Intronic Sequences Are Involved in Neural Targeting of Human Dopamine Transporter Gene Expression

doi:10.1006/bbrc.1997.7754

Biochemical and Biophysical Research Communications

Volume 240, Issue 3, 26 November 1997, Pages 807-811

https://doi.org/10.1006/bbrc.1997.7754 Get rights and content

Abstract

Dopamine transporter (DAT) plays a key role in terminating synaptic dopaminergic transmission. DAT acts exclusively on the plasma membrane of presynaptic dopaminergic neurons and DAT gene is an appropriate model for the study of dopaminergic neuron-specific regulation of gene activity. DAT represents an important target for widely used neuroleptic drugs and psychostimulants and for catecholamine-selective neurotoxins. Functional abnormalities of DAT have been implicated in diverse neurologic and psychiatric disorders. Understanding the mechanisms regulating human DAT gene activity is an important step towards elucidation of the molecular bases of a number of disorders and psychostimulant drug abuse and dependence. In this study we have cloned and characterised a 7-kb segment of the human DAT gene which includes at least 4 kb of its 5′-flanking region, localised its essential, or core-promoter, and identified the region involved in regulation of DAT neurospecific expression.

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Cited by (37)

Valproate increases dopamine transporter expression through histone acetylation and enhanced promoter binding of Nurr1
2017, Neuropharmacology
Citation Excerpt :
The dopamine transporter (DAT) is a transmembrane protein responsible for modulating dopaminergic transmission through selective uptake of the neurotransmitter dopamine (DA). Studies on the regulation of the DAT gene expression identified core promoter features and putative transcription factor binding motifs (Kouzmenko et al., 1997; Sacchetti et al., 1999), including that of the orphan nuclear receptor nuclear receptor related 1 protein (Nr4a2; Nurr1) and paired-like homeobox 3 protein (Pitx3) (Martinat et al., 2006; Riddle and Pollock, 2003; Smits and Smidt, 2006). In addition to transcription factor binding, the DAT promoter lacks a conserved TATA box, suggesting the potential for regulation by histone acetylation, similar to other TATA-less genes (Choi and Kim, 2008).
The dopamine transporter (DAT) is the key regulator of dopaminergic transmission and is a target of several xenobiotics, including pesticides and pharmacological agents. Previously, we identified a prominent role for histone deacetylases in the regulation of DAT expression. Here, we utilized a rat dopaminergic cell line (N27) to probe the responsiveness of DAT mRNA expression to inhibitors of histone acetylation. Inhibition of histone deacetylases (HDACs) by valproate, butyrate and Trichostatin A led to a 3–10-fold increase in DAT mRNA expression, a 50% increase in protein levels, which were accompanied by increased H3 acetylation levels. To confirm the mechanism of valproate-mediated increase in DAT mRNA, chromatin immunoprecipitation (ChIP) assays were used and demonstrated a significant increase in enrichment of acetylation of histone 3 on lysines 9 and 14 (H3K9/K14ac) in the DAT promoter. Expression of Nurr1 and Pitx3, key regulators of DAT expression, were increased following valproate treatment and Nurr1 binding was enriched in the DAT promoter. Together, these results indicate that histone acetylation and subsequent enhancement of transcription factor binding are plausible mechanisms for DAT regulation by valproate and, perhaps, by other xenobiotics.
Transgenic medaka fish which mimic the endogenous expression of neuronal kinesin, KIF5A
2012, Brain Research
Citation Excerpt :
Although medaka KIF5Aa and KIF5Ab were expressed at similar levels within neuronal tissues, specific neuronal gene expression appears to have been inherited only by the KIF5Aa gene; therefore, KIF5Aa is the more suitable gene for analyses pertaining to mechanisms which restrict expression to neurons. Previous reports have suggested that neuron-specific promoter activity is actualized by neuronal enhancers located upstream of the core promoter, and by non-neuronal repressors within the first intron (Uveges et al., 2002; Zhou et al., 2006; Kouzmenko et al., 1997). However, the latter case does not apply to the regulation of medaka KIF5Aa gene expression as the first intron is not necessary to mimic the endogenous expression pattern of the gene.
Intracellular transport is spatiotemporally controlled by microtubule-dependent motor proteins, including kinesins. In order to elucidate the mechanisms controlling kinesin expression, it is important to analyze their genomic regulatory regions. In this study, we cloned the neuronal tissue-specific kinesin in medaka fish and generated transgenic fish which mimic endogenous neuronal kinesin expression in order to elucidate the mechanisms which regulate kinesin expression. Searches for medaka neuronal orthologues by RT-PCR identified a candidate gene expressed only in neuronal tissues. Using BAC clones, we determined the cDNA sequence and the gene structure of the candidate neuronal kinesin. Evolutionary analysis indicated that the candidate gene encoded medaka KIF5Aa. The endogenous medaka orthologue was found to be expressed only in the nervous system, including the brain and spinal cord, while expression of KIF5Ab was not exclusive to neuronal tissues. Transgenic (Tg) medaka that expressed EGFP under the control of the 6.9 kbp 5′ and 1.9 kbp 3′ flanking regions of the KIF5Aa gene showed characteristic expression throughout the nervous system, including the brain, spinal cord, olfactory pit, eye and cranial nerve. Immunohistological analysis showed that EGFP expression in Tg fish co-localized with expression of HuC/D, a neuronal marker. These results demonstrate that the 6.9 kbp 5′ and 1.9 kbp 3′ flanking regions of medaka KIF5Aa have neuronal-specific promoter activity mimicking endogenous expression of medaka KIF5Ab. This transgenic fish strain will be useful for further functional analysis of the effects of these regulatory regions on gene expression.
Association between the dopamine transporter gene and the inattentive subtype of attention deficit hyperactivity disorder in Taiwan
2011, Progress in Neuro-Psychopharmacology and Biological Psychiatry
Citation Excerpt :
The DAT1 gene is expressed primarily in brain areas with dopaminergic circuitry, such as mesostriatal, mesolimbic, and mesocortical pathways (Ciliax et al., 1999), and this highly restricted pattern of DAT1 gene expression is presumably regulated by a unique combination of regulatory factors. Although intronic polymorphisms have no direct effect on the amino acid sequence, there are a growing number of examples of intronic sequences that play an important role in the tissue-specificity of DAT1 gene expression (Greenwood and Kelsoe, 2003; Kouzmenko et al., 1997). For example, data presented by Kouzmenko et al. (1997) indicated an involvement of the first intron sequences in restricting DAT1 gene expression to neuronal cells.
Attention deficit hyperactivity disorder (ADHD) is a common heritable childhood psychiatric disorder. Since methylphenidate, one of the main drugs used to treat ADHD, targets the dopamine transporter, this study examined the linkage disequilibrium (LD) structure of the dopamine transporter gene (DAT1) and investigated whether the DAT1 gene was associated with ADHD. This Chinese family-based association sample consisted of 273 DSM-IV diagnosed ADHD probands and their family members (n = 906). We screened 15 polymorphisms across the DAT1 gene, including 14 single nucleotide polymorphism (SNP) markers and the variable number of tandem repeat (VNTR) polymorphism in 3′-untranslated region (3′UTR). Calculations of pairwise LD revealed three main haplotype blocks (HBs): HB1 (intron 2 through intron 6), HB2 (intron 8 through intron 11), and HB3 (3′UTR). Family-Based Association Tests showed that no allele was significantly more transmitted than expected to the ADHD children for these 15 markers. Haplotype-Based Association Tests showed that a haplotype rs27048 (C)/rs429699 (T) was significantly associated with the inattentive subtype (P = 0.008). In quantitative analyses, this haplotype also demonstrated significant association with the inattention severity (P = 0.012). Our finding of the haplotype rs27048 (C)/rs429699 (T) as a novel genetic marker in the inattentive ADHD subtype suggests that variation in the DAT1 gene may primarily affect the inattentive subtype of ADHD.
Nicotine stimulates transcriptional activity of the human dopamine transporter gene
2010, Neuroscience Letters
Nicotine modulates dopaminergic activity in the central nervous system by acting on the reuptake system, including the dopamine transporter (DAT), although precisely remains unclear. Here we investigated the effect of nicotine on the transcriptional regulation of the human DAT (hDAT) gene by conducting luciferase reporter assays. Nicotine enhanced the transcription of hDAT gene constructs in transiently transfected SK-N-SH cells. Hexamethonium, a neuronal (ganglionic) nicotinic acetylcholine receptor antagonist, blocked the action of nicotine. Functional analyses placed the nicotine-responsive region −3.5 to −1.0 kb (from the transcription start site) upstream of the core promotor region. Deletion of intron 1, known as a silencer element of the hDAT gene, abolished nicotine's stimulatory effect. Nicotine failed to stimulate DAT promotor activity in non-neuronal CHO or COS-7 cells or in SK-N-AS cells, another neuronal cell line recently reported as a model for investigating DAT gene expression. These results suggest a nicotinic cholinergic mechanism to be involved in the nicotine-induced up-regulation of DAT gene expression.
Cis-acting elements responsible for dopaminergic neuron-specific expression of zebrafish slc6a3 (dopamine transporter) in vivo are located remote from the transcriptional start site
2009, Neuroscience
Citation Excerpt :
In vitro analysis of the human SLC6A3 promoter previously suggested that there is a strong basal promoter in the vicinity of the transcriptional start site, whose function is dependent on trans-activation by Sp1 and Sp3 (Wang and Bannon, 2005). Neural specificity of the human promoter in vitro is dictated by neuronal enhancers located upstream of the core promoter, and non-neuronal repressors including an element situated within the first intron (Kouzmenko et al., 1997). In light of this information, the presence of putative CpG island sequences within the first intron of the zebrafish slc6a3 gene, and the multiple known instances of genes with untranslated first exons, in which the first intron contains important regulatory sequences (Burton et al., 1999; Bai et al., 2007), we included intron 1 of slc6a3 in the transgene constructs used in this study.
The purpose of this study was to analyze the transcriptional regulation of the zebrafish solute carrier family 6 member 3 gene (slc6a3, dopamine transporter, dat), as a first step towards isolating regulatory sequences useful for driving transgene expression within dopaminergic neurons of the zebrafish CNS in vivo. We found that the 3.0 kb slc6a3 mRNA is expressed in each of the major groups of dopaminergic neurons previously identified in the zebrafish CNS. The slc6a3 gene spans >20 kb of genomic DNA and contains 15 exons. The genomic organization of slc6a3 is highly conserved with respect to its human orthologue, including the presence of an untranslated first exon. The promoter lacks a canonical TATA box and there are multiple transcriptional start sites. Functional analysis of cis-acting elements responsible for the expression pattern of slc6a3 was carried out by generating stable transgenic zebrafish lines expressing fluorescent reporters under transcriptional control of fragments of slc6a3 genomic sequence. The region between −2 kb and +5 kb with respect to the transcriptional start site contains the core slc6a3 promoter, in addition to neuronal enhancers and/or non-neuronal repressors that restrict expression to the CNS, but this region lacks cis-acting elements responsible for slc6a3 expression in dopaminergic neurons. The upstream sequence between −6 kb and −2 kb contains an enhancer element that drives slc6a3 expression in dopaminergic neurons of the pretectal region, and additional sequences that partially repress expression in non-dopaminergic neurons. However, expression of slc6a3 in dopaminergic neurons of the ventral diencephalon and telencephalon is dependent on elements that lie outside the region −6 kb to +5 kb. These data provide a detailed analysis of the slc6a3 gene and show that its expression in different populations of dopamine neurons is driven by discrete enhancers, rather than a single target sequence for a terminal factor involved in specifying neurochemical phenotype.
DAT, dopamine transporter
2007, xPharm: The Comprehensive Pharmacology Reference

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^☆: F. E. BloomD. J. Kupfer, Eds

¹: Corresponding author. Mental Health Research Institute, Locked Bag 11, Parkville, Victoria 3052, Australia. Fax: (+61-3)-9387 5061. E-mail: [email protected].

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Regular ArticleIntronic Sequences Are Involved in Neural Targeting of Human Dopamine Transporter Gene Expression☆

Abstract

Trends Pharmacol. Sci.

Mol. Brain Res.

Mol. Brain Res.

Neuron

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Psychopharmacology: The Fourth Generation of Progress

Regular Article
Intronic Sequences Are Involved in Neural Targeting of Human Dopamine Transporter Gene Expression☆