Cell-specific effects of variants of the 68-base pair tandem repeat on prodynorphin gene promoter activity

Abstract

A polymorphic 68-bp tandem repeat has been identified within the promoter of the human prodynorphin (PDYN) gene. We found that this 68-bp repeat in the PDYN promoter occurs naturally up to five times. We studied the effect of the number of 68-bp repeats, and of a SNP (rs61761346) found within the repeat on PDYN gene promoter activity. Thirteen promoter forms, different naturally occurring combinations of repeats and the internal SNP, were cloned upstream of the luciferase reporter gene, transfected into human SK-N-SH, H69, or HEK293 cells. Cells were then stimulated with TPA or caffeine. We found cell-specific effects of the number of 68-bp repeats on the transcriptional activity of the PDYN promoter. In SK-N-SH and H69 cells, three or four repeats led to lower expression of luciferase than did one or two repeats. The opposite effect was found in HEK293 cells. The SNP also had an effect on PDYN gene expression in both SK-N-SH and H69 cells; promoter forms with the A allele had significantly higher expression than promoter forms with the G allele. These results further our understanding of the complex transcriptional regulation of the PDYN gene promoter.

Figure 1

The 68-bp tandem repeat polymorphism (rs35286281) with the internal SNP (rs61761346) of the PDYN gene promoter, and schematic representation of the PDYN promoter construct used for the luciferase gene reporter assays. A. Sequence of the 68-bp tandem repeat showing the putative AP-1 binding site (bold) and the putative GATA-1 binding site when there is an A allele at the ninth position of the tandem repeat (bold). B. 3.1 kb fragment of the PDYN gene was cloned upstream of the firefly luciferase reporter gene. This fragment includes: 241 bp upstream of the 68-bp tandem repeat region, the 68-bp tandem repeat region (fuschia), 1250 bp upstream of exon 1 including the TATA box located 33 bp upstream of exon 1 and most of the transcription start sites, exon 1 (145 bp, green) containing a binding site for the transcription factor DREAM (red), intron 1 (1268 bp), exon 2 (60 bp) and 17 bp of intron 2. The restriction sites used to generate deletion constructs (Mlu I, Ase I, Nco I) are indicated. The major transcription start sites are indicated by □.

Figure 2

Deletion analysis of the PDYN gene promoter. Luciferase gene reporter assay of full-lenght and deletion constructs of the 1G variant of PDYN gene promoter in SK-N-SH, H69 and HEK293 cells under basal conditions (white bars), stimulated with 100 nM TPA (grey bars) or 10 mM caffeine (black bars). Data are shown as mean + SEM for three different DNA preparations, each performed in triplicate for each construct. Note the different scales in normalized fluorescence levels (r.lu) for three different cells.

Figure 3

Effect of the number of 68-bp tandem repeats (rs35286281) and of the internal SNP (rs61761346) on PDYN gene promoter activity in SK-N-SH cells. A. Luciferase gene reporter assay of 13 different naturally-occurring variants of PDYN gene promoter and a control promoter construct, with deletion of a ~1 kb fragment containing the repeats (NO), in SK-N-SH cells under basal conditions (white bars), stimulated with 100 nM TPA (grey bars) or 10 mM caffeine (black bars). Data are shown as mean + SEM of two experiments with three different DNA preparations, each performed in triplicate for each construct. B. Data have been grouped by number of tandem repeats, 1 represents 1A and 1G, 2 represents 2AA, 2GA and 2GG, 3 represents 3AAA, 3GAA, 3GGA and 3GGG, and 4 represents 4AAAA, 4GAAA, 4GGAA and 4GGGG. In SK-N-SH cells, constructs with 1 tandem repeat showed higher expression of luciferase reporter gene than constructs with 2, 3 or 4 tandem repeats. Constructs with 1 tandem repeat showed higher expression of luciferase reporter gene than constructs with 2, 3 or 4 tandem repeats. C. In SK-N-SH cells under caffeine stimulation, cells containing the A allele showed higher luciferase expression than those with the G allele (* p < 0.05, ** p < 0.005, *** p < 0.0005).

Figure 4

Effect of the number of 68-bp tandem repeats (rs35286281) and of the internal SNP (rs61761346) on PDYN gene promoter activity in H69 cells. A. Luciferase gene reporter assay of 13 different naturally-occurring variants of the PDYN gene promoter and a control promoter construct, with deletion of a ~1 kb fragment containing the repeats (NO), in H69 cells under basal conditions (white bars), stimulated with 100 nM TPA (grey bars) or 10 mM caffeine (black bars). Data are shown as mean + SEM of one experiment with three different DNA preparations, each performed in triplicate for each construct. B. Data have been grouped by number of tandem repeats, as in Figure 4. In H69 cells, constructs with 1 tandem repeat showed higher expression of luciferase reporter gene than constructs with 2, 3 or 4 tandem repeats. Constructs with 1 tandem repeat showed higher expression of luciferase reporter gene than constructs with 2, 3 or 4 tandem repeats. C. H69 cells containing only the A allele at rs61761346 showed higher expression of the luciferase reporter gene than those with the G allele (* p < 0.05, ** p < 0.005, *** p < 0.0005).

Figure 5

Effect of the number of 68-bp tandem repeats (rs35286281) and of the internal SNP (rs61761346) on PDYN gene promoter activity in HEK293 cells. A. Luciferase gene reporter assay of 13 different naturally-occurring variants of PDYN gene promoter and a control promoter construct, with deletion of a ~1 kb fragment containing the repeats (NO), in H69 cells under basal conditions (white bars), stimulated with 100 nM TPA (grey bars) or 10 mM caffeine (black bars). Data are shown as mean + SEM of one experiment with three different DNA preparations, each performed in triplicate for each construct. B. Data have been grouped by number of tandem repeats, as in Figure 4. Cells transfected with constructs containing 1, 2 or 3 copies of the 68-bp tandem repeat showed lower expression of the luciferase reporter gene than constructs containing 4 copies. C. In HEK293 cells, there was no difference of luciferase expression between cells containing only the A allele at rs61761346 and those with the G allele.

Figure 6

5’-RLM-RACE PCR analysis of transcription start sites of the PDYN gene promoter construct in SK-N-SH, H69 and HEK293 cell lines. A. Sequence of the PDYN gene promoter from −143 bp to +39 bp showing the previously identified transcription start sites located at positions −146, −7, −1, +1, +5, +16, +24 and +29 (bars below sequence). An ↓ indicates the transcription start site identified by 5’-RLM-RACE PCR. B. Sequence of the PDYN gene promoter from +880 to +1437 showing the previously identified transcription start located at positions +1206, +1244, +1250 and +1269 (bars below sequence). An ↓ indicate the transcription start site identified by 5’-RLM-RACE PCR in this study. C. Frequencies of used transcription start sites in SK-N-SH, H69 and HEK293 cells under basal conditions or after stimulation with either 100 nM TPA or 10 mM caffeine.