Figure 4. Human TFIIAγ gene mutations inhibited promoter activity and endogenous gene expression.

(A) A diagram showing the conservative amino acids between yeast TOA2 and human TFIIAγ (top panel) and the mutation sites within human TFIIAγ (bottom panel). (B) Western blot analysis for 293T stable cell lines expressing TFIIAγ shRNA and WT HA-TFIIAγ or its mutants. 293T cells were transfected with the plasmids expressing TFIIAγ shRNA and WT HA-TFIIAγ or its derivatives in the presence of the lentiviral packaging vectors. Expression of TFIIAγ and HA-TFIIAγ was analyzed by Western blot. (C) Mutations of TFIIAγ gene significantly inhibited the activity of AdML promoter. 293T stable cell lines were transfected with the AdML promoter-driving reporter gene vectors; after 48 h, the cells were harvested for luciferase assays. (D) The effect of the N63A mutant on the activities of natural promoters. 293T stable cell lines, including WT and N63A, were transfected using the vectors expressing a reporter gene that is driven by one of the natural promoters as indicated; after 48 h, the cells were harvested for luciferase assays. (E) RT-qPCR result showing the effect of the N63A mutant on expression of endogenous genes. Total RNA was extracted from stable cell lines, including WT and N63A, the cDNA for each sample was synthesized and detected by quantitative PCR. Each column in (C–E) represents the mean ± S.D. of three independent experiments. *, P<0.05; **, P<0.01; P-values were obtained with one way ANOVA.

Abbreviation: RLA, relative luciferase activity