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. 2012 May;39(5):5393-9.
doi: 10.1007/s11033-011-1339-4. Epub 2011 Dec 13.

Identification of an intronic cis-acting element in the human dopamine transporter gene

Ying Zhao  1 , Yanhong Zhou, Nian Xiong, Zhicheng Lin
Affiliations

Identification of an intronic cis-acting element in the human dopamine transporter gene

Ying Zhao et al. Mol Biol Rep. 2012 May.

Abstract

The human dopamine transporter gene (hDAT) encodes the dopamine transporter in dopamine (DA) neurons to regulate DA transmission. hDAT expression varies significantly from neuron to neuron, and from individual to individual so that dysregulation of hDAT is related to many neuropsychiatric disorders. It is critical to identify hDAT-specific cis-acting elements that regulate the hDAT expression. Previous studies showed that hDAT Intron 1 displayed inhibitory activity for reporter gene expression. Here we report that the hDAT Intron 1 contains a 121-bp fragment that down-regulated both SV40 and hDAT promoter activities by 80% in vitro. Subfragments of 121-bp still down-regulated the SV40 promoter but not the hDAT promoter, as supported by nuclear protein-binding activities. Collectively, 121-bp is a silencer in vitro that might coordinate with transcriptional activities both inside and outside 121-bp in regulation of hDAT.

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Figures

Fig. 1
Fig. 1
Endogenous hDAT expression in five human cell lines including four DA cell lines SK-N-AS, BE(2)-M17, IMR-32, SH-SY5Y and one non-neuronal cell line HEK293T, based on qRT-PCR that used two different pairs of hDAT primers (N = 3)
Fig. 2
Fig. 2
Promoter activity decreased by addition of (extending) a 121-bp fragment into Intron 1. a Schematic of 7.9 kb-Luc expression constructs and addition of a 121-bp fragment. b–d Addition of 121-bp fragment decreased the promoter activity in SK-N-AS, SH-SY5Y and HEK293T. Luc activity (arbitrary units) represents promoter activity of 7.9 kb hDAT fragment before (7.9 kb for pGL3-hDAT7.9kb) and after (7.9kb+121bp for pGL3-hDAT7.9kb+121bp) adding 121-bp. P < 0.0001 for SK-N-AS (N = 6) and SH-SY5Y (N = 14) or P = 0.0342 for HEK293T (N = 2 each in duplicate) all by t tests
Fig. 3
Fig. 3
Down-regulation of SV40 promoter activity by hDAT intronic 121-bp fragment. a Cloning of the 120-bp fragment into two different locations (indicated by two arrows) in pGL3-Promoter Vector (Vector), resulting in pGL3-SV40+121bp. 121-bp inhibition of SV40 promoter activity, based on Luc activity from Vector versus pGL3-SV40+121bp in two different human cell lines SK-N-AS and BE(2)-M17 (b, c). Arrows indicate two different insertion locations. ANOVA and Tukey tests: *P < 0.05; ***P < 0.001, compared to Vector (N = 4, each in duplicate)
Fig. 4
Fig. 4
Inhibition of SV40 promoter activity by subfragments of 121-bp. a DNA sequences of four subfragments (I–IV) throughout 121-bp. b Reporter pGL3-Promoter Vector. Gray arrow indicates subfragment insertion site. c–f Influences of subfragments on SV40 promoter activity in four different cell lines. SV40 pGL3-Promoter Vector; I pGL3-SV40-I; II pGL3-SV40-II; III pGL3-SV40-III; IV pGL3-SV40-IV. Indicated P values are based on ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; Student’s t test: #P < 0.05, comparing to SV40 (N = 4–5, each in duplicate)
Fig. 5
Fig. 5
EMSA of nuclear protein binding to four subfragments (I–IV) of 121-bp. Nuclear proteins were isolated from SK-N-AS, BE(2)-M17, SH-SY5Y and HEK293T, as indicated on top. DNA sequences of I–IV are shown in Fig. 4a and ds oligos were labeled by 35S-dATP. Competitor is 30-fold excess of unlabeled double-strand oligo. Arrow specific binding activity by comparing to next lane with competitor; lowest band poly(A/T, generated by 35S-A filling-in)-binding activity (control not shown)
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References

    1. Hitri A, Hurd YL, Wyatt RJ, Deutsch SI. Molecular, functional and biochemical characteristics of the dopamine transporter: regional differences and clinical relevance. Clin Neuropharmacol. 1994;17:1–22. - PubMed
    1. Ohyama K, Sogawa C, Sogawa N, Morita K, Dohi T, Kitayama S. Nicotine stimulates transcriptional activity of the human dopamine transporter gene. Neurosci Lett. 2010;471:34–37. - PubMed
    1. Stevens SE, Kumsta R, Kreppner JM, Brookes KJ, Rutter M, Sonuga-Barke EJ. Dopamine transporter gene polymorphism moderates the effects of severe deprivation on ADHD symptoms: developmental continuities in gene–environment interplay. Am J Med Genet B. 2009;150B:753–761. - PubMed
    1. Lin Z, Canales JJ, Björgvinsson T, Thomsen M, Qu H, Liu QR, Torres GE, Caine SB. Monoamine transporters vulnerable and vital doorkeepers. Prog Mol Biol Transl Sci. 2011;98:1–46. - PMC - PubMed
    1. Bannon MJ, Whitty CJ. Age-related and regional differences in dopamine transporter mRNA expression in human midbrain. Neurology. 1997;48:969–977. - PubMed

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