Figure 2.
Figure 2. Ttp promoter contains a functional GAS element that binds STAT1. (A) Schematic representation of the Ttp promoter as cloned into the reporter construct pGL2-TTP, comprising the region from –2025 to +25. The Ttp promoter contains a GAS element at the position –1976 to –1964. The reporter plasmid pGL2-TTP-GAS contains the TTP-GAS with flanking sequences (–2085 to –1891). The consensus for STAT1-binding GAS42 is shown (according to IUPAC nomenclature, H stands for A, C, or T; S stands for G or C; D stands for G, A, or T). Mutated TTP-GAS (TTP-GASMut) was obtained by introducing 2 point mutations. (B) p38α+/+ cells stably transfected with a reporter plasmid containing the luciferase gene under the control of a 2-kb fragment of the Ttp promoter (pGL2-TTP) were left untreated (w/o) or treated for 1 hour with IFN-γ(g), anisomycin (a), or both (a/g). Total RNA was isolated and luciferase expression was assayed by qRT-PCR. Error bars indicate SD. *P < .01 (a/g) versus (a) treatment by univariate linear regression models; n = 3 experiments. (C) For nuclear run-on assay p38α+/+ cells were left untreated (w/o) or treated for 25 minutes with IFN-γ (g), anisomycin (a), or both (a/g), nuclei were prepared and run-on reaction was performed. Nuclear RNA was isolated and hybridized to membranes containing cDNA of Gapdh (as a control) and Ttp. (D) Bac 1.2F5 mouse macrophages were stimulated for 30 minutes with IFN-γ (g) or left untreated (–), and whole-cell extracts were assayed for binding to a radioactively labeled TTP-GAS probe using EMSA. The results demonstrate IFN-γ–inducible binding of STAT1 to TTP-GAS but not to mutated TTP-GAS (TTP-GASMut). The presence of STAT1 in the complexes was confirmed by super-shift using STAT1 antibodies (lanes marked with +S1). (E) A luciferase reporter containing TTP-GAS (pGL-TTPGAS) was transfected into STAT1–/–MEFs reconstituted with STAT1 cDNA (STAT1+/+) and for control into the parental STAT1–/– cells. After the transfection, cells were divided in 2 halves and 24 hours later treated for 6 hours with IFN-γ (g) or left untreated. Error bars indicate SD. †P < .01 +/+ versus –/–MEFs by univariate linear regression models; n = 3 experiments. (F) For ChIP, STAT1+/+ MEFs were treated for 30 minutes with IFN-γ (g) or left untreated (–). Recruitment of STAT1 to the TTP promoter was assayed using STAT1 antibodies (+S1). A control ChIP was performed using rabbit preimmune serum (C). Equal amount of material used in the ChIP experiments was confirmed by PCR-amplification of the input DNA (input).

Ttp promoter contains a functional GAS element that binds STAT1. (A) Schematic representation of the Ttp promoter as cloned into the reporter construct pGL2-TTP, comprising the region from –2025 to +25. The Ttp promoter contains a GAS element at the position –1976 to –1964. The reporter plasmid pGL2-TTP-GAS contains the TTP-GAS with flanking sequences (–2085 to –1891). The consensus for STAT1-binding GAS42 is shown (according to IUPAC nomenclature, H stands for A, C, or T; S stands for G or C; D stands for G, A, or T). Mutated TTP-GAS (TTP-GASMut) was obtained by introducing 2 point mutations. (B) p38α+/+ cells stably transfected with a reporter plasmid containing the luciferase gene under the control of a 2-kb fragment of the Ttp promoter (pGL2-TTP) were left untreated (w/o) or treated for 1 hour with IFN-γ(g), anisomycin (a), or both (a/g). Total RNA was isolated and luciferase expression was assayed by qRT-PCR. Error bars indicate SD. *P < .01 (a/g) versus (a) treatment by univariate linear regression models; n = 3 experiments. (C) For nuclear run-on assay p38α+/+ cells were left untreated (w/o) or treated for 25 minutes with IFN-γ (g), anisomycin (a), or both (a/g), nuclei were prepared and run-on reaction was performed. Nuclear RNA was isolated and hybridized to membranes containing cDNA of Gapdh (as a control) and Ttp. (D) Bac 1.2F5 mouse macrophages were stimulated for 30 minutes with IFN-γ (g) or left untreated (–), and whole-cell extracts were assayed for binding to a radioactively labeled TTP-GAS probe using EMSA. The results demonstrate IFN-γ–inducible binding of STAT1 to TTP-GAS but not to mutated TTP-GAS (TTP-GASMut). The presence of STAT1 in the complexes was confirmed by super-shift using STAT1 antibodies (lanes marked with +S1). (E) A luciferase reporter containing TTP-GAS (pGL-TTPGAS) was transfected into STAT1–/–MEFs reconstituted with STAT1 cDNA (STAT1+/+) and for control into the parental STAT1–/– cells. After the transfection, cells were divided in 2 halves and 24 hours later treated for 6 hours with IFN-γ (g) or left untreated. Error bars indicate SD. †P < .01 +/+ versus –/–MEFs by univariate linear regression models; n = 3 experiments. (F) For ChIP, STAT1+/+ MEFs were treated for 30 minutes with IFN-γ (g) or left untreated (–). Recruitment of STAT1 to the TTP promoter was assayed using STAT1 antibodies (+S1). A control ChIP was performed using rabbit preimmune serum (C). Equal amount of material used in the ChIP experiments was confirmed by PCR-amplification of the input DNA (input).

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