Figure 6

Suppressed transcriptional responses of STAT1 mutants with high-affinity GAS binding. (A) Decreased reporter gene activation in U3A cells expressing STAT1 DNA-binding mutants as compared to wild-type protein. U3A cells were transiently transfected with expression plasmids coding for the indicated STAT1 variants, a luciferase reporter construct and a constitutively expressed β-galactosidase gene used for normalization. The three reporter constructs used in these experiments contained either a triple GAS site from the Ly6E promoter (3xLy6E) or a 339 base pair or 1352 base pair fragment from the native ICAM-1 promoter (pIC339 and pIC1352). On the next day, the cells were either left untreated (gray columns) or stimulated for 6 hours with 5 ng/ml IFNγ (black columns) before, in whole cell extracts, luciferase luminescence and ß-galactosidase activity were measured. (B) Reduced activation of three endogenous STAT1 target genes by the E411K and E421K mutants. RT-PCR assays were performed with U3A cells expressing either STAT1 wild-type, E411K or E421K. Expression levels of the irf1, mig1, gbp1, and for control stat1 gene before and after 6 hours stimulation with 5 ng/ml IFNγ (gray and black columns, respectively) are shown. Gene induction was normalized to the expression of the house-keeping gene gapdh. The data are presented as means and standard deviations from at least three independent experiments. Statistical significance between the groups of IFNγ−stimulated cells expressing the indicated STAT1 variants is marked by asterisks.