A polymorphism in the norepinephrine transporter gene alters promoter activity and is associated with attention-deficit hyperactivity disorder

Abstract

The norepinephrine transporter critically regulates both neurotransmission and homeostasis of norepinephrine in the nervous system. In this study, we report a previously uncharacterized and common A/T polymorphism at -3081 upstream of the transcription initiation site of the human norepinephrine transporter gene [solute carrier family 6, member 2 (SLC6A2)]. Using both homologous and heterologous promoter-reporter constructs, we found that the -3081(T) allele significantly decreases promoter function compared with the A allele. Interestingly, this T allele creates a new palindromic E2-box motif that interacts with Slug and Scratch, neural-expressed transcriptional repressors binding to the E2-box motif. We also found that both Slug and Scratch repress the SLC6A2 promoter activity only when it contains the T allele. Finally, we observed a significant association between the -3081(A/T) polymorphism and attention-deficit hyperactivity disorder (ADHD), suggesting that anomalous transcription factor-based repression of SLC6A2 may increase risk for the development of attention-deficit hyperactivity disorder and other neuropsychiatric diseases.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

The sequence between −4000 and −3018 of SLC6A2 promoter contains the noradrenergic cell-specific transcriptional element. The fragment between −4000 and −3018 was subcloned in either sense or antisense orientation 5′ of the pBLCAT2 or 5′ of the pNET133(i)CAT (20). The reporter constructs were transfected into the NET-positive SK-N-BE (2)C cells and NET-negative HeLa cells, and the normalized CAT activity of each construct was expressed relative to the vector pBLCAT2 (A) and pNET133(i)CAT (B), respectively.

Fig. 2.

Basal promoter activities of reporter constructs containing nucleotide A or T at position −3081. (A) The fragment between −4000 and −3018 containing either A or T at base pair −3081 was subcloned in antisense orientation 5′ of the pNET133(i)CAT. The normalized CAT activities driven by an A allele containing construct in each cell line was set to 100 to compare the relative strength of a T allele-containing construct. Significant differences between A and T alleles were evaluated by unpaired t test with two-tailed P values: ∗, P < 0.05; ∗∗, P < 0.005; ∗∗∗, P < 0.0005. (B) A 4-kb upstream sequence and the first intron of SLC6A2 were inserted upstream of the luciferase coding sequence in the pGL3 basic (Promega) vector. These constructs were designed to contain the A or T allele at position −3081 and transiently transfected into cell lines. (C) Six copies of the oligonucleotide containing A or T alleles were coupled to a heterologous TK promoter and a luciferase reporter gene. To compare luciferase activities between two constructs, luciferase activity driven by TK promoter was set to 100.

Fig. 3.

Allele-specific binding of protein to oligonucleotide surrounding polymorphic site. (A) EMSA were conducted by using nuclear extracts from SK-N-BE (2)C and HeLa cells with labeled probes for the allele A (lanes 1 and 3) and for the allele T (lanes 2 and 4). An allele-specific complex was indicated by the arrow at right. A nonspecific complex was shown by the asterisk. (B) EMSA were performed by using nuclear extracts from SK-N-BE (2)C cells with 32P-labeled oligonucleotide containing allele T. This DNA–protein complex was competed by unlabeled −3081(T) oligonucleotide but not by either −3081(A) or unrelated CRE and sp1 oligonucleotides. (C) Schematic representation of E2-box DNA sequence created by polymorphism at −3081.

Fig. 4.

Slug and Scratch, but not Snail, bind to the E2-box generated by polymorphism at −3081 of SLC6A2. The −3081(T) oligonucleotide radiolabeled probe was incubated with SK-N-BE (2)C nuclear extract in the absence (lane 1) or presence of antibodies (lanes 2–8). Coincubation of nuclear proteins with increasing amounts of Slug- and Scratch-specific antibodies [0.2 μg (lanes 3 and 6), 0.5 μg (lanes 4 and 7), and 1 μg (lanes 5 and 8)] resulted in the generation of a supershifted band in a dose-responsive manner. In addition, formation of DNA–protein complex was significantly diminished. In contrast, coincubation with Snail-specific antibody (1 μg) neither generated the supershifted band nor diminished formation of DNA–protein complex (lane 2).

Fig. 5.

Slug and Scratch transrepress the SLC6A2 promoter in an allele-specific manner. (A) SK-N-BE (2)C cells were transiently cotransfected with reporter constructs and Slug or Scratch expression vectors. To compare the transrepression directly, basal luciferase activity driven by each reporter construct was set to 100%. (B) Diagram of the overall structure of Snail family protein and dominant-negative construct. The activating forms of Slug and Scratch were constructed by replacing the N-terminal repressor domain with the activation domain of VP16 (Upper). The −3081(T) allele of the 4.0-kb SLC6A2 promoter-luciferase reporter constructs were cotransfected with the either Slug or Scratch expression vector and dominant-negative expression plasmids. Here, basal promoter activity driven by the 4.0-kb SLC6A2 promoter-luciferase reporter constructs containing the T allele at −3081 was set to 100%.