Figure 7
Figure 7. Antithetic effects of NFI-A on the β-globin and G-CSFR promoters in vivo. (A) Schematic representation of the β-globin proximal promoter. NFI-A DNA-binding site sequences are highlighted in bold characters. TSS indicates the transcriptional start site; arrows, the position of the primers used for the ChIP analysis. Mutant sites are numbered according to their vicinity to the TSS. (B) Relative quantification of NFI-A occupancy on the β-globin promoter. Chromatin was immunoprecipitated with an NFI-A antibody, and the bound DNA was analyzed by multiplex PCR using specific primers corresponding to the β-globin promoter region containing the NFI-A binding sites and an unrelated intergenic genomic region (UR) as an internal control. Histograms represent the mean ± SEM from 3 independent DNA preparations; PCR analyses were repeated at least 3 times. (C) Promoter assay showing a positive role of NFI-A in β-globin proximal promoter activation. Wild-type and mutant constructs were assayed for transcriptional activity relative to the endogenous NFI-A or in a cotransfection with an NFI-A–expressing plasmid. The firefly luciferase values were normalized to the Renilla luciferase values for each transfection, and the relative luciferase activity is represented as relative fold induction over the HB-Wt transfection (mean ± SEM values from 4 independent transfections). Each reading was repeated at least 2 times. (D) Schematic representation of the NFI-A binding sites on the G-CSFR promoter. (E) Relative quantification of NFI-A occupancy on the G-CSFR promoter. Chromatins were immunoprecipitated with an NFI-A antibody, and the bound DNA was analyzed by multiplex PCR using specific primers corresponding to the G-CSFR promoter region containing the NFI-A binding sites and an unrelated intergenic genomic region (UR) as an internal control. Histograms represent the mean ± SEM from 3 independent DNA preparations; multiplex PCR analysis was repeated at least 3 times. (F) Promoter assay showing NFI-A repressive activity on the G-CSFR promoter performed in K562 cells. Wild-type or mutant promoter constructs were transfected, and firefly luciferase activity was normalized to Renilla luciferase activity for each transfection. The relative luciferase activity is represented as relative fold induction over the GR-Wt transfection. Data represent mean ± SEM from 3 independent transfections. Each reading was repeated at least 2 times.

Antithetic effects of NFI-A on the β-globin and G-CSFR promoters in vivo. (A) Schematic representation of the β-globin proximal promoter. NFI-A DNA-binding site sequences are highlighted in bold characters. TSS indicates the transcriptional start site; arrows, the position of the primers used for the ChIP analysis. Mutant sites are numbered according to their vicinity to the TSS. (B) Relative quantification of NFI-A occupancy on the β-globin promoter. Chromatin was immunoprecipitated with an NFI-A antibody, and the bound DNA was analyzed by multiplex PCR using specific primers corresponding to the β-globin promoter region containing the NFI-A binding sites and an unrelated intergenic genomic region (UR) as an internal control. Histograms represent the mean ± SEM from 3 independent DNA preparations; PCR analyses were repeated at least 3 times. (C) Promoter assay showing a positive role of NFI-A in β-globin proximal promoter activation. Wild-type and mutant constructs were assayed for transcriptional activity relative to the endogenous NFI-A or in a cotransfection with an NFI-A–expressing plasmid. The firefly luciferase values were normalized to the Renilla luciferase values for each transfection, and the relative luciferase activity is represented as relative fold induction over the HB-Wt transfection (mean ± SEM values from 4 independent transfections). Each reading was repeated at least 2 times. (D) Schematic representation of the NFI-A binding sites on the G-CSFR promoter. (E) Relative quantification of NFI-A occupancy on the G-CSFR promoter. Chromatins were immunoprecipitated with an NFI-A antibody, and the bound DNA was analyzed by multiplex PCR using specific primers corresponding to the G-CSFR promoter region containing the NFI-A binding sites and an unrelated intergenic genomic region (UR) as an internal control. Histograms represent the mean ± SEM from 3 independent DNA preparations; multiplex PCR analysis was repeated at least 3 times. (F) Promoter assay showing NFI-A repressive activity on the G-CSFR promoter performed in K562 cells. Wild-type or mutant promoter constructs were transfected, and firefly luciferase activity was normalized to Renilla luciferase activity for each transfection. The relative luciferase activity is represented as relative fold induction over the GR-Wt transfection. Data represent mean ± SEM from 3 independent transfections. Each reading was repeated at least 2 times.

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