A Polymorphism Located in a Putative p53 Binding Site of the Murine Bax Promoter Affects Transcription: Implications of Bax as a Susceptibility Allele in Glaucoma

Abstract

Purpose:: Previously, we showed that DBA/2J mice (Bax +/-) were resistant to optic nerve crush while 129B6 (Bax +/-) were susceptible. Quantitative PCR revealed higher levels of latent Bax mRNA in 129B6 neurons which may account for the difference in susceptibilities (Semaan SJ, ARVO Abstract 1279, 2005). We hypothesized that polymorphisms located in the murine Bax promoter affect expression.

Methods:: The Bax promoter region (1.4 kb) from DBA/2J and 129B6 mouse lines was isolated and sequenced. Strain-specific reporter constructs were created by cloning the promoters into a luciferase expression vector (pGL3-Basic). Basal luciferase expression from DBA/2J-Luc and 129B6-Luc was measured in transient transfection assays using NIH 3T3 murine fibroblasts. Luciferase expression was also assessed 24 hrs and 48 hrs after serum deprivation in NIH 3T3 cells to test for inducible expression. The effect of genetic background on expression was evaluated by cross-strain transfection assays using primary fibroblasts.

Results:: Sequence analysis identified a single polymorphism in one of two p53 consensus binding sites of the Bax promoter. The DBA/2J promoter contains a perfect El-Deiry p53 binding motif, while the 129B6 promoter contains a C to a T variation at position 4. Basal luciferase expression in NIH 3T3 cells was higher from the 129B6 Bax promoter than the DBA/2J Bax promoter (t-test p = 0.0002). There was no significant change in luciferase expression from both promoters 24 hrs after serum deprivation (DBA/2J p = 0.30, 129B6 p = 0.10), and expression significantly decreased 48 hrs after serum deprivation (DBA/2J p = 0.00003, 129B6 p = 0.00005). Preliminary data suggest that genetic background does not affect expression.

Conclusions:: A single polymorphism located in a p53 binding site affects the transcription rate of the Bax gene. This polymorphism is located in a highly critical area of the consensus sequence that is required for p53 binding. This may account for the difference in latent Bax mRNA levels and suggests p53 attenuates Bax transcription. We are conducting further experiments using transient transfection assays and DNA binding assays to assess the role of p53-binding in regulating basal transcription of the Bax gene.

Keywords: apoptosis/cell death • gene/expression • transcription