Cholinergic Gene Locus Research Articles

Cis-regulatory elements of the cholinergic gene locus in the silkworm Bombyx mori.

Genes of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter are encoded in the same gene locus, called the cholinergic gene locus. They are essential in cholinergic neurons to maintain their functional phenotype. The genomic structure of the cholinergic gene locus is conserved among invertebrates to mammals. However, the cholinergic gene expression in a specific subset of neurons is unknown in insects except for Drosophila melanogaster. In this study, we analysed the upstream sequence of cholinergic gene locus in the silkworm Bombyx mori to identify specific cis-regulatory regions. We found multiple enhancer regions that are localized within 1 kb upstream of the cholinergic gene locus. The combination of promoter assays using small deletions and bioinformatic analysis among insect species illuminates two conserved sequences in the cis-regulatory region: TGACGTA and CCAAT, which are known as the cAMP response element and CAAT box, respectively. We found that dibutyryl-cAMP, an analogue of cAMP, influences the expression of ChAT in B. mori. Tissue-specific expression analysis of transcriptional factors identified potential candidates that control the cholinergic gene locus expression. Our investigation provides new insight into the regulation mechanism of cholinergic neuron-specific gene machinery in this lepidopteran insect.

Cholinergic axons regulate type I acini in salivary glands of Ixodes ricinus and Ixodes scapularis ticks

Regulatory factors controlling tick salivary glands (SGs) are direct upstream neural signaling pathways arising from the tick’s central nervous system. Here we investigated the cholinergic signaling pathway in the SG of two hard tick species. We reconstructed the organization of the cholinergic gene locus, and then used in situ hybridization to localize mRNA encoding choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in specific neural cells in the Ixodes synganglion. Immunohistochemical staining revealed that cholinergic axonal projections exclusively reached type I acini in the SG of both Ixodes species. In type I acini, the rich network of cholinergic axons terminate within the basolateral infoldings of the lamellate cells. We also characterized two types (A and B) of muscarinic acetylcholine receptors (mAChRs), which were expressed in Ixodes SG. We pharmacologically assessed mAChR-A to monitor intracellular calcium mobilization upon receptor activation. In vivo injection of vesamicol—a VAChT blocker—at the cholinergic synapse, suppressed forced water uptake by desiccated ticks, while injection of atropine, an mAChR-A antagonist, did not show any effect on water volume uptake. This study has uncovered a novel neurotransmitter signaling pathway in Ixodes SG, and suggests its role in water uptake by type I acini in desiccated ticks.

Unusual regulation of splicing of the cholinergic locus in Caenorhabditis elegans.

The essential neurotransmitter acetylcholine functions throughout the animal kingdom. In Caenorhabditis elegans, the acetylcholine biosynthetic enzyme [choline acetyltransferase (ChAT)] and vesicular transporter [vesicular acetylcholine transporter (VAChT)] are encoded by the cha-1 and unc-17 genes, respectively. These two genes compose a single complex locus in which the unc-17 gene is nested within the first intron of cha-1, and the two gene products arise from a common pre-messenger RNA (pre-mRNA) by alternative splicing. This genomic organization, known as the cholinergic gene locus (CGL), is conserved throughout the animal kingdom, suggesting that the structure is important for the regulation and function of these genes. However, very little is known about CGL regulation in any species. We now report the identification of an unusual type of splicing regulation in the CGL of C. elegans, mediated by two pairs of complementary sequence elements within the locus. We show that both pairs of elements are required for efficient splicing to the distal acceptor, and we also demonstrate that proper distal splicing depends more on sequence complementarity within each pair of elements than on the sequences themselves. We propose that these sequence elements are able to form stem-loop structures in the pre-mRNA; such structures would favor specific splicing alternatives and thus regulate CGL splicing. We have identified complementary elements at comparable locations in the genomes of representative species of other animal phyla; we suggest that this unusual regulatory mechanism may be a general feature of CGLs.

Nerve growth factor is primarily produced by GABAergic neurons of the adult rat cortex.

Within the cortex, nerve growth factor (NGF) mediates the innervation of cholinergic neurons during development, maintains cholinergic corticopetal projections during adulthood and modulates cholinergic function through phenotypic control of the cholinergic gene locus. Recent studies suggest NGF may also play an important role in cortical plasticity in adulthood. Previously, NGF-producing cells have been shown to colocalize with GABAergic cell markers within the hippocampus, striatum, and basal forebrain. Classification of cells producing NGF in the cortex is lacking, however, and cholinergic corticopetal projections have been shown to innervate both pyramidal and GABAergic neurons in the cortex. In order to clarify potential trophic interactions between cortical neurons and cholinergic projections, we used double-fluorescent immunohistochemistry to classify NGF-expressing cells in several cortical regions, including the prefrontal cortex, primary motor cortex, parietal cortex and temporal cortex. Our results show that NGF colocalizes extensively with GABAergic cell markers in all cortical regions examined, with >91% of NGF-labeled cells coexpressing GAD65/67. Conversely, NGF-labeled cells exhibit very little co-localization with the excitatory cell marker CaMKIIα (<5% of cells expressing NGF). NGF expression was present in 56% of GAD-labeled cells, suggesting that production is confined to a specific subset of GABAergic neurons. These findings demonstrate that GABAergic cells are the primary source of NGF production in the cortex, and likely support the maintenance and function of basal forebrain cholinergic projections in adulthood.

Cholinergic left-right asymmetry in the habenulo-interpeduncular pathway

The habenulo-interpeduncular pathway, a highly conserved cholinergic system, has emerged as a valuable model to study left-right asymmetry in the brain. In larval zebrafish, the bilaterally paired dorsal habenular nuclei (dHb) exhibit prominent left-right differences in their organization, gene expression, and connectivity, but their cholinergic nature was unclear. Through the discovery of a duplicated cholinergic gene locus, we now show that choline acetyltransferase and vesicular acetylcholine transporter homologs are preferentially expressed in the right dHb of larval zebrafish. Genes encoding the nicotinic acetylcholine receptor subunits α2 and β4 are transcribed in the target interpeduncular nucleus (IPN), suggesting that the asymmetrical cholinergic pathway is functional. To confirm this, we activated channelrhodopsin-2 specifically in the larval dHb and performed whole-cell patch-clamp recording of IPN neurons. The response to optogenetic or electrical stimulation of the right dHb consisted of an initial fast glutamatergic excitatory postsynaptic current followed by a slow-rising cholinergic current. In adult zebrafish, the dHb are divided into discrete cholinergic and peptidergic subnuclei that differ in size between the left and right sides of the brain. After exposing adults to nicotine, fos expression was activated in subregions of the IPN enriched for specific nicotinic acetylcholine receptor subunits. Our studies of the newly identified cholinergic gene locus resolve the neurotransmitter identity of the zebrafish habenular nuclei and reveal functional asymmetry in a major cholinergic neuromodulatory pathway of the vertebrate brain.

Asymmetric regulation by estrogen at the cholinergic gene locus in differentiated NG108-15 neuronal cells

Aims Estrogen acts as a neurogenerative and neuroprotective factor in the cholinergic system. Choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) are regarded as markers of cholinergic neurons. The genes coding these proteins are located at a common locus, the cholinergic gene locus. However, few details concerning activation of the locus have been obtained. We examined the effect of estrogen on the activation pattern of the locus using a cholinergic cell line. Main methods NG108-15 neuronal cells, as a model of cholinergic neurons, were used. Dose-dependent effects of estradiol (E 2) on the gene expression of ChAT and VAChT were quantitatively determined by a real-time RT-PCR. The expression of ChAT mRNA variants was qualitatively evaluated by RT-PCR using specific primers. Key findings The expression of ChAT and VAChT mRNA was strongly enhanced with the induction of differentiation. The enhanced expression of ChAT mRNA was further increased dose-dependently by E 2 (10 − 10 to 10 − 7 M), while that of VAChT mRNA did not respond to E 2. The up-regulation of ChAT mRNA expression by E 2 was abolished by co-treatment with a pure-antagonist of estrogen receptors. A qualitative analysis of ChAT mRNA variants revealed the R types, which share a common sequence with the VAChT gene, and type M ChAT mRNA to mainly be expressed, and that the appearance of these variants was not altered by E 2. Significance The cholinergic gene locus in differentiated NG108-15 neuronal cells is further activated by E 2, but the effect is restricted to the transcription of ChAT gene.

In situ hybridization study of the distribution of choline acetyltransferase mRNA and its splice variants in the mouse brain and spinal cord

Choline acetyltransferase is the enzyme that catalyzes the biosynthesis of the neurotransmitter acetylcholine. Seven types of mRNA for choline acetyltransferase that differ in the 5′-noncoding region are transcribed from the cholinergic gene locus from different promoter regions and produced by alternative splicing in the mouse. Digoxigenin-labeled riboprobes and in situ hybridization histochemistry were used to investigate the expression of N1, R1, R2, R3, R4 and total choline acetyltransferase mRNA in the mouse CNS. The relative levels of choline acetyltransferase transcripts differed dramatically in distinct subdivisions of the mature cholinergic nervous system. Neurons hybridizing with antisense riboprobes for all of the five investigated splice variants (R1, R2, R3, R4 and N1) as well as those hybridizing with riboprobe for the common protein-coding region were found in a number of expected regions in the CNS. They include the basal forebrain, striatum, pontomesencephalic tegmentum, motor and autonomic nuclei of the brainstem, and spinal cord. Neurons with a moderate to very high level of expression of R1 and R2 splice variants were distributed in both the forebrain and brainstem nuclei. On the other hand, R3, R4 and N1 splice variants revealed a moderate to high level of expression in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord compared to a low expression level in forebrain cholinergic structures. No expression of the N1, R1, R2, R3 and R4 splice variants was detectable in the neurons of the cerebral cortex, hippocampus and medial habenular nucleus. With the riboprobe for the common protein-coding region, the neurons of the medial habenular nucleus could be labeled at high level, while intrinsic cortical neurons were labeled at low level. Hippocampus revealed no significant hybridization for total choline acetyltransferase mRNA. These findings strongly suggested that: (1) R1 and R2 were the major splice variants expressed in the neurons of forebrain nuclei; (2) R1, R2, R3, R4 and N1 splice variants were almost equally expressed in the brainstem motor and autonomic nuclei and ventral and lateral horns of the spinal cord; (3) inferring from a paucity of other isoforms, M type choline acetyltransferase mRNA is a splice variant predominantly expressed in the cerebral cortex and medial habenular nucleus.

Distinct regional differences in the expression pattern of the transcripts of the cholinergic gene locus

The cholinergic gene locus (CGL) was first identified in 1994 as the site (human chromosome 10q11.2) at which choline acetyltransferase and a functional vesicular acetylcholine transporter are co-localized. Here, we present recent neuroanatomical, developmental, and evolutionary insights into the chemical coding of cholinergic neurotransmission that have been gleaned from the study of the CGL, and its protein products VAChT and ChAT, which comprise a synthesis-sequestration pathway that functionally defines the cholinergic phenotype.Le locus génétique cholinergique (cholinergic gene locus, CGL) a été identifié en 1994 et regroupe les gènes de la choline acétyltransférase et d'un récepteur vésiculaire d'acétylcholine (chromosome humain 10q11.2). Nous présentons ici des données sur la neuroanatomie, le développement et l'évolution de la neurotransmission cholinergique obtenus à partir de l'étude du CGL et de ses produits protéiques, VAChT et ChAT: ce système de synthèse-séquestration définit fonctionnellement le phénotype cholinergique.

The role of GDNF family ligand signalling in the differentiation of sympathetic and dorsal root ganglion neurons

The diversity of neurons in sympathetic ganglia and dorsal root ganglia (DRG) provides intriguing systems for the analysis of neuronal differentiation. Cell surface receptors for the GDNF family ligands (GFLs) glial cell-line-derived neurotrophic factor (GDNF), neurturin and artemin, are expressed in subpopulations of these neurons prompting the question regarding their involvement in neuronal subtype specification. Mutational analysis in mice has demonstrated the requirement for GFL signalling during embryonic development of cholinergic sympathetic neurons as shown by the loss of expression from the cholinergic gene locus in ganglia from mice deficient for ret, the signal transducing subunit of the GFL receptor complex. Analysis in mutant animals and transgenic mice overexpressing GFLs demonstrates an effect on sensitivity to thermal and mechanical stimuli in DRG neurons correlating at least partially with the altered expression of transient receptor potential ion channels and acid-sensitive cation channels. Persistence of targeted cells in mutant ganglia suggests that the alterations are caused by differentiation effects and not by cell loss. Because of the massive effect of GFLs on neurite outgrowth, it remains to be determined whether GFL signalling acts directly on neuronal specification or indirectly via altered target innervation and access to other growth factors. The data show that GFL signalling is required for the specification of subpopulations of sensory and autonomic neurons. In order to comprehend this process fully, the role of individual GFLs, the transduction of the GFL signals, and the interplay of GFL signalling with other regulatory pathways need to be deciphered.

The cholinergic gene locus in amphioxus: Molecular characterization and developmental expression patterns

The cholinergic gene locus (CGL), consisting of the vesicular acetylcholine transporter (VAChT)/choline acetyltransferase (ChAT) gene, encodes two specific cholinergic neuronal markers used extensively to study cholinergic transmission. In the present work, we isolated the amphioxus homologs of VAChT and ChAT and examined their expression during development. Analysis of the 5' untranslated region of VAChT and ChAT suggests that the splicing of the VAChT/ChAT mRNA has been evolutionarily conserved in amphioxus and mammals. By double whole-mount in situ hybridization, we demonstrate that VAChT and ChAT are coexpressed in the same cells. They are first expressed in four pairs of differentiating cells in the neural plate. Their later expression is primarily in the anterior nerve cord in several types of motoneurons, some of the interneurons and in the receptor cells of the larval ocellus.

Cholinergic Differentiation Occurs Early in Mouse Sympathetic Neurons and Requires Phox2b

The generation of neurotransmitter identity in the autonomic nervous system is a classical model system to study the development of neuronal diversity. Analysis of the expression of genes coding for enzymes of noradrenaline biosynthesis in the sympathoadrenal system allowed the characterization of factors involved in the differentiation of the noradrenergic transmitter phenotype. The development of cholinergic properties in the autonomic system is less well understood. Here we show that expression of mRNAs for choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT), both encoded by the cholinergic gene locus, is induced in mouse sympathetic ganglia at embryonic day 11 (E11). Positive cells amount to more than 50% of Phox2b-positive sympathetic cells at cervical levels. The proportion declines caudally, decreasing to approximately 20% of Phox2b-positive cells at lower thoracic levels. In the adrenal anlage, ChAT and VAChT mRNA are largely undetectable at E11 and E13. In mice homozygous for a mutational inactivation of the transcription factor Phox2b, ChAT and VAChT mRNA expression is absent from sympathetic ganglia. The data show that expression from the cholinergic gene locus is regulated differently in sympathetic neurons and adrenal chromaffin cells. Phox2b is required for development of cholinergic neurons but does not suffice to support cholinergic properties in chromaffin cells.

Characterization of the REST/NRSF‐interacting LIM domain protein (RILP): localization and interaction with REST/NRSF

We previously identified a nuclear envelope protein repressor element-1 silencing transcription factor (REST)/neuron-restrictive silencer factor (NRSF)-interacting Lin-11, Isl-1 and Mec-3 (LIM) domain protein (RILP) that we proposed functions in the nuclear translocation of the transcriptional repressor REST/NRSF. In this study we assessed the functionality of the prenylation motif, protein kinase A (PKA) phosphorylation sites and nuclear localization sequences (NLSs) of RILP. [(3)H]-mevalonolactone labeled endogenous RILP, showing that RILP is indeed prenylated, while phosphorylation analysis showed that the two PKA sites are phosphorylated. Blocking RILP prenylation, mutating the NLSs or mutating the PKA phosphorylation sites caused RILP to mislocalize to the cytosol. Concurrent with this mislocalization of RILP, REST/NRSF and REST4, which are normally found in the nucleus, co-localized in the cytosol with the RILP mutants. This provides additional evidence that RILP interacts with REST/NRSF and REST4 in vivo, and is involved in the nuclear localization of REST/NRSF and REST4. Reporter gene analysis using the promoter region of the human cholinergic gene locus revealed that these RILP mutants prevented repression of the reporter gene. By trapping REST/NRSF in the cytosol, the RILP mutants prevented translocation to the nucleus where REST/NRSF binds to an RE-1/NRSE element to repress gene transcription. These results show that RILP is required for REST/NRSF nuclear targeting and function.

Expression of the cholinergic gene locus in the rat placenta.

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the "cholinergic gene locus" in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.

Regulation of the cholinergic gene locus by the repressor element-1 silencing transcription factor/neuron restrictive silencer factor (REST/NRSF)

The cholinergic gene locus is comprised of two genes, the choline acetyltransferase gene and the vesicular acetylcholine transporter gene. The vesicular acetylcholine transporter gene is located within the first intron of the choline acetyltransferase gene. This arrangement permits coordinate regulation of the locus. Protein kinase A regulates expression of the cholinergic gene locus in PC12 cells. This regulation was found to be dependent on the presence of a 21-bp DNA sequence known as the repressor element-1 (RE-1)/neuron-restrictive silencer element (NRSE). Repressor element-1 silencing transcription factor (REST)/ neuron-restrictive silencer factor (NRSF), which binds to the RE-1/NRSE, is a zinc finger containing transcriptional repressor that blocks the expression of many neuronal RE-1/NRSE containing genes in nonneuronal cells. However, REST/NRSF expression has also been observed in neurons as well as the PC12 cell line used in these studies. REST/NRSF truncated isoforms were expressed in neuronal cells, suggesting they also function in regulating neuronal gene expression. A study of REST4, one of the REST/NRSF isoforms, suggests that it regulates transcription of the cholinergic gene locus by blocking the repressor activity of REST/NRSF. Protein kinase A regulation of the cholinergic gene locus in PC12 cells can thus be attributed, at least in part, to increased synthesis of REST4, which in turn derepresses the repressor activity of REST/NRSF.

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

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 H-type, 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.

Identification of a region from the human cholinergic gene locus that targets expression of the vesicular acetylcholine transporter to a subset of neurons in the medial habenular nucleus in transgenic mice.

We use a transgenic mouse model system to elucidate the regulatory regions within the human cholinergic gene locus responsible for vesicular acetylcholine transporter gene expression in vivo. In this report we characterized two transgenes for their ability to confer cholinergic-specific expression of the encoded vesicular acetylcholine transporter. An 11.2 kb transgene (named hV11.2) that spanned from about 5 kb upstream of the start of vesicular acetylcholine transporter translation down to the first choline acetyltransferase coding exon gave expression in the somatomotor neurons and a subpopulation of cholinergic neurons in the medial habenular nucleus. The second transgene (named hV6.7), a 5-prime truncated version of hV11.2 that was devoid of 4.5 kb of gene-regulatory sequences completely lacked vesicular acetylcholine transporter expression in vivo. Our data indicate that vesicular acetylcholine transporter expression in somatomotor neurons and in the medial habenular nucleus is uniquely specified within the cholinergic gene locus, and separable from cholinergic expression elsewhere. The identification of these two subdivisions of the cholinergic nervous system suggests that other cholinergic neurons in the CNS and PNS are similarly regulated by additional discrete domains within the cholinergic gene locus.

VAChT-Cre. Fast and VAChT-Cre.Slow: postnatal expression of Cre recombinase in somatomotor neurons with different onset.

The cholinergic gene locus (CGL) consists of the genes encoding the choline acetyltransferase (ChAT) and the vesicular acetylcholine transporter (VAChT). To establish a cholinergic-specific Cre-expressing mouse, we constructed a transgene expression vector (VAChT-Cre) with 11.3 kb human CGL in which a Cre-IRES-EGFP unit was inserted in the VAChT open reading frame. The activity of Cre, whose expression was driven by the VAChT promoter, was examined by crossing a reporter mouse (CAG-CAT-Z) in which expression of LacZ is activated upon Cre-mediated recombination. Transgenic lines with the VAChT-Cre construct displayed the restricted Cre expression in a subset of cholinergic neurons in the somatomotor nuclei and medial habenular nucleus, but absent in visceromotor and other central and peripheral cholinergic neurons. Cre expression was first observed at postnatal day 7 and later detected in approximately 40-60% of somatomotor neurons. Based on the onset of Cre expression, we generated two mouse lines (two alleles; VAChT-Cre. Fast and VAChT-Cre.Slow) in which Cre expression reaches maximal levels fast and slow, respectively. The use of VAChT-Cre mice should allow us to deliver Cre to a subset of postnatal motor neurons, thereby bypassing lethality and facilitating analysis of gene function in adult motor neurons.

Regulation of cholinergic gene expression by the neuron restrictive silencer factor/repressor element-1 silencing transcription factor

The role of protein kinase A in regulating transcription of the cholinergic gene locus, which contains both the vesicular acetylcholine transporter gene and the choline acetyltransferase gene, was investigated in PC12 cells and a protein kinase A deficient PC12 mutant, A126.1B2 in which transcription of the locus is reduced. The site of action of protein kinase A was localized to a neuron restrictive silencer element/repressor element-1 (NRSE/RE-1) within the upstream region of the cholinergic gene locus. The neuron restrictive silencer factor/repressor element-1 silencing transcription factor (NRSF/REST), the transcription factor which binds to NRSE/RE-1, was expressed at similar levels in both PC12 and A126.1B2. Although nuclear extracts containing NRSF/REST from A126.1B2 exhibited binding to NRSE/RE-1, nuclear extracts from PC12 cells did not. The NRSF/REST isoform repressor element-1 silencing transcription factor-4 (REST4) was found to be expressed in PC12 cells, but not in the protein kinase A deficient PC12 cell line. REST4 inhibited the binding of NRSF/REST to NRSE/RE-1 as determined by gel mobility shift assays. Co-immunoprecipitation was used to demonstrate interaction between NRSF/REST and REST4. Expression of recombinant REST4 in the protein kinase A deficient PC12 cell line was sufficient to transcriptionally activate the cholinergic gene locus. Thus in PC12 cells protein kinase A promotes the production of REST4, which in turn de-represses of the cholinergic gene locus by inactivating the transcription repressor NRSF/REST.

Abnormal chemosensory jump 6 is a positive transcriptional regulator of the cholinergic gene locus in Drosophila olfactory neurons.

Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5' flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in Drosophila and other animals. Approximately 300 bases of proximal 5' flanking DNA control expression in Drosophila CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study we identify the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of acj6 mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5' flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional trans factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6.

Abnormal Chemosensory Jump 6 Is a Positive Transcriptional Regulator of the Cholinergic Gene Locus inDrosophilaOlfactory Neurons

Cholinergic neurons acquire their neurotransmitter phenotype, in part, by expressing the cholinergic gene locus. Previous studies have indicated that the 5′ flanking DNA of the locus contains both positive and negative regulatory elements important for expression in different subsets of cholinergic neurons in <i>Drosophila</i> and other animals. Approximately 300 bases of proximal 5′ flanking DNA control expression in <i>Drosophila</i> CNS neurons essential for viability, whereas more distal regulatory elements are important for expression in PNS sensory neurons. In this study we identify the POU domain transcription factor abnormal chemosensory jump 6 (Acj6) as a necessary positive transcriptional regulator for cholinergic locus expression in primary olfactory neurons. Choline acetyltransferase enzyme activity, protein levels, mRNA, and a fluorescent cholinergic reporter gene are all decreased in olfactory neurons of<i>acj6</i> mutants. Decreased cholinergic expression was observed in both adults and larvae. The presence of a specific Acj6 binding site has been identified in the cholinergic locus 5′ flanking DNA, suggesting that Acj6 may play a direct role in specifying the cholinergic neurotransmitter phenotype of most olfactory neurons. Transgenic expression of two different isoforms of Acj6 restricted to olfactory neurons indicates that additional <i>trans</i>factors may be required for cholinergic locus expression. Transgenic expression in all cholinergic neurons, however, results in lethality when a POU IV box element is absent but is essentially benign when present, indicating the importance of this motif in specifying different functional roles for Acj6.