Figure 3
Figure 3. FOXO1 directly activates the PRDM1α promoter. (A) Positive correlation between expression levels of FOXO1 and PRDM1 in microdissected HRS cells. p, 1-tailed Student t test; r, Pearson correlation coefficient. Data were mined from http://www.ncbi.nlm.nih.gov/geo/; GSE39133. (B) Schematic diagram showing the promoter region of PRDM1α and PRDM1β. The FOXO1-binding motif TRTTTAY is indicated as F. The PRDM1β promoter is located within intron 3 of PRDM1α. Promoter regions were amplified using primers indicated as arrows and cloned into pGL4.20 luciferase reporter plasmid. (C) FOXO1 activates PRDM1α promoter. L428 cells expressing FOXO1(A3)ER were transiently transfected with empty pGL4.20 vector or vector containing PRDM1 promoter regions, together with ubi-Renilla as reference vector. A total of 2 hours later, cells were treated or were not treated with 4-OHT. Relative luciferase activity was measured 24 hours after transfection. A total of 3 independent experiments were performed. Data are shown as mean ±SD. P value was determined using Student t test. (D) EMSA indicates DNA-binding activity of FOXO1 to PRDM1α promoter. Nuclear extracts were obtained from HEK-293T cells expressing EV or the FOXO1 DNA-binding domain (Fhbox). The probes used contained wild-type (wt) or mutated (mut) FOXO1-binding motifs flanked by PRDM1α promoter region. Position of motifs is indicated. A representative of 2 independent experiments is shown. (E) Supershift assay using Flag antibody or nonspecific IRS2 antibody confirmed binding of Fhbox to wt probe −832. A representative of 2 independent experiments is shown. (F) FOXO1 binds to the PRDM1α promoter. L428 cells were transfected with bFOXO1 and BirA or with EV and BirA. Twenty-four hours later, cells were harvested and ChIP was performed as described in the “Chromatin Immunoprecipitation” section of the supplemental Experimental Procedures. The tested PRDM1α promoter regions are indicated. A FOXP1 enhancer region was used as positive control. The precipitated chromatin was quantified by Q-PCR. Data are shown as fold enrichment compared with the negative control region located on chromosome 12 (Chr12) (see also supplemental Experimental Procedures, “Chromatin Immunoprecipitation”). A total of 5 independent experiments were performed. Data are shown as the mean ±SD. For FOXP1, results from 4 experiments are shown.

FOXO1 directly activates the PRDM1α promoter. (A) Positive correlation between expression levels of FOXO1 and PRDM1 in microdissected HRS cells. p, 1-tailed Student t test; r, Pearson correlation coefficient. Data were mined from http://www.ncbi.nlm.nih.gov/geo/; GSE39133. (B) Schematic diagram showing the promoter region of PRDM1α and PRDM1β. The FOXO1-binding motif TRTTTAY is indicated as F. The PRDM1β promoter is located within intron 3 of PRDM1α. Promoter regions were amplified using primers indicated as arrows and cloned into pGL4.20 luciferase reporter plasmid. (C) FOXO1 activates PRDM1α promoter. L428 cells expressing FOXO1(A3)ER were transiently transfected with empty pGL4.20 vector or vector containing PRDM1 promoter regions, together with ubi-Renilla as reference vector. A total of 2 hours later, cells were treated or were not treated with 4-OHT. Relative luciferase activity was measured 24 hours after transfection. A total of 3 independent experiments were performed. Data are shown as mean ±SD. P value was determined using Student t test. (D) EMSA indicates DNA-binding activity of FOXO1 to PRDM1α promoter. Nuclear extracts were obtained from HEK-293T cells expressing EV or the FOXO1 DNA-binding domain (Fhbox). The probes used contained wild-type (wt) or mutated (mut) FOXO1-binding motifs flanked by PRDM1α promoter region. Position of motifs is indicated. A representative of 2 independent experiments is shown. (E) Supershift assay using Flag antibody or nonspecific IRS2 antibody confirmed binding of Fhbox to wt probe −832. A representative of 2 independent experiments is shown. (F) FOXO1 binds to the PRDM1α promoter. L428 cells were transfected with bFOXO1 and BirA or with EV and BirA. Twenty-four hours later, cells were harvested and ChIP was performed as described in the “Chromatin Immunoprecipitation” section of the supplemental Experimental Procedures. The tested PRDM1α promoter regions are indicated. A FOXP1 enhancer region was used as positive control. The precipitated chromatin was quantified by Q-PCR. Data are shown as fold enrichment compared with the negative control region located on chromosome 12 (Chr12) (see also supplemental Experimental Procedures, “Chromatin Immunoprecipitation”). A total of 5 independent experiments were performed. Data are shown as the mean ±SD. For FOXP1, results from 4 experiments are shown.

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