Pro-apoptotic Bax is essential for RGC (retinal ganglion cell) death. Gene dosage experiments in mice, yielding a single wild-type Bax allele, indicated that genetic background was able to influence the cell death phenotype. DBA/2J(Bax+/-) mice exhibited complete resistance to nerve damage after 2 weeks (similar to Bax(-/-) mice), but 129B6(Bax+/-) mice exhibited significant cell loss (similar to wild-type mice). The different cell death phenotype was associated with the level of Bax expression, where 129B6 neurons had twice the level of endogenous Bax mRNA and protein as DBA/2J neurons. Sequence analysis of the Bax promoters between these strains revealed a single nucleotide polymorphism (T(129B6) to C(DBA/2J)) at position -515. A 1.5- to 2.5-fold increase in transcriptional activity was observed from the 129B6 promoter in transient transfection assays in a variety of cell types, including RGC5 cells derived from rat RGCs. Since this polymorphism occurred in a p53 half-site, we investigated the requirement of p53 for the differential transcriptional activity. Differential transcriptional activity from either 129B6 or DBA/2J Bax promoters were unaffected in p53(-/-) cells, and addition of exogenous p53 had no further effect on this difference, thus a role for p53 was excluded. Competitive electrophoretic mobility-shift assays identified two DNA-protein complexes that interacted with the polymorphic region. Those forming Complex 1 bound with higher affinity to the 129B6 polymorphic site, suggesting that these proteins probably comprised a transcriptional activator complex. These studies implicated quantitative expression of the Bax gene as playing a possible role in neuronal susceptibility to damaging stimuli.
Keywords: Bax; C/EBP, CCAAT/enhancer-binding protein; DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electrophoretic mobility-shift assay; FBS, fetal bovine serum; FOXI1, Forkhead box I1; Luc, luciferase; ONC, optic nerve crush; RGC, retinal ganglion cell; gene expression; glaucoma; neuronal apoptosis; retinal ganglion cells; susceptibility allele; βgal, β-galactosidase.