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The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders
European Neuropsychopharmacology 11 (2001) 449–455 www.elsevier.com / locate / euroneuro
The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders Michael J. Bannon*, Sharon K. Michelhaugh, Jun Wang, Paola Sacchetti Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Rm 2309 Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201, USA
Abstract The dopamine transporter is a plasma membrane protein that controls the spatial and temporal domains of dopamine neurotransmission through the accumulation of extracellular dopamine. The dopamine transporter may play a role in numerous dopamine-linked neuropsychiatric disorders. We review the cloning and organization of the human dopamine transporter gene, polymorphisms in its coding and noncoding sequence, and emerging data on its transcriptional regulation. 2001 Elsevier Science B.V. All rights reserved. Keywords: Gene expression; Transcriptional regulation; Gene polymorphisms; Neuron-restrictive silencer; nurr1; Variable number tandem repeat
1. Introduction Neurons utilizing dopamine (DA) as a neurotransmitter constitute a rare neurochemical phenotype (approximately one of every 10 6 CNS neurons), but nevertheless play an important role in regulating locomotion, motivation, cognition and hormone release. The DA transporter (DAT) is a plasma membrane transport protein that influences the spatio-temporal domains of DA neurotransmission by rapidly reaccumulating DA that has been released into the extracellular space (Fig. 1). It is a member of a large family of sodium- and chloride-dependent transporters, including the closely related norepinephrine transporter and serotonin transporter (SERT) (Amara and Sonders, 1998). DAT gene expression is limited exclusively to a subset of CNS (and retinal) DA neurons, yet robustly expressed within the midbrain (several hundred thousand DA neurons in the human). The DAT gene thus exhibits a truly extraordinary level of cellular specificity of expression — much more so than other genes related to DA phenotype (e.g. tyrosine hydroxylase, aromatic amino acid decarboxylase, vesicular monoamine transporter) — making the DAT an invaluable gene in which to characterize neuron-specific enhancers and silencers. A wide spectrum of neurological and psychiatric dis*Corresponding author. Tel.: 11-313-577-5949; fax: 11-313-9934269. E-mail address: [email protected] (M.J. Bannon).
orders, including Parkinson’s disease, schizophrenia, drug abuse, affective disorders, Tourette’s syndrome, and attention deficit hyperactivity disorder (ADHD) is thought to involve DA systems and the DAT in some manner (Bannon et al., 1998). The DAT is an important target for
Fig. 1. Schematic representation of dopamine transporter (DAT) function in a dopaminergic synapse. Dopamine (DA) is released into the synapse, where it binds to postsynaptic DA receptors or diffuses into the surrounding extracellular space. DA transported back into the presynaptic terminal via DAT may be repackaged into vesicles.
0924-977X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 01 )00122-5
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therapeutic and illicit drugs (e.g. methylphenidate, buproprion, amphetamine, and cocaine), and serves as the point of entry for DA-specific neurotoxins (e.g. MPP 1 ) (Amara and Sonders, 1998). DAT radioligand binding may provide an in vivo measure of DA cell integrity and the efficacy of therapeutic interventions in neurodegenerative disease (Stoof et al., 1999). A greater understanding of the regulation of the DAT is likely to impact the prevention, diagnosis and treatment of a number of neuropsychiatric disorders. Herein we review the cloning and organization of the human dopamine transporter (hDAT) gene, polymorphisms in its coding and non-coding sequence, and emerging data on its transcriptional regulation.
2. Structure of the hDAT gene
2.1. Cloning and exon /intron organization hDAT cDNAs were first isolated nearly a decade ago, using highly homologous rat DAT or human norepinephrine transporter cDNA sequences (Bannon et al., 1992; Giros et al., 1992; Vandenbergh et al., 1992). As expected, hDAT coding sequence exhibits .90% identity with rat DAT at the amino acid level. Like all members of this gene family, the hDAT is thought to contain 12 transmembrane domains, cytoplasmic amino- and carboxy-termini, and a large glycosylated second extracellular loop. When compared with the rat sequence, the hDAT possesses one less potential N-glycosylation site within this loop and one additional amino acid in the third extracellular loop. The functional importance of specific amino acids and domains within the hDAT has been the subject of intensive investigation, and has been reviewed in detail elsewhere (Chen and Reith, 2000). The 39 untranslated region of the hDAT cDNA is longer than in the rat and, unlike the rat, contains a very interesting repetitive element (see below). The hDAT gene (SLC6A3, also termed DAT1) is localized to chromosome 5p15.3 (Giros et al., 1992; Vandenbergh et al., 1992). The organization of the entire gene has been reported and many potential cis-elements identified by sequence analysis (Donovan et al., 1995; Kawarai et al., 1997; Kouzmenko et al., 1997; Sacchetti et al., 1999). The hDAT gene spans .60 kb and consists of 15 exons separated by 14 introns, with consensus se-
Fig. 3. Nucleotide sequence alignment of human and mouse DAT core promoter. The putative cis-acting regulatory elements are boxed.
quences for RNA splicing at each intron–exon junction (Fig. 2). The protein-coding portion of the gene begins within exon 2 and ends near the beginning of exon 15. Many individual exons encode a single intra- or extracellular domain and transmembrane domain (Kawarai et al., 1997). 59-Rapid Amplification of cDNA Ends (RACE) and RNase protection experiments have revealed a single transcription start site for the hDAT gene (Vandenbergh et al., 1992; Kawarai et al., 1997). There is no evidence at present for hDAT RNA splice variants or the use of multiple polyadenylation sites. Within the 39 non-coding region of hDAT lies a 40-nt repeat polymorphism, termed a variable number of tandem repeat (VNTR) polymorphism, which is discussed below.
2.2. Sequence analysis of hDAT regulatory region The 59-flanking sequences thought to control transcription of the hDAT gene are interesting in a number of regards (Donovan et al., 1995; Kawarai et al., 1997; Kouzmenko et al., 1997; Sacchetti et al., 1999). No canonical TATA and CAAT boxes are found immediately upstream of the transcription start site, suggesting that hDAT may be a TATA-less gene. Approximately 180 bp of GC-rich proximal 59 sequence containing multiple Sp1 sites is conserved between mouse and human DAT, and could possibly serve to direct transcription (Fig. 3). On the other hand, the single transcription start site and lack of initiator motif in hDAT are more consistent with a TATAcontaining promoter, and a conserved TATA-like TAAGA sequence located at 232 bp (relative to the start site; Fig.
Fig. 2. Organization of the DAT gene. Exons 1, 2 and 15 contain non-coding sequences (shaded boxes). Exons 2–15 contain the coding sequences for DAT (black boxes). The 39 untranslated region contains the variable number of tandem repeats (white boxes), with the 40 base consensus sequence for each repeat indicated above.
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3) may serve as a TATA box, although this has not been confirmed experimentally. GC-rich promoters lacking canonical TATA boxes were once thought to be indicative of ‘housekeeping’ genes, but a number of neuronal gene core promoters with properties similar to the hDAT have been reported (Kawarai et al., 1997). A limited number of potential transcription factor response elements (most prominently E box sequences) are found within the hDAT proximal 59-flanking sequence. To date, these sites have not been assessed for function. The relative paucity of response elements for activity-dependent factors (CREB, AP-1) may be in keeping with the general unresponsiveness of the DAT gene in vivo to a variety of stimuli (Bannon et al., 1998). Recently, over 8 kb of 59-flanking sequence have been sequenced (Sacchetti et al., 1999). As would be expected, many potential response elements are suggested on the basis of sequence analysis, but only a few of these have been characterized for their effects on hDAT transcription (see below).
3. hDAT gene polymorphisms
3.1. VNTR The quantitatively most important hDAT polymorphism occurs in the 39 untranslated region, just upstream of the polyadenylation site. Alleles of this 40-bp variable number of tandem repeat (VNTR) sequence range from three to 13 repeats, with the nine-repeat and ten-repeat alleles by far the most common (Fig. 2) (Vandenbergh et al., 1992). Perhaps the best evidence for association of the hDAT with a neuropsychiatric disorder comes from the study of ADHD. ADHD appears to be a heritable, polygenetic disorder. The therapeutic effects of DAT ligands (e.g. methylphenidate and amphetamine) in the treatment of ADHD have prompted consideration of hDAT as a candidate gene for the disorder. The interpretation of population-based association analyses is rendered difficult because of ethnic variations in frequencies of hDAT VNTR alleles (Vandenbergh et al., 1992). A number of investigators have evaluated the DAT and ADHD using haplotype-based haplotype relative risk analyses for family based association studies (Cook et al., 1995; Gill et al., 1997; Waldman et al., 1998; Daly et al., 1999). Although the ‘high-risk’ allele is the ten-copy VNTR repeat, this is also the highest frequency allele in the general population, suggesting that this may be a modest risk factor in a polygenetic disorder. A recent report (Winsberg and Comings, 1999) suggests that homozygosity of the ten-repeat is associated with poor response to methylphenidate. Two studies have also examined the relationship between in vivo binding of the DAT (and SERT) ligand 123 I-b-CIT (as seen by single photon emission computerized tomography) and DAT VNTR genotype. While one study (Jacobsen et al., 2000)
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found that ten-repeat allele subjects had lower DAT binding than nine-repeat subjects, the other study (Heinz et al., 2000) reported that ten-repeat subjects had higher DAT binding than nine- / ten-repeat subjects. Finally, smaller numbers of studies have investigated the possible association between this hDAT polymorphism and bipolar disorder, schizophrenia, Tourette’s syndrome, drug abuse and drug-induced paranoia, alcoholism and alcohol withdrawal, and Parkinson’s disease, with mixed results (Bannon et al., 1998; Vandenbergh et al., 2000). The question remains as to how allelic differences in the VNTR might affect hDAT function. Since the VNTR is in the 39 non-coding region, allelic variants cannot result in structural or functional differences in the hDAT protein. On the other hand, recent evidence suggests that VNTRs can function as transcriptional and translational regulators (Nakamura et al., 1998). The closely related SERT gene contains a VNTR within a non-coding (intronic) region and allelic differences in SERT VNTR copy number are associated with susceptibility to a variety of anxiety-related disorders (Fiskerstrand et al., 1999; MacKenzie and Quinn, 1999 and references therein). Recent provocative studies have demonstrated that the SERT VNTR can functional as an enhancer of gene transcription and, furthermore, that allelic variants of the SERT VNTR exhibit quantitatively and qualitatively different activities (Fiskerstrand et al., 1999; MacKenzie and Quinn, 1999). It is plausible that the hDAT VNTR functions in a similar manner, although this awaits direct experimental analysis.
3.2. Protein coding and intron sequences Alternatively, it is possible that the VNTR polymorphism is not directly involved in the etiology of any disorder but is in linkage disequilibrium with another variant or variants within the coding, intron or 59 flanking region of the gene. Polymorphisms in these regions have been examined in a recent series of papers, using single strand conformation analysis, restriction fragment length polymorphism and direct sequencing of PCR products. The identification of polymorphisms scattered throughout the hDAT gene should help to clarify any association between the hDAT and DA-related neuropsychiatric disorders. One report (Grunhage et al., 2000) screened the complete coding region and exon–intron boundaries of the hDAT in bipolar disorder and control subjects. Five variants of exon-flanking intron sequences were identified, but none altered RNA splice sites. Within the coding region, three single nucleotide polymorphisms that did not change the encoded amino acid were detected. Two rare missense mutations within the coding region were identified in single bipolar patients: one conservative mutation (Ala559Val) not co-segregating with disease, and one nonconservative mutation (Glu602Gly) inherited from an affected father, leaving at least the possibility that it may be a rare cause of bipolar disorder.
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Another group (Vandenbergh et al., 2000) used similar methods to analyze the hDAT gene in a large group of Tourette’s disorder, alcoholism, ADHD, and control subjects. They identified a number of silent single nucleotide changes in four different exons and the 39 untranslated region, as well a new VNTR polymorphism in intron 8. Two rare conservative mutations in coding sequence (Val55Ala and Val 382Ala) were also identified. All polymorphisms were distinct from those reported by Grunhage et al. (2000). No non-conservative mutations were observed in any subjects. No significant differences were found in the frequencies of sequence variants across diagnostic groups. A third group (Morino et al., 2000) screened the exon sequences of a small group of Parkinson’s disease and control subjects, identifying only two silent single nucleotide polymorphisms, both distinct from those polymorphisms reported by the other groups. A larger cohort of Parkinson’s disease and control subjects were examined for these two variants. The silent 1215A / G polymorphism was reportedly more frequent in controls than affected subjects. In summary, the polymorphisms identified within the coding region of hDAT were nearly all either silent single nucleotide changes or rare conservative amino acid substitutions. The strong conservation of hDAT coding sequences is consistent with the overall conservation of gene and protein structure among related members of this transporter family. Importantly, the conservation of hDAT coding sequence suggests that individual differences in hDAT expression, if identified, must arise from variations in hDAT regulatory sequences and / or the trans factors acting upon them.
3.3. 59 -Flanking sequence Some 59-flanking sequences of the hDAT gene (21586 to 197 bp) have been examined recently for polymorphisms (Rubie et al., 2001). Five single nucleotide polymorphisms were identified: T-67A, G-660C, C-839T, C1169G and T-1476G; the first, third, and fourth of these constitute common alleles. The investigators suggested that most of the 59 polymorphic sequences they identified fall within putative transcription factor binding sites, and that the alleleic variants would impact the nature of these response elements. This possibility highlights the need to characterize the transcriptional regulation of the hDAT gene.
sequence is capable of driving reporter construct expression at a level equivalent to that seen with an SV40 promoter (Kouzmenko et al., 1997). Likewise, 800 bp of proximal hDAT promoter drives reporter gene expression at a level 10–100% of that seen with SV40 promoter and 10–150 times the activity of a promoterless construct (Sacchetti et al., 1999). The core and proximal hDAT promoter are without neurospecificity, being strongly expressed in both neurally-derived (NC14.4.6E, NC14.9.1, SH-SY5Y, PC12) and non-neuronal (HeLa, JAR, HEH293T, Y-1, AtT20) cell lines (Kouzmenko et al., 1997; Sacchetti et al., 1999).
4.2. Multiple mechanisms are likely involved in hDAT silencing The non-selective activity of the proximal hDAT promoter suggests that one or more silencing elements outside of this core region must contribute to the exquisite cellular specificity of native hDAT gene expression observed in vivo. Sequences within intron 1 reportedly contribute to hDAT silencing within non-neuronal cells (Fig. 4) (Kouzmenko et al., 1997). Other studies suggest complex silencing mechanisms involving 59-flanking sequences: the addition of 2000 bp of upstream hDAT sequence to the strong proximal promoter partially silences reporter construct expression within both neural and non-neuronal cells lines, suggesting that some element(s) within this sequence might silence hDAT expression in any DAT-negative cell. Inclusion of additional upstream sequence appears to further silence construct expression within non-neuronal (but not neuronal) cells (Fig. 4) (Sacchetti et al., 1999). This additional sequence includes a potential neural restrictive silencing element (NRSE) at 23042 bp. The NRSE is the best-characterized neuronal silencing element, being involved in neural-specific expression of many genes (Schoenherr et al., 1996). Studies are underway to determine the functionality of the putative hDAT NRSE. Overall, the data available suggest that several strong elements act in a combinatorial manner to completely silence the strong basal activity of the core hDAT promoter, not only in non-neuronal cells, but within non-DA neurons as well (Fig. 4). The recent description of a DAT-expressing cell line (Son et al., 1999) may facilitate the identification and analysis of these elements.
4. Transcriptional regulation of the hDAT gene
4.1. Strong, non-selective proximal promoter activity The hDAT gene core promoter seems to be located between 2240 and 145 bp relative to the start site. This
Fig. 4. Putative silencing domains within hDAT gene. Nucleotide numbering is in relation to the transcriptional start site (defined as 11). Shaded boxes indicate the proposed silencing elements.
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4.3. DAT is NOT-regulated The transcription factor NOT (termed nurr1 in the mouse) is an orphan receptor of the hormone receptor superfamily. NOT / nurr1 expression largely parallels the distribution of DA neurons, and homologous recombination experiments have shown that this transcription factor is essential for the proper development of mesencephalic DA cells (Zetterstrom et al., 1997; Castillo et al., 1998; Saucedo-Cardenas et al., 1998). NOT / nurr1 expression persists in adult brain (Xiao et al., 1996) and may play a role in the maintenance of DA phenotype as well. NOT / nurr1 typically binds to an extended half-hormone response element termed NGFI-B responsive element (NBRE; Wilson et al., 1991; Murphy et al., 1996). The hDAT promoter contains a canonical NBRE and several NBRE-like sequences within 59-flanking regions of the gene (Sacchetti et al., 1999). NOT / nurr1 activates transcription of hDAT gene constructs whereas other members
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of the NGFI-B subfamily of nuclear receptors have lesser or no effect (Sacchetti et al., 2001), suggesting a rather specific role for NOT / nurr1 in the transcriptional regulation of hDAT, consistent with the distinct spatial patterns of expression of NGFI-B family members (Zetterstrom et al., 1996). Although, under some conditions, NOT / nurr1 can heterodimerize with another hormone receptor, the retinoid X receptor, NOT / nurr1 activation of the hDAT gene is retinoid X receptor-independent (Sacchetti et al., 2001). Examining the responsiveness of a series of hDAT reporter constructs encompassing different lengths of 59flanking region unexpectedly revealed that NOT / nurr1 activates hDAT gene transcription via an NBRE-independent mechanism (Sacchetti et al., 2001). Precedent for such an indirect mechanism exists for other members of the nuclear receptor superfamily (Glass and Rosenfeld, 2000). Further studies are underway to determine the precise cis- and trans-elements mediating the activation of hDAT gene transcription by NOT / nurr1. Other recent
Fig. 5. Biolistic transfection of DA neurons in organotypic slice cultures. Midbrain slices from postnatal day 5 rat pups were biolistically transfected with a plasmid containing a non-coding sequence from the DAT gene driving the expression of green fluorescent protein. (A) Detection of green fluorescent protein-labeled neuron 48 h after transfection. (B) Tyrosine hydroxylase immunohistochemistry detected with a cy3-conjugated secondary antibody (red). (C) Computer-merged image of A and B. Overlap of green and red signal is depicted as yellow. Arrow indicates tyrosine hydroxylase-positive cell that was not transfected. (D) Computer-merged image of A and a transmission scan of the same microscopic field. Arrow indicates the gold microbead that carried the plasmid into the transfected DA cell.
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experiments indicate that hDAT and NOT gene expression change in parallel in certain pathological conditions (unpublished data), suggesting that NOT plays a critical role in controlling hDAT gene expression in vivo.
5. Emerging strategies for studying hDAT regulatory elements in situ Developing a complete understanding of the mechanisms underlying the extraordinary cellular specificity of hDAT gene expression is a daunting task. This task is made more difficult by the general unavailability of neuronal cell lines stably expressing a strong DAT phenotype. Any methodologies for introducing hDAT gene constructs into authentic DA neurons could facilitate the analysis of hDAT regulatory elements in the appropriate cellular context. Transfection of DA neurons is being attempted using viral vectors (Pothos et al., 2000) and receptor-mediated DNA internalization (Martinez-Fong et al., 1999). Our laboratory has developed a methodology for particle-mediated (biolistic) transfection of DA neurons in organotypic slice culture (Fig. 5). This approach will expedite the assessment of numerous hDAT constructs and transcriptional elements, as a prelude to confirmatory studies of selected constructs in transgenic animals.
6. Conclusion The regulation of DAT gene expression may be relevant to neuropsychiatric diseases in a number of ways. First, although the DAT gene may not be convincingly linked to most brain disorders at the present time, the DAT could contribute to disease processes as a component of a polygenetic disorder. Second, the DAT is an obligatory target for some disease-inducing factors, such as Parkinson’s disease-inducing neurotoxins and addictive psychostimulants. Finally, the alterations in DAT gene expression that accompanying a variety of pathological processes (Bannon et al., 1998) may represent homeostatic responses to dysfunction of DA cells or associated neural circuits. As an example, in Parkinson’s disease, which entails extensive DA cell loss, the DAT gene expression per surviving DA cell is down-regulated (Blanchard et al., 1994; Harrington et al., 1996; Joyce et al., 1997). A diminished capacity to recapture extracellular DA via the DAT may represent an adaptive mechanism to augment DA neurotransmission in the Parkinsonian brain. A greater understanding of the transcriptional regulation of the hDAT gene may provide novel therapeutic strategies for the treatment of Parkinson’s disease and other DA-linked neurological and psychiatric disorders.
Acknowledgements The work of the authors was supported by NIH grants DA06470, MH47181, MH60854, NS34935, and DA07310.
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The human dopamine transporter gene: gene organization, transcriptional regulation, and potential involvement in neuropsychiatric disorders Michael J. Bannon*, Sharon K. Michelhaugh, Jun Wang, Paola Sacchetti Department of Psychiatry and Behavioral Neurosciences, Wayne State University School of Medicine, Rm 2309 Scott Hall, 540 E. Canfield Ave., Detroit, MI 48201, USA
Abstract The dopamine transporter is a plasma membrane protein that controls the spatial and temporal domains of dopamine neurotransmission through the accumulation of extracellular dopamine. The dopamine transporter may play a role in numerous dopamine-linked neuropsychiatric disorders. We review the cloning and organization of the human dopamine transporter gene, polymorphisms in its coding and noncoding sequence, and emerging data on its transcriptional regulation. 2001 Elsevier Science B.V. All rights reserved. Keywords: Gene expression; Transcriptional regulation; Gene polymorphisms; Neuron-restrictive silencer; nurr1; Variable number tandem repeat
1. Introduction Neurons utilizing dopamine (DA) as a neurotransmitter constitute a rare neurochemical phenotype (approximately one of every 10 6 CNS neurons), but nevertheless play an important role in regulating locomotion, motivation, cognition and hormone release. The DA transporter (DAT) is a plasma membrane transport protein that influences the spatio-temporal domains of DA neurotransmission by rapidly reaccumulating DA that has been released into the extracellular space (Fig. 1). It is a member of a large family of sodium- and chloride-dependent transporters, including the closely related norepinephrine transporter and serotonin transporter (SERT) (Amara and Sonders, 1998). DAT gene expression is limited exclusively to a subset of CNS (and retinal) DA neurons, yet robustly expressed within the midbrain (several hundred thousand DA neurons in the human). The DAT gene thus exhibits a truly extraordinary level of cellular specificity of expression — much more so than other genes related to DA phenotype (e.g. tyrosine hydroxylase, aromatic amino acid decarboxylase, vesicular monoamine transporter) — making the DAT an invaluable gene in which to characterize neuron-specific enhancers and silencers. A wide spectrum of neurological and psychiatric dis*Corresponding author. Tel.: 11-313-577-5949; fax: 11-313-9934269. E-mail address: [email protected] (M.J. Bannon).
orders, including Parkinson’s disease, schizophrenia, drug abuse, affective disorders, Tourette’s syndrome, and attention deficit hyperactivity disorder (ADHD) is thought to involve DA systems and the DAT in some manner (Bannon et al., 1998). The DAT is an important target for
Fig. 1. Schematic representation of dopamine transporter (DAT) function in a dopaminergic synapse. Dopamine (DA) is released into the synapse, where it binds to postsynaptic DA receptors or diffuses into the surrounding extracellular space. DA transported back into the presynaptic terminal via DAT may be repackaged into vesicles.
0924-977X / 01 / $ – see front matter 2001 Elsevier Science B.V. All rights reserved. PII: S0924-977X( 01 )00122-5
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therapeutic and illicit drugs (e.g. methylphenidate, buproprion, amphetamine, and cocaine), and serves as the point of entry for DA-specific neurotoxins (e.g. MPP 1 ) (Amara and Sonders, 1998). DAT radioligand binding may provide an in vivo measure of DA cell integrity and the efficacy of therapeutic interventions in neurodegenerative disease (Stoof et al., 1999). A greater understanding of the regulation of the DAT is likely to impact the prevention, diagnosis and treatment of a number of neuropsychiatric disorders. Herein we review the cloning and organization of the human dopamine transporter (hDAT) gene, polymorphisms in its coding and non-coding sequence, and emerging data on its transcriptional regulation.
2. Structure of the hDAT gene
2.1. Cloning and exon /intron organization hDAT cDNAs were first isolated nearly a decade ago, using highly homologous rat DAT or human norepinephrine transporter cDNA sequences (Bannon et al., 1992; Giros et al., 1992; Vandenbergh et al., 1992). As expected, hDAT coding sequence exhibits .90% identity with rat DAT at the amino acid level. Like all members of this gene family, the hDAT is thought to contain 12 transmembrane domains, cytoplasmic amino- and carboxy-termini, and a large glycosylated second extracellular loop. When compared with the rat sequence, the hDAT possesses one less potential N-glycosylation site within this loop and one additional amino acid in the third extracellular loop. The functional importance of specific amino acids and domains within the hDAT has been the subject of intensive investigation, and has been reviewed in detail elsewhere (Chen and Reith, 2000). The 39 untranslated region of the hDAT cDNA is longer than in the rat and, unlike the rat, contains a very interesting repetitive element (see below). The hDAT gene (SLC6A3, also termed DAT1) is localized to chromosome 5p15.3 (Giros et al., 1992; Vandenbergh et al., 1992). The organization of the entire gene has been reported and many potential cis-elements identified by sequence analysis (Donovan et al., 1995; Kawarai et al., 1997; Kouzmenko et al., 1997; Sacchetti et al., 1999). The hDAT gene spans .60 kb and consists of 15 exons separated by 14 introns, with consensus se-
Fig. 3. Nucleotide sequence alignment of human and mouse DAT core promoter. The putative cis-acting regulatory elements are boxed.
quences for RNA splicing at each intron–exon junction (Fig. 2). The protein-coding portion of the gene begins within exon 2 and ends near the beginning of exon 15. Many individual exons encode a single intra- or extracellular domain and transmembrane domain (Kawarai et al., 1997). 59-Rapid Amplification of cDNA Ends (RACE) and RNase protection experiments have revealed a single transcription start site for the hDAT gene (Vandenbergh et al., 1992; Kawarai et al., 1997). There is no evidence at present for hDAT RNA splice variants or the use of multiple polyadenylation sites. Within the 39 non-coding region of hDAT lies a 40-nt repeat polymorphism, termed a variable number of tandem repeat (VNTR) polymorphism, which is discussed below.
2.2. Sequence analysis of hDAT regulatory region The 59-flanking sequences thought to control transcription of the hDAT gene are interesting in a number of regards (Donovan et al., 1995; Kawarai et al., 1997; Kouzmenko et al., 1997; Sacchetti et al., 1999). No canonical TATA and CAAT boxes are found immediately upstream of the transcription start site, suggesting that hDAT may be a TATA-less gene. Approximately 180 bp of GC-rich proximal 59 sequence containing multiple Sp1 sites is conserved between mouse and human DAT, and could possibly serve to direct transcription (Fig. 3). On the other hand, the single transcription start site and lack of initiator motif in hDAT are more consistent with a TATAcontaining promoter, and a conserved TATA-like TAAGA sequence located at 232 bp (relative to the start site; Fig.
Fig. 2. Organization of the DAT gene. Exons 1, 2 and 15 contain non-coding sequences (shaded boxes). Exons 2–15 contain the coding sequences for DAT (black boxes). The 39 untranslated region contains the variable number of tandem repeats (white boxes), with the 40 base consensus sequence for each repeat indicated above.
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3) may serve as a TATA box, although this has not been confirmed experimentally. GC-rich promoters lacking canonical TATA boxes were once thought to be indicative of ‘housekeeping’ genes, but a number of neuronal gene core promoters with properties similar to the hDAT have been reported (Kawarai et al., 1997). A limited number of potential transcription factor response elements (most prominently E box sequences) are found within the hDAT proximal 59-flanking sequence. To date, these sites have not been assessed for function. The relative paucity of response elements for activity-dependent factors (CREB, AP-1) may be in keeping with the general unresponsiveness of the DAT gene in vivo to a variety of stimuli (Bannon et al., 1998). Recently, over 8 kb of 59-flanking sequence have been sequenced (Sacchetti et al., 1999). As would be expected, many potential response elements are suggested on the basis of sequence analysis, but only a few of these have been characterized for their effects on hDAT transcription (see below).
3. hDAT gene polymorphisms
3.1. VNTR The quantitatively most important hDAT polymorphism occurs in the 39 untranslated region, just upstream of the polyadenylation site. Alleles of this 40-bp variable number of tandem repeat (VNTR) sequence range from three to 13 repeats, with the nine-repeat and ten-repeat alleles by far the most common (Fig. 2) (Vandenbergh et al., 1992). Perhaps the best evidence for association of the hDAT with a neuropsychiatric disorder comes from the study of ADHD. ADHD appears to be a heritable, polygenetic disorder. The therapeutic effects of DAT ligands (e.g. methylphenidate and amphetamine) in the treatment of ADHD have prompted consideration of hDAT as a candidate gene for the disorder. The interpretation of population-based association analyses is rendered difficult because of ethnic variations in frequencies of hDAT VNTR alleles (Vandenbergh et al., 1992). A number of investigators have evaluated the DAT and ADHD using haplotype-based haplotype relative risk analyses for family based association studies (Cook et al., 1995; Gill et al., 1997; Waldman et al., 1998; Daly et al., 1999). Although the ‘high-risk’ allele is the ten-copy VNTR repeat, this is also the highest frequency allele in the general population, suggesting that this may be a modest risk factor in a polygenetic disorder. A recent report (Winsberg and Comings, 1999) suggests that homozygosity of the ten-repeat is associated with poor response to methylphenidate. Two studies have also examined the relationship between in vivo binding of the DAT (and SERT) ligand 123 I-b-CIT (as seen by single photon emission computerized tomography) and DAT VNTR genotype. While one study (Jacobsen et al., 2000)
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found that ten-repeat allele subjects had lower DAT binding than nine-repeat subjects, the other study (Heinz et al., 2000) reported that ten-repeat subjects had higher DAT binding than nine- / ten-repeat subjects. Finally, smaller numbers of studies have investigated the possible association between this hDAT polymorphism and bipolar disorder, schizophrenia, Tourette’s syndrome, drug abuse and drug-induced paranoia, alcoholism and alcohol withdrawal, and Parkinson’s disease, with mixed results (Bannon et al., 1998; Vandenbergh et al., 2000). The question remains as to how allelic differences in the VNTR might affect hDAT function. Since the VNTR is in the 39 non-coding region, allelic variants cannot result in structural or functional differences in the hDAT protein. On the other hand, recent evidence suggests that VNTRs can function as transcriptional and translational regulators (Nakamura et al., 1998). The closely related SERT gene contains a VNTR within a non-coding (intronic) region and allelic differences in SERT VNTR copy number are associated with susceptibility to a variety of anxiety-related disorders (Fiskerstrand et al., 1999; MacKenzie and Quinn, 1999 and references therein). Recent provocative studies have demonstrated that the SERT VNTR can functional as an enhancer of gene transcription and, furthermore, that allelic variants of the SERT VNTR exhibit quantitatively and qualitatively different activities (Fiskerstrand et al., 1999; MacKenzie and Quinn, 1999). It is plausible that the hDAT VNTR functions in a similar manner, although this awaits direct experimental analysis.
3.2. Protein coding and intron sequences Alternatively, it is possible that the VNTR polymorphism is not directly involved in the etiology of any disorder but is in linkage disequilibrium with another variant or variants within the coding, intron or 59 flanking region of the gene. Polymorphisms in these regions have been examined in a recent series of papers, using single strand conformation analysis, restriction fragment length polymorphism and direct sequencing of PCR products. The identification of polymorphisms scattered throughout the hDAT gene should help to clarify any association between the hDAT and DA-related neuropsychiatric disorders. One report (Grunhage et al., 2000) screened the complete coding region and exon–intron boundaries of the hDAT in bipolar disorder and control subjects. Five variants of exon-flanking intron sequences were identified, but none altered RNA splice sites. Within the coding region, three single nucleotide polymorphisms that did not change the encoded amino acid were detected. Two rare missense mutations within the coding region were identified in single bipolar patients: one conservative mutation (Ala559Val) not co-segregating with disease, and one nonconservative mutation (Glu602Gly) inherited from an affected father, leaving at least the possibility that it may be a rare cause of bipolar disorder.
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Another group (Vandenbergh et al., 2000) used similar methods to analyze the hDAT gene in a large group of Tourette’s disorder, alcoholism, ADHD, and control subjects. They identified a number of silent single nucleotide changes in four different exons and the 39 untranslated region, as well a new VNTR polymorphism in intron 8. Two rare conservative mutations in coding sequence (Val55Ala and Val 382Ala) were also identified. All polymorphisms were distinct from those reported by Grunhage et al. (2000). No non-conservative mutations were observed in any subjects. No significant differences were found in the frequencies of sequence variants across diagnostic groups. A third group (Morino et al., 2000) screened the exon sequences of a small group of Parkinson’s disease and control subjects, identifying only two silent single nucleotide polymorphisms, both distinct from those polymorphisms reported by the other groups. A larger cohort of Parkinson’s disease and control subjects were examined for these two variants. The silent 1215A / G polymorphism was reportedly more frequent in controls than affected subjects. In summary, the polymorphisms identified within the coding region of hDAT were nearly all either silent single nucleotide changes or rare conservative amino acid substitutions. The strong conservation of hDAT coding sequences is consistent with the overall conservation of gene and protein structure among related members of this transporter family. Importantly, the conservation of hDAT coding sequence suggests that individual differences in hDAT expression, if identified, must arise from variations in hDAT regulatory sequences and / or the trans factors acting upon them.
3.3. 59 -Flanking sequence Some 59-flanking sequences of the hDAT gene (21586 to 197 bp) have been examined recently for polymorphisms (Rubie et al., 2001). Five single nucleotide polymorphisms were identified: T-67A, G-660C, C-839T, C1169G and T-1476G; the first, third, and fourth of these constitute common alleles. The investigators suggested that most of the 59 polymorphic sequences they identified fall within putative transcription factor binding sites, and that the alleleic variants would impact the nature of these response elements. This possibility highlights the need to characterize the transcriptional regulation of the hDAT gene.
sequence is capable of driving reporter construct expression at a level equivalent to that seen with an SV40 promoter (Kouzmenko et al., 1997). Likewise, 800 bp of proximal hDAT promoter drives reporter gene expression at a level 10–100% of that seen with SV40 promoter and 10–150 times the activity of a promoterless construct (Sacchetti et al., 1999). The core and proximal hDAT promoter are without neurospecificity, being strongly expressed in both neurally-derived (NC14.4.6E, NC14.9.1, SH-SY5Y, PC12) and non-neuronal (HeLa, JAR, HEH293T, Y-1, AtT20) cell lines (Kouzmenko et al., 1997; Sacchetti et al., 1999).
4.2. Multiple mechanisms are likely involved in hDAT silencing The non-selective activity of the proximal hDAT promoter suggests that one or more silencing elements outside of this core region must contribute to the exquisite cellular specificity of native hDAT gene expression observed in vivo. Sequences within intron 1 reportedly contribute to hDAT silencing within non-neuronal cells (Fig. 4) (Kouzmenko et al., 1997). Other studies suggest complex silencing mechanisms involving 59-flanking sequences: the addition of 2000 bp of upstream hDAT sequence to the strong proximal promoter partially silences reporter construct expression within both neural and non-neuronal cells lines, suggesting that some element(s) within this sequence might silence hDAT expression in any DAT-negative cell. Inclusion of additional upstream sequence appears to further silence construct expression within non-neuronal (but not neuronal) cells (Fig. 4) (Sacchetti et al., 1999). This additional sequence includes a potential neural restrictive silencing element (NRSE) at 23042 bp. The NRSE is the best-characterized neuronal silencing element, being involved in neural-specific expression of many genes (Schoenherr et al., 1996). Studies are underway to determine the functionality of the putative hDAT NRSE. Overall, the data available suggest that several strong elements act in a combinatorial manner to completely silence the strong basal activity of the core hDAT promoter, not only in non-neuronal cells, but within non-DA neurons as well (Fig. 4). The recent description of a DAT-expressing cell line (Son et al., 1999) may facilitate the identification and analysis of these elements.
4. Transcriptional regulation of the hDAT gene
4.1. Strong, non-selective proximal promoter activity The hDAT gene core promoter seems to be located between 2240 and 145 bp relative to the start site. This
Fig. 4. Putative silencing domains within hDAT gene. Nucleotide numbering is in relation to the transcriptional start site (defined as 11). Shaded boxes indicate the proposed silencing elements.
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4.3. DAT is NOT-regulated The transcription factor NOT (termed nurr1 in the mouse) is an orphan receptor of the hormone receptor superfamily. NOT / nurr1 expression largely parallels the distribution of DA neurons, and homologous recombination experiments have shown that this transcription factor is essential for the proper development of mesencephalic DA cells (Zetterstrom et al., 1997; Castillo et al., 1998; Saucedo-Cardenas et al., 1998). NOT / nurr1 expression persists in adult brain (Xiao et al., 1996) and may play a role in the maintenance of DA phenotype as well. NOT / nurr1 typically binds to an extended half-hormone response element termed NGFI-B responsive element (NBRE; Wilson et al., 1991; Murphy et al., 1996). The hDAT promoter contains a canonical NBRE and several NBRE-like sequences within 59-flanking regions of the gene (Sacchetti et al., 1999). NOT / nurr1 activates transcription of hDAT gene constructs whereas other members
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of the NGFI-B subfamily of nuclear receptors have lesser or no effect (Sacchetti et al., 2001), suggesting a rather specific role for NOT / nurr1 in the transcriptional regulation of hDAT, consistent with the distinct spatial patterns of expression of NGFI-B family members (Zetterstrom et al., 1996). Although, under some conditions, NOT / nurr1 can heterodimerize with another hormone receptor, the retinoid X receptor, NOT / nurr1 activation of the hDAT gene is retinoid X receptor-independent (Sacchetti et al., 2001). Examining the responsiveness of a series of hDAT reporter constructs encompassing different lengths of 59flanking region unexpectedly revealed that NOT / nurr1 activates hDAT gene transcription via an NBRE-independent mechanism (Sacchetti et al., 2001). Precedent for such an indirect mechanism exists for other members of the nuclear receptor superfamily (Glass and Rosenfeld, 2000). Further studies are underway to determine the precise cis- and trans-elements mediating the activation of hDAT gene transcription by NOT / nurr1. Other recent
Fig. 5. Biolistic transfection of DA neurons in organotypic slice cultures. Midbrain slices from postnatal day 5 rat pups were biolistically transfected with a plasmid containing a non-coding sequence from the DAT gene driving the expression of green fluorescent protein. (A) Detection of green fluorescent protein-labeled neuron 48 h after transfection. (B) Tyrosine hydroxylase immunohistochemistry detected with a cy3-conjugated secondary antibody (red). (C) Computer-merged image of A and B. Overlap of green and red signal is depicted as yellow. Arrow indicates tyrosine hydroxylase-positive cell that was not transfected. (D) Computer-merged image of A and a transmission scan of the same microscopic field. Arrow indicates the gold microbead that carried the plasmid into the transfected DA cell.
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experiments indicate that hDAT and NOT gene expression change in parallel in certain pathological conditions (unpublished data), suggesting that NOT plays a critical role in controlling hDAT gene expression in vivo.
5. Emerging strategies for studying hDAT regulatory elements in situ Developing a complete understanding of the mechanisms underlying the extraordinary cellular specificity of hDAT gene expression is a daunting task. This task is made more difficult by the general unavailability of neuronal cell lines stably expressing a strong DAT phenotype. Any methodologies for introducing hDAT gene constructs into authentic DA neurons could facilitate the analysis of hDAT regulatory elements in the appropriate cellular context. Transfection of DA neurons is being attempted using viral vectors (Pothos et al., 2000) and receptor-mediated DNA internalization (Martinez-Fong et al., 1999). Our laboratory has developed a methodology for particle-mediated (biolistic) transfection of DA neurons in organotypic slice culture (Fig. 5). This approach will expedite the assessment of numerous hDAT constructs and transcriptional elements, as a prelude to confirmatory studies of selected constructs in transgenic animals.
6. Conclusion The regulation of DAT gene expression may be relevant to neuropsychiatric diseases in a number of ways. First, although the DAT gene may not be convincingly linked to most brain disorders at the present time, the DAT could contribute to disease processes as a component of a polygenetic disorder. Second, the DAT is an obligatory target for some disease-inducing factors, such as Parkinson’s disease-inducing neurotoxins and addictive psychostimulants. Finally, the alterations in DAT gene expression that accompanying a variety of pathological processes (Bannon et al., 1998) may represent homeostatic responses to dysfunction of DA cells or associated neural circuits. As an example, in Parkinson’s disease, which entails extensive DA cell loss, the DAT gene expression per surviving DA cell is down-regulated (Blanchard et al., 1994; Harrington et al., 1996; Joyce et al., 1997). A diminished capacity to recapture extracellular DA via the DAT may represent an adaptive mechanism to augment DA neurotransmission in the Parkinsonian brain. A greater understanding of the transcriptional regulation of the hDAT gene may provide novel therapeutic strategies for the treatment of Parkinson’s disease and other DA-linked neurological and psychiatric disorders.
Acknowledgements The work of the authors was supported by NIH grants DA06470, MH47181, MH60854, NS34935, and DA07310.
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