Differential effect of cataract-associated mutations in MAF on transactivation of MAF target genes

Three mutations in the highly conserved DNA-binding region of c-MAF (R288P, K297R, and R299S) are associated with phenotypically distinct forms of autosomal dominant congenital cataract. However, the molecular mechanisms underlying this phenotypic diversity remain unclear. In this work, we have investigated the hypothesis that differential transactivation of MAF target genes could be one factor determining the phenotypic differences. Promoter constructs were generated for four human crystallin genes with conserved half-site MAF responsive elements (MARE). MAF expression constructs were constructed with the wildtype MAF sequence and with each of the three known mutations, i.e., R288P (associated with pulverulent cataract), K297R (associated with cerulean cataract), and R299S (associated with the most severe phenotype, congenital cataract, and microcornea syndrome). Transactivation was measured using luciferase reporter assays following cotransfection in HEK cells. Responsiveness to wildtype c-MAF was established for each of the four crystallin promoter constructs. The same constructs were then investigated using c-MAF mutants corresponding to each of the three mutations. A differential response was noted for each of the tested crystallin genes. The mutation R288P significantly reduced the expression of the CRYGA and CRYBA1 constructs but had no significant effect on the other two constructs. K297R did not lead to a significant reduction in expression of any of the four constructs, although there was a tendency toward reduced expression especially for the CRYGA construct. R299S, which is associated with the most severe phenotype, congenital cataract, and microcornea syndrome, was associated with the most severe overall effect on the transactivation of the four crystallin expression constructs. Our findings suggest that differential effects of mutations on the transactivation potential of c-MAF could be a molecular correlate of the striking genotype-phenotype correlations seen in cataract forms caused by mutations in the MAF gene.