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

BMC Neuroscience

John P Quinn²  Gabriele A Macho¹  Kate Haddley²  Vivien J Bubb²  Ursula M Paredes³ 

[1] Institut Català de Paleontologia, Campus de la Universitat Autònoma de Barcelona, 08193 Cerdanyola del Vallès, Barcelona, Spain;Institute of Translational Medicine, University of Liverpool, Ashton Street, Liverpool, L69 3GE, UK;MRC centre for Social, Genetic and Developmental Psychiatry Centre, Institute of Psychiatry, King's College, University of London, PO 80, DeCrespigny Park, London, SE5 8AF, UK

关键词: enhancer;    ECR;    sequence conservation;    dopamine;    transcriptional;    DRD4;   

Others  :  1174784
DOI  :  10.1186/1471-2202-12-46

 received in 2010-07-28, accepted in 2011-05-20,  发布年份 2011

【 摘 要 】

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.

【 授权许可】

   
2011 Paredes et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]MacKenzie A, Quinn JP: Post-genomic approaches to exploring neuropeptide gene mis-expression in disease. Neuropeptides 2004, 38:1-15.
• [2]Wagner GP, Fried C, Prohaska SJ, Stadler PF: Divergence of conserved non-coding sequences: rate estimates and relative rate tests. Mol Biol Evol 2004, 21:2116-2121.
• [3]Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N: A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 2003, 425:917-925.
• [4]Noain D, Avale ME, Wedemeyer C, Calvo D, Peper M, Rubinstein M: Identification of brain neurons expressing the dopamine D4 receptor gene using BAC transgenic mice. Eur J Neurosci 2006, 24:2429-2438.
• [5]Van Tol HH, Bunzow JR, Guan HC, Sunahara RK, Seeman P, Niznik HB, Civelli O: Cloning of the gene for a human dopamine D4 receptor with high affinity for the antipsychotic clozapine. Nature 1991, 350:610-614.
• [6]Arbelle S, Benjamin J, Golin M, Kremer I, Belmaker RH, Ebstein RP: Relation of shyness in grade school children to the genotype for the long form of the serotonin transporter promoter region polymorphism. Am J Psychiatry 2003, 160:671-676.
• [7]Comings DE, Gade-Andavolu R, Gonzalez N, Wu S, Muhleman D, Chen C, Koh P, Farwell K, Blake H, Dietz G, MacMurray JP, Lesieur HR, Rugle LJ, Rosenthal RJ: The additive effect of neurotransmitter genes in pathological gambling. Clin Genet 2001, 60:107-116.
• [8]Gornick MC, Addington A, Shaw P, Bobb AJ, Sharp W, Greenstein D, Arepalli S, Castellanos FX, Rapoport JL: Association of the dopamine receptor D4 (DRD4) gene 7-repeat allele with children with attention-deficit/hyperactivity disorder (ADHD): an update. Am J Med Genet B Neuropsychiatr Genet 2007, 144:379-382.
• [9]Higuchi S, Muramatsu T, Arai H, Hayashida M, Sasaki H, Trojanowski JQ: Polymorphisms of dopamine receptor and transporter genes and Parkinson's disease. J Neural Transm Park Dis Dement Sect 1995, 10:107-113.
• [10]Mage RG, Newman BA, Harindranath N, Bernstein KE, Becker RS, Knight KL: Evolutionary conservation of splice sites in sterile C mu transcripts and of immunoglobulin heavy chain (IgH) enhancer region sequences. Mol Immunol 1989, 26:1007-1010.
• [11]Prabhakar S, Poulin F, Shoukry M, Afzal V, Rubin EM, Couronne O, Pennacchio LA: Close sequence comparisons are sufficient to identify human cis-regulatory elements. Genome Res 2006, 16:855-863.
• [12]Shashikant CS, Bolanowsky SA, Anderson SM: Comparison of diverged Hoxc8 early enhancer activities reveals modification of regulatory interactions at conserved cis-acting elements. J Exp Zoolog B Mol Dev Evol 2007, 308:242-249.
• [13]Davidson S, Miller KA, Dowell A, Gildea A, Mackenzie A: A remote and highly conserved enhancer supports amygdala specific expression of the gene encoding the anxiogenic neuropeptide substance-P. Mol Psychiatry 2006, 11(4):410-421. 323
• [14]Ronai Z, Guttman A, Keszler G, Sasvari-Szekely M: Capillary electrophoresis study on DNA-protein complex formation in the polymorphic 5' upstream region of the dopamine D4 receptor (DRD4) gene. Curr Med Chem 2004, 11:1023-1029.
• [15]Schoots O, Van Tol HH: The human dopamine D4 receptor repeat sequences modulate expression. Pharmacogenomics 2003, 3:343-348. J
• [16]Herregodts P, Velkeniers B, Ebinger G, Michotte Y, Vanhaelst L, Hooghe-Peters E: Development of monoaminergic neurotransmitters in fetal and postnatal rat brain: analysis by HPLC with electrochemical detection. J Neurochem 1990, 55:774-779.
• [17]Tarazi FI, Baldessarini RJ: Comparative postnatal development of dopamine D(1), D(2) and D(4) receptors in rat forebrain. Int J Dev Neurosci 2000, 18:29-37.
• [18]Nair VD, Mishra RK: Ontogenic development of dopamine D4 receptor in rat brain. Brain Res Dev Brain Res 1995, 90:180-183.
• [19]Nayyar T, Zawia NH, Hood DB: Transplacental effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on the temporal modulation of Sp1 DNA binding in the developing cerebral cortex and cerebellum. Exp Toxicol Pathol 2002, 53:461-468.
• [20]Seaman MI, Fisher JB, Chang F, Kidd KK: Tandem duplication polymorphism upstream of the dopamine D4 receptor gene (DRD4). Am J Med Genet 1999, 88(6):705-709.
• [21]Szantai E, Szmola R, Sasvari-Szekely M, Guttman A, Ronai Z: The polymorphic nature of the human dopamine D4 receptor gene: a comparative analysis of known variants and a novel 27 bp deletion in the promoter region. BMC Genet 2005, 28(6):39.
• [22]Kamakura S, Iwaki A, Matsumoto M, Fukumaki Y: Cloning and characterization of the 5'-flanking region of the human dopamine D4 receptor gene. Biochem Biophys Res Commun 1997, 235(2):321-326.
• [23]Zhou Q, Kindlundh AM, Hallberg M, Nyberg F: The substance P (SP) heptapeptide fragment SP1-7 alters the density of dopamine receptors in rat brain mesocorticolimbic structures during morphine withdrawal. Peptides 2004, 25:1951-1957.
• [24]Wang J, Bannon MJ: Sp1 and Sp3 activate transcription of the human dopamine transporter gene. J Neurochem 2005, 93:474-482.