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.

Tissue-specific DNA methylation of the human prodynorphin gene in post-mortem brain tissues and PBMCs

“…Thus, the methylation rate of the CpG island in brain regions was not associated directly with PDYN expression levels. The promoter region of the human PDYN containing the CpG island was shown to be implicated in up-regulation [44] or down-regulation [45] of basal and forskolin-induced PDYN promoter activity in vitro depending on cell culture type. Therefore, the PDYN CpG island may be implicated in a tissue-specific regulation of the gene expression.…”

κ-opioid receptor/dynorphin system: genetic and pharmacotherapeutic implications for addiction

“…One of the most studied PDYN polymorphism is a 68-base pair nucleotide tandem repeat polymorphism (rs35286281) located 1250 bp upstream of exon 1 [6] (Table 1). This polymorphism, which contains a putative activator protein-1 (AP-1) transcription complex (including c-Fos/c-Jun) binding site, is found in 1–5 copies [59]. With careful phenotyping, it was found that individuals with three or four copies of the 68-bp tandem repeat showed an increased vulnerability to develop cocaine dependence or cocaine/alcohol codependence, in African-American subjects only [60, 61].…”

“…rs35286281) produced approximately 1.5 greater levels of forskolin-induced transcriptional activity compared with constructs with one or two copies of the repeat [67]. A more recent study in human cell lines transfected with reporter gene expression constructs containing the PDYN promoter with the thirteen most common combinations of the tandem repeat region and the internal SNP (rs61761346) in a control human population, explored this influence more broadly [59]. In neuronal SK-N-SH and H69 cell lines, three or four repeats led to lower expression of luciferase than one or two repeats, which would suggest lower levels of PDYN message and potentially peptide production.…”

The dynorphin/κ-opioid receptor system and its role in psychiatric disorders

“…1) [63][64][65]. Among these, attempts have been made to associate rs35286281 (copy number variant) (unsuccessful) and rs1997794 (SNP) (successful) with have been associated with allelic imbalance (differential expression of alleles at one or more loci) in the cortex and cerebellum [52, 64,66,67].…”