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Identification of functional DNA variants in the constitutive promoter region of MDM2.

ABSTRACT: Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 and is p53-responsive, and an upstream constitutive promoter (P1), which is p53-independent. Both promoter regions contain DNA variants that could influence the expression levels of MDM2, including the well-studied single nucleotide polymorphism (SNP) SNP309, which is located in the promoter P2; i.e., upstream of exon 2. In this report, we screened the promoter P1 for DNA variants and assessed the functional impact of the corresponding SNPs. Using the dbSNP database and genotyping validation in individuals of European descent, we identified three common SNPs (-1494?G?>?A; indel 40?bp; and -182?C?>?G). Three major promoter haplotypes were inferred by using these three promoter SNPs together with rs2279744 (SNP309). Following subcloning into a gene reporter system, we found that two of the haplotypes significantly influenced MDM2 promoter activity in a haplotype-specific manner. Site-directed mutagenesis experiments indicated that the 40?bp insertion/deletion variation is causing the observed allelic promoter activity. This study suggests that part of the variability in the MDM2 expression levels could be explained by allelic p53-independent P1 promoter activity.

SUBMITTER: Lalonde ME

PROVIDER: S-EPMC3500213 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Identification of functional DNA variants in the constitutive promoter region of MDM2.

Lalonde Marie-Eve ME Ouimet Manon M Larivière Mathieu M Kritikou Ekaterini A EA Sinnett Daniel D

Human genomics 20120901

Although mutations in the oncoprotein murine double minute 2 (MDM2) are rare, MDM2 gene overexpression has been observed in several human tumors. Given that even modest changes in MDM2 levels might influence the p53 tumor suppressor signaling pathway, we postulated that sequence variation in the promoter region of MDM2 could lead to disregulated expression and variation in gene dosage. Two promoters have been reported for MDM2; an internal promoter (P2), which is located near the end of intron 1 ...[more]

PMID: 23244604

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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.

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Identification of functional DNA variants in the constitutive promoter region of MDM2.

Publications

Identification of functional DNA variants in the constitutive promoter region of MDM2.

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