Allele-specific expression of the serotonin transporter and its transcription factors following lamotrigine treatment in vitro.

Lamotrigine, a mood stabilizer used clinically in the treatment of bipolar disorder, is thought to exert actions on the serotonin system. However lamotrigine's exact mechanism of action remains unclear. The current study investigated whether lamotrigine might exert its effects through altering the expression of the serotonin transporter (5-HTT) gene and its regulatory transcription factors Y box binding protein 1 (YB-1) and CCCTC-binding factor (CTCF). We further considered whether functional variable number tandem repeat (VNTR) polymorphisms in the promoter region of 5-HTT, (5-HTTLPR) and within intron 2 (Stin2) of the gene, moderated any putative gene expression changes. The study employed an in vitro design carried out in human lymphoblastoid cell lines (LCLs) to investigate the effects of lamotrigine treatment at 0.04, 0.2, and 0.4 mM doses for 24 hr on the mRNA expression of 5-HTT, YB-1, and CTCF. LCLs were selected based on combinations of haplotypes of the two VNTRs in the serotonin transporter gene; creating low-expressing and high-expressing LCL groups. Ubiquitin C (UBC) and topoisomerase I (TOP1) genes were found to be the most stably expressed housekeeping genes in drug-treated LCLs. Subsequently, quantitative PCR revealed that higher doses of lamotrigine significantly lowered 5-HTT expression and increased CTCF expression. Haplotype-specific differences in CTCF expression were found in response to lamotrigine, with strongest expression changes observed in the high-expressing LCLs. These data provide an allele-specific in vitro model for examining the molecular targets of lamotrigine, and support the important role of the serotonin transporter gene in its clinical mechanism of action.

The SLC6A4 VNTR genotype determines transcription factor binding and epigenetic variation of this gene in response to cocaine in vitro.

We demonstrated that the genotype of the variable number tandem repeats (VNTRs) in the linked polymorphic region (LPR) of the 5' promoter and in the intron 2 (Stin2) transcriptional regulatory domains of the serotonin transporter SLC6A4 gene determined its promoter interactions with transcription factors and co-activators in response to cocaine in the JAr cell line. The LPR variants contain 14 (short, s) or 16 (long, l) copies of a 22-23 bp repeat element, whereas the Stin2 VNTR exists as three variants containing 9, 10 or 12 copies of a 16-17 bp repeat. We observed a differential effect of cocaine on the association of the promoter with the transcription factor CTCF, which bound to both LPR alleles prior to cocaine exposure but only to the l-allele following exposure. Significantly, this differential effect of cocaine was correlated with the binding of the transcriptional regulator MeCP2 specifically to the s-allele and recruiting the histone deacetylase complex (HDAC). Concurrently, cocaine increased the association of positive histone marks over the SLC6A4 gene locus. At the Stin2 domain, we lost binding of the transcription factor YB-1, while CTCF remained bound. Our biochemical data are consistent with differential reporter gene activity directed by the individual or dual domains in response to cocaine in an Epstein-Barr virus-based episome model of stable transfections. These observations suggest that exposure of JAr cells to cocaine may result in differential binding of transcription factors and activators based on a specific genotype that might alter epigenetic parameters affecting gene expression after the initial challenge.

An evolutionary conserved region (ECR) in the human dopamine receptor D4 gene supports reporter gene expression in primary cultures derived from the rat cortex.

BACKGROUND: Detecting functional variants contributing to diversity of behaviour is crucial for dissecting genetics of complex behaviours. At a molecular level, characterisation of variation in exons has been studied as they are easily identified in the current genome annotation although the functional consequences are less well understood; however, it has been difficult to prioritise regions of non-coding DNA in which genetic variation could also have significant functional consequences. Comparison of multiple vertebrate genomes has allowed the identification of non-coding evolutionary conserved regions (ECRs), in which the degree of conservation can be comparable with exonic regions suggesting functional significance. RESULTS: We identified ECRs at the dopamine receptor D4 gene locus, an important gene for human behaviours. The most conserved non-coding ECR (D4ECR1) supported high reporter gene expression in primary cultures derived from neonate rat frontal cortex. Computer aided analysis of the sequence of the D4ECR1 indicated the potential transcription factors that could modulate its function. D4ECR1 contained multiple consensus sequences for binding the transcription factor Sp1, a factor previously implicated in DRD4 expression. Co-transfection experiments demonstrated that overexpression of Sp1 significantly decreased the activity of the D4ECR1 in vitro. CONCLUSION: Bioinformatic analysis complemented by functional analysis of the DRD4 gene locus has identified a) a strong enhancer that functions in neurons and b) a transcription factor that may modulate the function of that enhancer.

Combinatorial interaction between two human serotonin transporter gene variable number tandem repeats and their regulation by CTCF.

Two distinct variable number tandem repeats (VNTRs) within the human serotonin transporter gene (SLC6A4) have been implicated as predisposing factors for CNS disorders. The linked polymorphic region in the 5'-promoter exists as short (s) and long (l) alleles of a 22 or 23 bp elements. The second within intron 2 (Stin2) exists as three variants containing 9, 10 or 12 copies of a 16 or 17 bp element. These VNTRs, individually or in combination, supported differential reporter gene expression in rat neonate prefrontal cortical cultures. The level of reporter gene activity from the dual VNTR constructs indicated combinatorial action between the two domains. Chromatin immunoprecipitation demonstrated that both these VNTR domains can bind the CCCTC-binding factor and this correlated with the ability of exogenously supplied CCCTC-binding factor to modulate the expression supported by these reporter gene constructs. We suggest that the potential for interaction between multiple polymorphic domains should be incorporated into genetic association studies.

A proximal E-box modulates NGF effects on rat PPT-A promoter activity in cultured dorsal root ganglia neurones.

The rat preprotachykinin A (rtPPTA) promoter fragment spanning -865+92, relative to the major transcriptional start, has previously been demonstrated to be nerve growth factor (NGF) responsive in primary cultures of rat dorsal root ganglion (DRG) neurones [Harrison, P.T., Dalziel, R.G., Ditchfield, N.A., Quinn, J.P., 1999. Neuronal-specific and nerve growth factor-inducible expression directed by the preprotachykinin-A promoter delivered by an adeno-associated virus vector. Neuroscience 94, 997-1003]. In this communication, we demonstrate that an E box element at -60, in part, regulates the activity of this rtPPT-A promoter fragment in DRG neurones in response to NGF. Differential regulation of the promoter is observed in the presence or absence of NGF when the E Box site is present. Under basal conditions binding of proteins to this -60 element may antagonise promoter activity. Hence, in the absence of NGF, mutation of the -60 E box increased reporter gene expression. Further, comparison of levels of reporter gene expression supported by both WT and mutated promoter indicate that in the presence of NGF the -60 E box element also plays a role as an activator domain. This represents a novel mechanism for NGF regulation of rtPPT-A. Similarly, an important role for this signalling pathway was observed in neonate rat DRG neuronal cultures, which require NGF for their survival, namely mutation of the -60 element resulted in higher levels of reporter gene expression.

Mood-stabilizers differentially affect housekeeping gene expression in human cells.

Recent studies have revealed that antidepressants affect the expression of constitutively expressed "housekeeping genes" commonly used as normalizing reference genes in quantitative polymerase chain reaction (qPCR) experiments. There has yet to be an investigation however on the effects of mood-stabilizers on housekeeping gene stability. The current study utilized lymphoblastoid cell lines (LCLs) derived from patients with mood disorders to investigate the effects of a range of doses of lithium (0, 1, 2 and 5 mM) and sodium valproate (0, 0.06, 0.03 and 0.6 mM) on the stability of 12 housekeeping genes. RNA was extracted from LCLs and qPCR was used to generate cycle threshold (Ct ) values which were input into RefFinder analyses. The study revealed drug-specific effects on housekeeping gene stability. The most stable housekeeping genes in LCLs treated: acutely with sodium valproate were ACTB and RPL13A; acutely with lithium were GAPDH and ATP5B; chronically with lithium were ATP5B and CYC1. The stability of GAPDH and B2M were particularly affected by duration of lithium treatment. The study adds to a growing literature that the selection of appropriate housekeeping genes is important for the accurate normalization of target gene expression in experiments investigating the molecular effects of mood disorder pharmacotherapies.

Activity-dependent neuroprotective protein modulates its own gene expression.

We investigated whether activity-dependent neuroprotective protein (ADNP) could autoregulate its own expression. Both the endogenous ADNP gene and reporter gene constructs were analysed in response to overexpression of ADNP, supplied either as wild-type ADNP or a mutant form lacking the NAP motif, a motif which has neuroprotective properties. Overexpression of these two forms of ADNP resulted in both decreased endogenous ADNP expression and repressed ADNP promoter-directed reporter gene activity. Chromatin immunoprecipitation demonstrated the ability of ADNP to bind to its own promoter which is consistent with its action as a repressor of both promoter-supported and endogenous ADNP expression.

Intronic tandem repeat in the serotonin transporter gene in Old World monkeys: a new transcriptional regulator?

The serotonin transporter gene (SLC6A4) is heavily involved in the regulation of social behaviour of primates. Old World monkeys (e.g. macaques, baboons) have been used to study interactions between variation in the SLC6A4 gene and behaviour. Correlations of variation at one polymorphism located in the promoter region (known as 5HTTLPR) and variation at SLC6A4 expression levels, serotonin turnover and behaviour has been widely studied. In Old World monkeys, the third intron of the SLC6A4 gene also presents a tandem repeat, which sequence varies across species by a few point substitutions. We predict that in these species, this repeated region also acts as transcriptional regulatory domain and that sequence variation at this polymorphic locus might result in differential levels of expression in gene-environment interactions. For testing these hypotheses, the tandem repeat of Mandrillus sphinx and Cercopithecus aethiops from the third intron were cloned into a reporter gene vector and delivered to either primary cultures of rat neonate frontal cortex or the human cell line (JAr) to analyse their transcriptional activities. These repeated sequences supported significantly different levels of gene expression only when delivered into frontal cortex cultures. Furthermore, we tested in silico if such substitutions could have an effect on their binding profile to RNA- and DNA-binding proteins and on splicing. Taken together our results suggest that the tandem repeat in the third intron of the SLC6A4 gene of Old World monkeys could constitute a second transcriptional regulator as suggested for the 5HTTLPR and therefore contribute to diversification of serotonin-related behaviour in these primates.

Lithium chloride regulation of the substance P encoding preprotachykinin a, Tac1 gene in rat hippocampal primary cells.

In rat hippocampal cultures, the preprotachykinin A (PPTA/Tac1) gene, which encodes the neuropeptide substance P, is regulated by the action of lithium. We used reporter gene and expression constructs to demonstrate that this mechanism of action of lithium is mediated via a previously characterised cis-regulatory Ebox element in the proximal promoter, which binds members of the basic Helix-Loop-Helix family of transcription factors. Consistent with this, in hippocampal cells, both the expression of the endogenous gene and the function of this promoter element are differentially regulated by the basic Helix-Loop-Helix factors, upstream stimulatory factor 1 and 2 (USF1/2). In addition, the genes for USF1 and USF2 are differentially regulated by lithium in these cells. Our data implicate USF1 as a major regulator of the action of lithium on the proximal PPTA promoter.

Distinct gene expression profiles directed by the isoforms of the transcription factor neuron-restrictive silencer factor in human SK-N-AS neuroblastoma cells.

Neuron-restrictive silencer factor (NRSF) and its isoforms are differentially regulated in rodent models of self-sustaining status epilepticus (SSSE). NRSF isoforms regulate genes associated with SSSE, including the proconvulsant tachykinins, brain-derived neurotrophic factor and multiple ion channels. NRSF isoforms may direct distinct gene expression patterns during SSSE, and the ratio of each isoform may be a causative factor in traumatic damage to the central nervous system. Here, we analysed global gene expression changes by microarray in human SK-N-AS neuroblastoma cells following the over-expression of NRSF and a truncated isoform, HZ4. We used bioinformatics software to analyse the microarray dataset and correlated these data with epilepsy candidate gene pathways. Findings were validated by reverse transcriptase-polymerase chain reaction. We demonstrated that NRSF and HZ4 direct overlapping as well as distinct gene expression patterns, and that they differentially modulated gene expression patterns associated with epilepsy. Finally, we revealed that NRSF gene expression may be modulated by the anticonvulsant, phenytoin. We have interpreted our data to reflect altered gene expression directed by NRSF that might be relevant for SSSE.

Assessing the impact of genetic variation on transcriptional regulation in vitro.

Two alleles of a gene that contain polymorphic cis-regulatory regions can contribute differently to expression levels. Evolutionary changes in such cis-regulatory domains are believed to have participated in the cognitive evolution of H. sapiens as well as phenotypic diversity. There have been many studies that associate genetic variations to individual's susceptibility to behavioural and affective disorders. Cis-acting regulatory polymorphisms can effect gene expression at many levels, such as transcription, mRNA processing efficiency, pre-mRNA splicing, and mRNA stability. Trans-acting modulators (such as transcription factors) also play a major role in determining mRNA concentration of a specific allele. Several studies have demonstrated that VNTRs within various genes can support differential gene expression based on copy number and that the function of the VNTR as a transcriptional regulator can be modulated, in part, by transcription factors.A better understanding of the pathways regulating expression mediated by the VNTRs would complement clinical studies, demonstrating how these domains may be mechanistically involved in the progression of the disorder and may supply more defined targets for pharmaceutical intervention.

NO-cGMP mediated galanin expression in NGF-deprived or axotomized sensory neurons.

Leukaemia inhibitory factor (LIF) and nerve growth factor (NGF) are well characterized regulators of galanin expression. However, LIF knockout mice containing the rat galanin 5' proximal promoter fragment (- 2546 to + 15 bp) driving luciferase responded to axotomy in the same way as control mice. Also, LIF had no effect on reporter gene expression in vitro, neither in the presence or absence of NGF, suggesting that other factors mediate an axotomy response from the galanin promoter. We then addressed the role of nitric oxide (NO) using NGF-deprived rat dorsal root ganglion (DRG) neuron cultures infected with viral vectors containing the above-mentioned construct, and also studied endogenous galanin expression in axotomized DRG in vivo. Blocking endogenous NO in NGF-deprived DRG cultures suppressed galanin promoter activity. Consistent with this, axotomized/NGF-deprived DRG neurons expressed high levels of neuronal NO synthase (nNOS) and galanin. Further, using pharmacological NOS blockers, or adenoviral vectors expressing dominant-negative either for nNOS or soluble guanylate cyclase in vivo and in vitro, we show that the NO-cGMP pathway induces endogenous galanin in DRG neurons. We propose that both LIF and NO, acting at different promoter regions, are important for the up-regulation of galanin, and for DRG neuron survival and regeneration after axotomy.

A dopamine transporter gene functional variant associated with cocaine abuse in a Brazilian sample.

The dopamine (DA) transporter DAT1 is a major target bound by cocaine in brain. We examined the influence of functional genetic variants in DAT1 on cocaine addiction. Repeat polymorphisms, including a 30-bp variable-number tandem repeat (VNTR) in intron 8 (Int8 VNTR) with two common alleles, were genotyped in cocaine-dependent abusers (n = 699) and in controls with no past history of drug abuse (n = 866) from São Paulo, Brazil. Positive association was observed with allele 3 of the Int8 VNTR and cocaine abuse (allele odds ratio = 1.2, 95% confidence interval = 1.01-1.37, P = 0.036; 3/3 homozygote odds ratio = 1.45, 95% confidence interval = 1.18-1.78, P = 0.0008). Population stratification was assessed and did not affect the results. Haplotypic analyses using additional polymorphisms indicated that the Int8 VNTR is responsible for the observed association. Functional analyses in reporter-gene constructs, demonstrated that allele 3 mediates significant (P < 0.05) but small reduced expression compared with the "protective" allele 2. This difference increased when 1 and 10 muM cocaine was added to the cell culture ( approximately 40% reduction of the 3 allele expression versus the 2 allele). The 3 allele also demonstrated approximately 3-fold-increased expression over the 2 allele in response to KCl plus forskolin challenge. We demonstrate a robust association between cocaine dependence and a VNTR allele in SLC6A3, conferring a small but detectable effect, and we show that this VNTR may be functional. This study suggests that DAT1 gene variation may play a role in cocaine dependence etiology.