Figure 6.
Figure 6. Ikaros recruits IRF4 to the ZICEs and inhibits IRF4-mediated gene activation. (A) ZICE sequences of indicated regions. ZICE, GGGAANNNGAAA indicated with blue box; ISRE, GAAANNGAAA indicated with red box. (B) Luciferase assays using the ZICE containing reporter genes. 293T cells were transiently transfected with indicated reporter and effector plasmids. The amounts of plasmids were as follows: luciferase reporter (1.0 μg), Ikaros (100 ng), and IRF4 (100 ng). The reporters used are shown above each panel. The average luciferase activity and SD are from 3 independent experiments. *P < .05; **P < .01; ***P < .001. (C) Binding of Ikaros/IRF4 complexes to the ZICE motif. EMSA with the Ebf1 E1 sequence as probe. α-Ikaros or α-IRF4 or control antibodies were used in supershift assays to confirm Ikaros/IRF4 complexes. (D) Competition assay using the Ebf1 E1 probe. Increased amounts of competitor DNAs, WT or mutant IRF (IRFmut), were included as indicated. (E-F) Oligonucleotide precipitation assay using the Ebf1 E1 oligonucleotide. Biotinylated WT or mutant (MT) oligonucleotide was incubated with nuclear extracts as indicated. MT carries mutation in both a zinc finger and an IRF motifs. Oligonucleotide-protein complex was immunoprecipitated with streptavidin beads followed by immunoblot. As input, 10% of immunoprecipitation (IP) for Ikaros or 50% of IP for IRF4 were loaded. Immunoblot was performed using α-FLAG (M2) for detecting Ikaros or α-IRF4. (G) Binding of IRF4 homodimer to an ISRE motif within the ZICE of Haao E1 probe. α-IRF4 or control antibodies were used in supershift assays to confirm IRF4/IRF4 complexes. (H) The effective recruitment of IRF4 to the ZICE sequence in the presence of Ikaros rather than to the ISRE sequence. All binding reactions contain a Haao E1 probe. IRF4 concentration was increased in twofold increments as indicated in the presence or absence of Ikaros. For (C-H), nuclear extracts were prepared from 293T cells transfected with pcDNA3 HA-IRF4 or Flag-Ikaros expressing vector, respectively. Red asterisk, Ikaros specific band; red bracket, Ikaros/IRF4 complexes; red box, IRF4/IRF4 complexes; black asterisk, nonspecific (NS). (I) Schematic representation of IRF4 recruitment to the ZICEs. ZICE, GGGAANNNGAAA underlined and indicated with blue box; ISRE, GAAANNGAAA indicated with red box. The ZICEs embed the ISRE motif, and IRF4 enables to bind the ZICE sequence as a heterodimer with Ikaros or the ISRE sequence as a homodimer. However, IRF4 is effectively recruited to the ZICE sequence in the presence of Ikaros with lower IRF4 concentration. Therefore, the Ikaros/IRF4 complex binds the ZICEs for repressing target genes.

Ikaros recruits IRF4 to the ZICEs and inhibits IRF4-mediated gene activation. (A) ZICE sequences of indicated regions. ZICE, GGGAANNNGAAA indicated with blue box; ISRE, GAAANNGAAA indicated with red box. (B) Luciferase assays using the ZICE containing reporter genes. 293T cells were transiently transfected with indicated reporter and effector plasmids. The amounts of plasmids were as follows: luciferase reporter (1.0 μg), Ikaros (100 ng), and IRF4 (100 ng). The reporters used are shown above each panel. The average luciferase activity and SD are from 3 independent experiments. *P < .05; **P < .01; ***P < .001. (C) Binding of Ikaros/IRF4 complexes to the ZICE motif. EMSA with the Ebf1 E1 sequence as probe. α-Ikaros or α-IRF4 or control antibodies were used in supershift assays to confirm Ikaros/IRF4 complexes. (D) Competition assay using the Ebf1 E1 probe. Increased amounts of competitor DNAs, WT or mutant IRF (IRFmut), were included as indicated. (E-F) Oligonucleotide precipitation assay using the Ebf1 E1 oligonucleotide. Biotinylated WT or mutant (MT) oligonucleotide was incubated with nuclear extracts as indicated. MT carries mutation in both a zinc finger and an IRF motifs. Oligonucleotide-protein complex was immunoprecipitated with streptavidin beads followed by immunoblot. As input, 10% of immunoprecipitation (IP) for Ikaros or 50% of IP for IRF4 were loaded. Immunoblot was performed using α-FLAG (M2) for detecting Ikaros or α-IRF4. (G) Binding of IRF4 homodimer to an ISRE motif within the ZICE of Haao E1 probe. α-IRF4 or control antibodies were used in supershift assays to confirm IRF4/IRF4 complexes. (H) The effective recruitment of IRF4 to the ZICE sequence in the presence of Ikaros rather than to the ISRE sequence. All binding reactions contain a Haao E1 probe. IRF4 concentration was increased in twofold increments as indicated in the presence or absence of Ikaros. For (C-H), nuclear extracts were prepared from 293T cells transfected with pcDNA3 HA-IRF4 or Flag-Ikaros expressing vector, respectively. Red asterisk, Ikaros specific band; red bracket, Ikaros/IRF4 complexes; red box, IRF4/IRF4 complexes; black asterisk, nonspecific (NS). (I) Schematic representation of IRF4 recruitment to the ZICEs. ZICE, GGGAANNNGAAA underlined and indicated with blue box; ISRE, GAAANNGAAA indicated with red box. The ZICEs embed the ISRE motif, and IRF4 enables to bind the ZICE sequence as a heterodimer with Ikaros or the ISRE sequence as a homodimer. However, IRF4 is effectively recruited to the ZICE sequence in the presence of Ikaros with lower IRF4 concentration. Therefore, the Ikaros/IRF4 complex binds the ZICEs for repressing target genes.

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