Figure 2
Figure 2. Direct activation of the SOX4 promoter by FRA-2 and JUND. (A) A schematic depiction of the SOX4 promoter region from –768 to +102 bp with potential regulatory elements. (B) Deletion analysis. Luciferase reporter assays were performed using reporter constructs containing the SOX4 promoter fragment from –768 to +102 bp and its successive 5′-truncated fragments. The region from –103 to –57 bp was found to be necessary and sufficient for the reporter gene expression in ST1 cells. Data are presented as mean ± SEM of 3 separate experiments. *P < .05. (C) Mutation analysis. Luciferase reporter assays were performed in ST1 cells using reporter constructs containing the SOX4 promoter fragment from –768 to +102 bp with an AP-1 site at –92 to –82 bp being wild-type or mutated (ΔAP-1, from CATGAGAAGC to CATTGGACTC). Data are presented as mean ± SEM of 3 separate experiments. *P < .05. (D) Reconstitution experiment. Raji cells were cotransfected with pGL3-SOX4 (–768/+102) with the wild-type AP-1 site or mutated AP-1 site (ΔAP-1) and expression vectors for c-FOS, FRA-2, JUND, or control vector as indicated. After 24 hours, luciferase assays were performed in triplicate. Promoter activation was shown as fold induction of luciferase activity in cells transfected with indicated AP-1 member expression vectors vs cells transfected with the control vector. Transfection efficiency was normalized by β-galactosidase activity. Data are presented as mean ± SEM of 3 separate experiments. *P < .05. (E) ChIP assay. Chromatins prepared from normal resting and activated CD4+ T cells and 2 ATL cell lines (HUT102 and ST1) were fragmented and immunoprecipitated with anti-histone H3 (positive control), anti-FRA-2, anti-JUND, or control IgG (negative control). Real-time PCR was performed to quantify the AP-1 site of the SOX4 promoter in precipitated chromatin fragments relative to total input DNA (% input). Data are presented as mean ± SEM of 3 separate experiments. *P < .05; **P < .01. (F) Effect of HTLV-1 HBZ on the SOX4 promoter. ST1 cells were cotransfected with pGL3-SOX4 (–768/+102) and an expression vector for the spliced form of HBZ (sHBZ) or control vector as indicated. After 24 hours, luciferase assays were performed in triplicate. Transfection efficiency was normalized by β-galactosidase activity. Data are presented as mean ± SEM of 3 separate experiments.

Direct activation of the SOX4 promoter by FRA-2 and JUND. (A) A schematic depiction of the SOX4 promoter region from –768 to +102 bp with potential regulatory elements. (B) Deletion analysis. Luciferase reporter assays were performed using reporter constructs containing the SOX4 promoter fragment from –768 to +102 bp and its successive 5′-truncated fragments. The region from –103 to –57 bp was found to be necessary and sufficient for the reporter gene expression in ST1 cells. Data are presented as mean ± SEM of 3 separate experiments. *P < .05. (C) Mutation analysis. Luciferase reporter assays were performed in ST1 cells using reporter constructs containing the SOX4 promoter fragment from –768 to +102 bp with an AP-1 site at –92 to –82 bp being wild-type or mutated (ΔAP-1, from CATGAGAAGC to CATTGGACTC). Data are presented as mean ± SEM of 3 separate experiments. *P < .05. (D) Reconstitution experiment. Raji cells were cotransfected with pGL3-SOX4 (–768/+102) with the wild-type AP-1 site or mutated AP-1 site (ΔAP-1) and expression vectors for c-FOS, FRA-2, JUND, or control vector as indicated. After 24 hours, luciferase assays were performed in triplicate. Promoter activation was shown as fold induction of luciferase activity in cells transfected with indicated AP-1 member expression vectors vs cells transfected with the control vector. Transfection efficiency was normalized by β-galactosidase activity. Data are presented as mean ± SEM of 3 separate experiments. *P < .05. (E) ChIP assay. Chromatins prepared from normal resting and activated CD4+ T cells and 2 ATL cell lines (HUT102 and ST1) were fragmented and immunoprecipitated with anti-histone H3 (positive control), anti-FRA-2, anti-JUND, or control IgG (negative control). Real-time PCR was performed to quantify the AP-1 site of the SOX4 promoter in precipitated chromatin fragments relative to total input DNA (% input). Data are presented as mean ± SEM of 3 separate experiments. *P < .05; **P < .01. (F) Effect of HTLV-1 HBZ on the SOX4 promoter. ST1 cells were cotransfected with pGL3-SOX4 (–768/+102) and an expression vector for the spliced form of HBZ (sHBZ) or control vector as indicated. After 24 hours, luciferase assays were performed in triplicate. Transfection efficiency was normalized by β-galactosidase activity. Data are presented as mean ± SEM of 3 separate experiments.

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