Figure 6
From: Characterization of the upstream and intron promoters of the gene encoding TAR DNA-binding protein
hnRNP K regulates the TARDBP promoter via the predicted i-motif region. (a) Conservation of C-rich sequences in predicted i-motif regions between humans and mice. The squares and asterisks indicate C-rich and conserved sequences, respectively. (b) Schematic representation and luciferase assay of the TARDBP promoter construct with deletion of the predicted i-motif (P17). n = 6 replicates; Welch’s t-test relative to P1. (c) Immunoblot analysis of FLAG-hnRNP K overexpression in HeLaS3 cells. (d) Luciferase assay of P1 and P17 promoter activity with dose-dependent hnRNP K overexpression. Each reporter plasmid was co-transfected with 0, 0.5, 1 and 2 μg of hnRNP K/pFLAG-CMV2. n = 3 replicates; two-way ANOVA with post hoc Dunnett’s test relative to 0 μg of hnRNP K/pFLAG-CMV2. All samples transfected 2 μg of reporter vector and 2 μg of pFLAG-CMV2 vector (empty vector + hnRNP K). All error bars indicate the means ± SEMs. *P < 0.05, ****P < 0.0001. (e) ChIP assay of overexpressed FLAG-hnRNP K (top) or endogenous hnRNP K (bottom) binding to the TARDBP promoter in HEK293T cells. Immunoprecipitation was performed using anti-FLAG antibody (for overexpressed FLAG-TDP-43) or anti-hnRNP K antibody (for endogenous hnRNP K), then PCR was conducted using primer detecting TARDBP − 721 to − 431 (290 bp). Control IgG and Histone H3 antibody are used for negative or positive control for immunoprecipitation.
