Dataset Information

Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region.

ABSTRACT: The delta-opioid receptor mediates rewarding effects of many substances of abuse. We reported an increased frequency of the minor G-allele of single-nucleotide polymorphism (SNP) rs569356 (the only variant identified so far in the promoter region of the delta-opioid receptor gene (OPRD1)) in subjects with opioid dependence. In this study, we examined the functional significance of this variant. OPRD1 promoter region harboring SNP rs569356 was amplified by PCR and inserted into a firefly luciferase reporter vector. HEK293 cells were co-transfected with these constructs and a renilla luciferase vector to control for transfection efficiency. Expression of firefly luciferase (driven by the OPRD1 promoter) was measured by a dual luciferase reporter assay and normalized by renilla luciferase expression. Moreover, alleles altering expression were further assessed for binding of human brain nuclear proteins by electrophoretic mobility shift assay (EMSA). The minor G-allele was associated with significantly greater expression levels of firefly luciferase than the major A-allele of SNP rs569356 (P=0.003). EMSA also showed specific gel shift bands, suggesting that SNP rs569356 is situated in the binding site of potential transcription factors. These results suggest that the minor G-allele of SNP rs569356 may enhance transcription factor binding and increase OPRD1 expression.

SUBMITTER: Zhang H

PROVIDER: S-EPMC2876206 | biostudies-literature | 2010 May

REPOSITORIES: biostudies-literature

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Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region.

Zhang Huiping H Gelernter Joel J Gruen Jeffrey R JR Kranzler Henry R HR Herman Aryeh I AI Simen Arthur A AA

Journal of human genetics 20100319 5

The delta-opioid receptor mediates rewarding effects of many substances of abuse. We reported an increased frequency of the minor G-allele of single-nucleotide polymorphism (SNP) rs569356 (the only variant identified so far in the promoter region of the delta-opioid receptor gene (OPRD1)) in subjects with opioid dependence. In this study, we examined the functional significance of this variant. OPRD1 promoter region harboring SNP rs569356 was amplified by PCR and inserted into a firefly lucifera ...[more]

PMID: 20300121

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Project description:Background and Purpose- The E3 ubiquitin ligase MDM2 (murine double minute 2) is the main negative regulator of the p53 protein-a key player in neuronal apoptosis after ischemia. A functional single-nucleotide polymorphism in the human MDM2 gene promoter (rs2279744) regulates MDM2 protein expression. We investigated whether the MDM2 SNP309, by controlling p53-mediated apoptosis, determines functional outcome after stroke. Methods- Primary cortical neurons were subjected to oxygen and glucose deprivation. Mice were subjected to ischemic (transient middle cerebral artery occlusion) or hemorrhagic (collagenase injection) stroke models. Protein and mRNA levels of MDM2 and p53 were measured in both neuronal and brain extracts. The interaction of MDM2 with p53 was disrupted by neuronal treatment with nutlin-3a. siRNA was used to knockdown MDM2 expression. We analyzed the link between the MDM2 SNP309 and functional outcome, measured by the modified Rankin Scale scores, in 2 independent hospital-based stroke cohorts: ischemic stroke cohort (408 patients) and intracerebral hemorrhage cohort (128 patients). Results- Experimental stroke and oxygen and glucose deprivation induced the expression of MDM2 in the brain and neurons, respectively. Moreover, oxygen and glucose deprivation promoted MDM2 binding with p53 in neurons. Disruption of the MDM2-p53 interaction with nutlin-3a, or MDM2 knockdown by siRNA, triggered p53 accumulation, which increased neuronal susceptibility to oxygen and glucose deprivation-induced apoptosis. Finally, we showed that patients harboring the G allele in the MDM2 promoter had higher MDM2 protein levels and showed better functional outcome after stroke than those harboring the T/T genotype. The T/T genotype was also associated with large infarct volume in ischemic stroke and increased lesion volume in patients with intracerebral hemorrhage. Conclusions- Our results reveal a novel role for the MDM2-p53 interaction in neuronal apoptosis after ischemia and show that the MDM2 SNP309 determines the functional outcome of patients after stroke.

Project description:The Distal-less 3 (homeobox protein DLX-3), a transcription factor, is critical for the development of hair follicle and hair formation and regeneration. We previously identified and found that four SNPs (c. *118T>C, c. *228T>C, c. *688A>G and c. *1,038_1,039 insC) in 3' untranslated region (UTR) of sheep DLX3 were in high linkage disequilibrium with each other and significantly associated with wool crimp (P<0.05), however, the underlying mechanisms by which these SNPs affect the wool crimp remains unknown. In the present study, we performed association analysis between these four identified SNPs and DLX3 gene expression in sheep skin using quantitative real-time RT-PCR. The results showed that these SNPs were significantly associated with sheep skin DLX3 mRNA expression levels. Then, we constructed DLX3 3'UTR luciferase reporters and validated the association. The reporter assays showed that the three major haplotypes, derived from the four SNPs, had significantly different effects on luciferase reporter activity and the four SNPs also had significantly different allelic effects on the luciferase reporter activity (p < 0.05). Bioinformatics analysis showed that the SNP (c. *1,038_1,039 insC) was located within a potential miR-188 binding site of the 3'UTR of sheep DLX3 mRNA. This SNP may affect miR-188-mediated DLX3 gene expression and result in phenotypic variation. To test the hypothesis, we investigated the effects of miR-188 mimic and inhibitor on the activity of the DLX3 3'UTR luciferase reporter with different SNP alleles. The results showed that in both sheep fetal fibroblasts (SFFs) and human HaCaT cells, miR-188 mimic could significantly decrease the allele D (deletion) luciferase reporter activity (p < 0.05), but miR-188 inhibitor could increased the reporter activitiy. However, neither miR-188 mimc nor inhibitor could influence the allele I (insertion) reporter activity. In addition, transfection of miR-188 mimic dramatically decreased the endogenous expression of DLX3 in SFFs (p < 0.05). Taken together, we demonstrated that DLX3 is a target gene of miR-188 and the SNP (c. *1,038_1,039 insC) is a functional SNP, and affects miR-188-mediated gene regulation of sheep DLX3. Our finding may in part explain allelic difference in gene expression and wool crimp in our tested sheep population.

Dataset Information

Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region.

Publications

Functional impact of a single-nucleotide polymorphism in the OPRD1 promoter region.

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