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

Marie-Eve Lalonde,Daniel Sinnett,Mathieu Larivière,Manon Ouimet,Ekaterini A Kritikou

doi:10.1186/1479-7364-6-15

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.

Highlights

The p53 tumor suppressor has a key role in orchestrating cellular responses to various types of stresses, including DNA damage and oncogene activation with apoptosis, cell-cycle arrest, senescence, DNA repair, cell metabolism, or autophagy [1,2]
Oligonucleotide probes specific for each promoter single nucleotide polymorphism (SNP) were used for allele-specific oligonucleotides (ASOs) analysis and are available upon request
The search for SNPs in the constitutive P1 promoter of murine double minute 2 (MDM2) led to the identification of eight promoter SNPs (pSNPs), including a 40 bp indel

Summary

Introduction

The p53 tumor suppressor has a key role in orchestrating cellular responses to various types of stresses, including DNA damage and oncogene activation with apoptosis, cell-cycle arrest, senescence, DNA repair, cell metabolism, or autophagy [1,2]. Malfunction and mutations of p53 have been found in most human cancers, leading to a deregulated p53 activity that allows cells to proliferate and survive [3]. MDM2 can regulate p53 activity in different ways and even modest modifications of MDM2 levels can affect the p53 pathway [4]. MDM2 directly binds to the p53 transactivation domain, mutations in MDM2 are rare, MDM2 overexpression is observed in a number of human tumors due to various mechanisms including gene amplification [8,9,10] and increased transcription [11,12]. Because MDM2 is an important negative regulator of p53 activity, overexpression of MDM2 can result in the inhibition of p53-mediated-transcriptional activation, thereby promoting human carcinogenesis

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