cholinergic phenotype requires the expression of the vesicular acetylcholine transporter and choline acetyltransferase proteins. Both genes are encoded at one chromosomal location called the cholinergic gene locus. We have identi¢ed by in situ hybridization histochemistry distinct patterns of transcription from the cholinergic gene locus in the subdivisions of the rat cholinergic nervous system. The vesicular acetylcholine transporter and choline acetyltransferase are co-expressed in cholinergic neurons at all developmental stages in all major types of cholinergic neurons. The relative levels of vesicular acetylcholine transporter and choline acetyltransferase transcripts, however, change substantially during development in the CNS. They also di¡er dramatically in distinct subdivisions of the mature cholinergic nervous system, with vesicular acetylcholine transporter mRNA expressed at high levels relative to choline acetyltransferase mRNA in the peripheral nervous system, but at equivalent levels in the CNS. Expression of the R-exon, the presumptive ¢rst non-coding exon common to both the vesicular acetylcholine transporter and choline acetyltransferase, was not detectable at any developmental stage in any of the cholinergic neuronal subtypes in the rat nervous system. Thus, in contrast to less complex metazoan organisms, production of the vesicular acetylcholine transporter and choline acetyltransferase via a common di¡erentially spliced transcript does not seem to occur to a signi¢cant extent in the rat. We suggest that separate transcriptional start sites within the cholinergic gene locus control vesicular acetylcholine transporter and choline acetyltransferase transcription, while additional elements are responsible for the speci¢c transcriptional control of the entire locus in cholinergic versus non-cholinergic neurons. Independent transcription of the vesicular acetylcholine transporter and choline acetyltransferase genes provides a mechanism for regulating the relative expression of these two proteins to ¢ne-tune acetylcholine quantal size in di¡erent types of cholinergic neurons, both centrally and peripherally.

Comparative study of gene expression of cholinergic system-related molecules in the human spinal cord and term placenta

Neuroscience, 2004

By reverse transcription-polymerase chain reaction, Southern blot analysis, direct sequencing, and immunohistochemistry, we studied the expression of cholinergic neuronal markers (choline acetyltransferase [ChAT], vesicular acetylcholine transporter [VAChT], and a high-affinity choline transporter [CHT1]), and gene regulatory molecules (repressor element-1 silencing transcription factor/neuron-restrictive silencer factor [REST/NRSF] and CoREST) in the human spinal cord and term placenta, both of which are well known to contain cells synthesizing acetylcholine. H-type, M-type, N2-type, and R-type ChAT mRNAs, VAChT mRNA, and CHT1 mRNA were detected in the spinal cord, but only H-type, M-type, and N2-type ChAT mRNAs, in the term placenta. REST/NRSF and CoREST were detected in the spinal cord and the placenta, but the amounts of both mRNAs were greater in the placenta than in the spinal cord. Further microdissection analyses revealed that the placental trophoblastic cells contained more REST/NRSF and CoREST transcripts than the spinal large motor neurons. Large motor neurons in the anterior horn of the spinal cord were immunohistochemically stained for ChAT and VAChT. In the placenta, stromal fibroblasts, endothelial cells, and trophoblastic cells of the chorionic villi were positively stained with anti-ChAT antibody but not with anti-VAChT antibody. These findings suggest that transcriptions of the R-type ChAT and VAChT mRNAs are coordinately suppressed in the human term placenta, which might be regulated in part by a REST/NRSF complex that binds to a consensus sequence of repressor element 1/neuron-restrictive silencer element (RE1/NRSE) in the 5= region upstream from exon R, whereas transcriptions of the Htype, M-type, and N2-type ChAT mRNAs might be independent of control by RE1/NRSE. It is possible that at least two separate regulatory mechanisms of gene expression are present for the human cholinergic gene locus, which might be selected by different combinations of DNA motifs and binding proteins to function in neuronal and non-neuronal cells.

Elements between the protein-coding regions of the adjacent ?4 and ?3 acetylcholine receptor genes direct neuron-specific expression in the central nervous system

Journal of Neurobiology, 1997

elements are positioned between the b4 and a3 coding ABSTRACT: The expression patterns of three regions, we investigated the activity of 02732 //47 in clustered neuronal nicotinic acetylcholine receptor vivo. Transgenic mice were generated, which carry the (nAchR) subunit genes ordered b4, a3, and a5 over-lacZ gene fused downstream of 02732 //47. Expreslap extensively in the peripheral nervous system sion of the lacZ transgene is restricted to neurons of (PNS) but only partially in the central nervous system the CNS; no expression was detected in the PNS or (CNS). We have begun to investigate cell type-spein nonneural tissues. LacZ -positive cells were detected cific cis elements regulating these genes by analyzing virtually exclusively in a subset of CNS nuclei that in both cell culture and transgenic mice, a 2.8-kb fragtranscribe the endogenous a3 gene. Some overlap was ment (02732//47) containing the a3 promoter reseen with the b4 gene, but nearly none with the a5 gion, the b4/a3 intergenic region, and a portion of the gene. Our results demonstrate that cis elements posi-b4 3-untranslated exon. The 02732 //47 fragment is tioned between the a3 and b4 coding regions are impreferentially active in PC12 cells relative to nonneuportant for establishing part of the restricted CNS ral cell lines. Deletion analysis revealed a cell typepatterns of b4, a3, and a5 gene transcription. ᭧ 1997 specific positive transcriptional element positioned in John Wiley & Sons, Inc. J Neurobiol 32: 311-324, 1997 the b4 3-untranslated exon. The positive element is Keywords: neuronal nicotinic receptors; cis-acting elelikely to be an enhancer and not a second a3 proments; neuron-specific regulation; transgenic mice; moter, because no a3 exons are present in this region. gene regulation; ligand-gated ion channel gene Having shown in cell culture that cell-type specific cis

Elements between the protein‐coding regions of the adjacent β4 and α3 acetylcholine receptor genes direct neuron‐specific expression in the central nervous system

Journal of Neurobiology, 1997

Partial cloning of the rat choline acetyltransferase gene and in situ localization of its transcripts in the cell body of cholinergic neurons in the brain stem and spinal cord

Molecular Brain Research, 1993

We have isolated recombinant lambda (A) phages which contain a part of the rat choline acetyltransferase (CHAT) gene. Restriction and Southern blot analyses using synthetic oligonucleotides indicate that these clones overlap one another and contain at least four exons which reside in 16.4 kb of sequence encoding from the middle to the 3' end, but not the 5'-region, of the rat ChAT gene. Partial sequence analyses revealed that the clones contain an exon whose nucleotide sequence corresponds to a highly conserved region of ChAT during evolution. RNase protection mapping experiments show that sequences represented by this exon are expressed at high levels in the spinal cord of adult rats and at low but detectable levels in PCI2 cells. By using the genomic sequences, including the exon, as a hybridization probe, we have detected ChAT mRNAs in situ in rat tissues. In situ hybridization experiments using radioactive and non-radioactive probes revealed that cholinergic motoneurons in the spinal cord, the laterodorsal tegmental nucleus as well as the hypoglossal nucleus in the brain stem were labeled, suggesting that the genomic sequence can be used as a probe to measure the ChAT mRNA levels in those cholinergic neurons. The results also indicate that the non-radioactive method gives a better resolution in localizing the expression of ChAT transcripts in the cytoplasm of cholinergic neurons.

Somatomotor neuron-specific expression of the human cholinergic gene locus in transgenic mice

Neuroscience, 2000

We examined the expression pattern of the vesicular acetylcholine transporter in the mouse nervous system, using rodent-specific riboprobes and antibodies, prior to comparing it with the distribution of vesicular acetylcholine transporter expressed from a human transgene in the mouse, using riboprobes and antibodies specific for human. Endogenous vesicular acetylcholine transporter expression was high in spinal and brainstem somatomotor neurons, vagal visceromotor neurons, and postganglionic parasympathetic neurons, moderate in basal forebrain and brainstem projection neurons and striatal interneurons, and low in intestinal intrinsic neurons. Vesicular acetylcholine transporter expression in intrinsic cortical neurons was restricted to the entorhinal cortex. The sequence of the mouse cholinergic gene locus to 5.1 kb upstream of the start of transcription of the vesicular acetylcholine transporter gene was determined and compared with the corresponding region of the human gene. Cisregulatory domains implicated previously in human or rat cholinergic gene regulation are highly conserved in mouse, indicating their probable relevance to the regulation of the mammalian cholinergic gene locus in vivo. Mouse lines were established containing a human transgene that included the vesicular acetylcholine transporter gene and sequences spanning 5 kb upstream and 1.8 kb downstream of the vesicular acetylcholine transporter open reading frame. In this transgene, the intact human vesicular acetylcholine transporter was able to act as its own reporter. This allowed elements within the vesicular acetylcholine transporter open reading frame itself, shown previously to affect transcription in vitro, to be assessed in vivo with antibodies and riboprobes that reliably distinguished between human and mouse vesicular acetylcholine transporters and their messenger RNAs. Expression of the human vesicular acetylcholine transporter was restricted to mouse cholinergic somatomotor neurons in the spinal cord and brainstem, but absent from other central and peripheral cholinergic neurons. The mouse appears to be an appropriate model for the study of the genetic regulation of the cholinergic gene locus, and the physiology and neurochemistry of the mammalian cholinergic nervous system, although differences exist in the distribution of cortical cholinergic neurons between the mouse and other mammals. The somatomotor neuron-specific expression pattern of the transgenic human vesicular acetylcholine transporter suggests a mosaic model for cholinergic gene locus regulation in separate subdivisions of the mammalian cholinergic nervous system.

Upstream sequencing and functional characterization of the human cholinergic gene locus (original) (raw)

Related papers