Figure 5
Figure 5. Binding of Ikaros to the il2 promoter induces H4 deacetylation and inhibits il2 expression. (A) Jurkat cells were transfected with an il2–luciferase reporter plasmid and increasing amounts of an Ikaros expression plasmid. Twenty-four hours after transfection, cells were stimulated with PMA and ionomycin, and luciferase activity was determined. (B) Western blot using an anti-Ikaros antibody was performed with samples from RV-GFP–infected control resting (C) and ionomycin-treated (Io) cells and cells infected with RV-Ikaros-IRES-GFP (Ik). Infected cells were stimulated with anti-CD3 and anti-CD28 for 24 hours and IL-2 expression determined by ELISA. Bars show mean + SEM of 3 independent experiments. (C) Th1 cells transduced with virus expressing HA-Ikaros (Ik) and GFP or control GFP-virus were sorted and ChIPs performed using an anti-HA antibody. PCR products amplified with primers for the il2 promoter are shown for immunoprecipitated complexes and inputs. Gel shows one of 3 experiment with similar results. Numbers below input bands represent relative amount (to the value in RV-GFP–infected cells) quantified using qPCR. Graph shows relative band intensity (to the intensity in GFP-infected control cells) of immunoprecipitated complexes from all experiments (mean + SEM). (D) ChIP assays were performed using an antiacetylated H4 antibody on samples from transduced Th1 cells expressing GFP or HA-Ikaros and GFP (Ik). PCR products amplified with primers for the il2 promoter are shown for immunoprecipitated complexes and inputs. One experiment of 3 with similar results is shown. Numbers below input bands represent relative amount (to the value in RV-GFP–infected cells) quantified using qPCR. Graph shows relative band intensity of amplified immunoprecipitated complexes from all experiments (mean + SEM). Samples were also amplified using primers for the CD3ϵ promoter. (E) Infected Th1 cells with retroviruses expressing either GFP (control) or Ikaros and GFP (Ikaros) were treated with TSA from day 2 after infection. Four days later, cells were stimulated with anti-CD3 and anti-CD28 for 24 hours and IL-2 expression was determined by ELISA. Bars show mean + SEM of 3 different samples.

Binding of Ikaros to the il2 promoter induces H4 deacetylation and inhibits il2 expression. (A) Jurkat cells were transfected with an il2–luciferase reporter plasmid and increasing amounts of an Ikaros expression plasmid. Twenty-four hours after transfection, cells were stimulated with PMA and ionomycin, and luciferase activity was determined. (B) Western blot using an anti-Ikaros antibody was performed with samples from RV-GFP–infected control resting (C) and ionomycin-treated (Io) cells and cells infected with RV-Ikaros-IRES-GFP (Ik). Infected cells were stimulated with anti-CD3 and anti-CD28 for 24 hours and IL-2 expression determined by ELISA. Bars show mean + SEM of 3 independent experiments. (C) Th1 cells transduced with virus expressing HA-Ikaros (Ik) and GFP or control GFP-virus were sorted and ChIPs performed using an anti-HA antibody. PCR products amplified with primers for the il2 promoter are shown for immunoprecipitated complexes and inputs. Gel shows one of 3 experiment with similar results. Numbers below input bands represent relative amount (to the value in RV-GFP–infected cells) quantified using qPCR. Graph shows relative band intensity (to the intensity in GFP-infected control cells) of immunoprecipitated complexes from all experiments (mean + SEM). (D) ChIP assays were performed using an antiacetylated H4 antibody on samples from transduced Th1 cells expressing GFP or HA-Ikaros and GFP (Ik). PCR products amplified with primers for the il2 promoter are shown for immunoprecipitated complexes and inputs. One experiment of 3 with similar results is shown. Numbers below input bands represent relative amount (to the value in RV-GFP–infected cells) quantified using qPCR. Graph shows relative band intensity of amplified immunoprecipitated complexes from all experiments (mean + SEM). Samples were also amplified using primers for the CD3ϵ promoter. (E) Infected Th1 cells with retroviruses expressing either GFP (control) or Ikaros and GFP (Ikaros) were treated with TSA from day 2 after infection. Four days later, cells were stimulated with anti-CD3 and anti-CD28 for 24 hours and IL-2 expression was determined by ELISA. Bars show mean + SEM of 3 different samples.

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