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Functional interplay between the RK motif and linker segment dictates Oct4-DNA recognition.

Kong X, Liu J, Li L, Yue L, Zhang L, Jiang H, Xie X, Luo C - Nucleic Acids Res. (2015)

Bottom Line: Intriguingly, computational simulations reveal that the function of the RK motif may be finely tuned by H-bond interactions with the partially disordered linker segment and that breaking these interactions significantly enhances the DNA binding and reprogramming activities of Oct4.These findings uncover a self-regulatory mechanism for specific Oct4-DNA recognition and provide insights into the functional crosstalk at the molecular level that may illuminate mechanistic studies of the Oct protein family and possibly transcription factors in the POU family.Our gain-of-function Oct4 mutants might also be useful tools for use in reprogramming and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

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The RKRKR motif mutants of Oct4 lose transcriptional activity independent of localization. (A) Schematic diagram of the Oct4 mutants at the RKRKR motif with or without an additional nuclear localization sequence (NLS). (B) Cellular localization of Oct4 mutants with or without an additional NLS. Immunofluorescence staining with an anti-HA antibody was carried out 48 h after transfection in HEK293 cells. Hoechst stain was used to mark the nuclei. Scale bar: 10 μM. (C) Statistical analysis of the nuclear localization of various Oct4 proteins presented in (B). Data are presented as means ± SEM (n = 10). ***P < 0.001 versus WT Oct4; ###P < 0.001 versus corresponding mutants without an additional NLS at the C terminal. (D) The transcriptional activities of WT and mutant Oct4 with or without an additional NLS were measured by luciferase reporter assay. Luciferase activities were measured 24 h after transfection and the control group was transfected with the reporter only. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4. (E) iPSCs were induced with three factors (various Oct4 mutant, Sox2 and Klf4) and the number of GFP-positive colonies was counted at day 16 following infection. Representative images of iPSC colonies are presented; scale bar: 5 mm. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4.
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Figure 3: The RKRKR motif mutants of Oct4 lose transcriptional activity independent of localization. (A) Schematic diagram of the Oct4 mutants at the RKRKR motif with or without an additional nuclear localization sequence (NLS). (B) Cellular localization of Oct4 mutants with or without an additional NLS. Immunofluorescence staining with an anti-HA antibody was carried out 48 h after transfection in HEK293 cells. Hoechst stain was used to mark the nuclei. Scale bar: 10 μM. (C) Statistical analysis of the nuclear localization of various Oct4 proteins presented in (B). Data are presented as means ± SEM (n = 10). ***P < 0.001 versus WT Oct4; ###P < 0.001 versus corresponding mutants without an additional NLS at the C terminal. (D) The transcriptional activities of WT and mutant Oct4 with or without an additional NLS were measured by luciferase reporter assay. Luciferase activities were measured 24 h after transfection and the control group was transfected with the reporter only. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4. (E) iPSCs were induced with three factors (various Oct4 mutant, Sox2 and Klf4) and the number of GFP-positive colonies was counted at day 16 following infection. Representative images of iPSC colonies are presented; scale bar: 5 mm. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4.

Mentions: Previous studies have identified the RK-motif as a nuclear localization signal (NLS) for Oct4 and mutation of the RK motif results in a random distribution of Oct4 throughout the cell (45). Immunofluorescence staining revealed that single point mutations (R225A, R227A) had very limited impact on the nuclear localization of Oct4 (Figure 3A–C). However, these mutants were almost completely defective in transactivating the 6W-luciferase reporter and in generating iPSCs (Figure 3D and E). Similarly, the 30–60% reduction in nuclear localization of Oct4s carrying double or multi-site mutations (R225A/R227A, R225E/R227E, RKA and RKE) could not account for the total ablation of its transactivation and reprogramming capabilities. This finding suggests that the RK motif not only functions as an NLS but also strongly regulates the transcriptional activity of Oct4 by mediating Oct4–DNA binding. Nevertheless, to unambiguously define the role of the RK motif in modulating the transcription activity of Oct4, a known NLS was fused to the C-terminus of each Oct4 mutant to restore their nuclear localization (Figure 3A). The exogenous NLS indeed re-directed the RK-motif mutant proteins exclusively to the nuclei (Figure 3A–C), as previously observed (45). However, the restoration of nuclear localization did not rescue the abolished transactivation and reprogramming functions, and virtually no improvement in transcriptional activity was obtained for the Oct4 mutants with an exogenous NLS (Figure 3D and E). Notably, similar expression levels of the mutant Oct4 proteins were observed in these assays (Supplementary Figure S4). Therefore, our data establish that the transcriptional regulatory function of the RK-motif is independent of its nuclear localization activity.


Functional interplay between the RK motif and linker segment dictates Oct4-DNA recognition.

Kong X, Liu J, Li L, Yue L, Zhang L, Jiang H, Xie X, Luo C - Nucleic Acids Res. (2015)

The RKRKR motif mutants of Oct4 lose transcriptional activity independent of localization. (A) Schematic diagram of the Oct4 mutants at the RKRKR motif with or without an additional nuclear localization sequence (NLS). (B) Cellular localization of Oct4 mutants with or without an additional NLS. Immunofluorescence staining with an anti-HA antibody was carried out 48 h after transfection in HEK293 cells. Hoechst stain was used to mark the nuclei. Scale bar: 10 μM. (C) Statistical analysis of the nuclear localization of various Oct4 proteins presented in (B). Data are presented as means ± SEM (n = 10). ***P < 0.001 versus WT Oct4; ###P < 0.001 versus corresponding mutants without an additional NLS at the C terminal. (D) The transcriptional activities of WT and mutant Oct4 with or without an additional NLS were measured by luciferase reporter assay. Luciferase activities were measured 24 h after transfection and the control group was transfected with the reporter only. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4. (E) iPSCs were induced with three factors (various Oct4 mutant, Sox2 and Klf4) and the number of GFP-positive colonies was counted at day 16 following infection. Representative images of iPSC colonies are presented; scale bar: 5 mm. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4.
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Figure 3: The RKRKR motif mutants of Oct4 lose transcriptional activity independent of localization. (A) Schematic diagram of the Oct4 mutants at the RKRKR motif with or without an additional nuclear localization sequence (NLS). (B) Cellular localization of Oct4 mutants with or without an additional NLS. Immunofluorescence staining with an anti-HA antibody was carried out 48 h after transfection in HEK293 cells. Hoechst stain was used to mark the nuclei. Scale bar: 10 μM. (C) Statistical analysis of the nuclear localization of various Oct4 proteins presented in (B). Data are presented as means ± SEM (n = 10). ***P < 0.001 versus WT Oct4; ###P < 0.001 versus corresponding mutants without an additional NLS at the C terminal. (D) The transcriptional activities of WT and mutant Oct4 with or without an additional NLS were measured by luciferase reporter assay. Luciferase activities were measured 24 h after transfection and the control group was transfected with the reporter only. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4. (E) iPSCs were induced with three factors (various Oct4 mutant, Sox2 and Klf4) and the number of GFP-positive colonies was counted at day 16 following infection. Representative images of iPSC colonies are presented; scale bar: 5 mm. Data are presented as means ± SEM (n = 3). ***P < 0.001 versus WT Oct4.
Mentions: Previous studies have identified the RK-motif as a nuclear localization signal (NLS) for Oct4 and mutation of the RK motif results in a random distribution of Oct4 throughout the cell (45). Immunofluorescence staining revealed that single point mutations (R225A, R227A) had very limited impact on the nuclear localization of Oct4 (Figure 3A–C). However, these mutants were almost completely defective in transactivating the 6W-luciferase reporter and in generating iPSCs (Figure 3D and E). Similarly, the 30–60% reduction in nuclear localization of Oct4s carrying double or multi-site mutations (R225A/R227A, R225E/R227E, RKA and RKE) could not account for the total ablation of its transactivation and reprogramming capabilities. This finding suggests that the RK motif not only functions as an NLS but also strongly regulates the transcriptional activity of Oct4 by mediating Oct4–DNA binding. Nevertheless, to unambiguously define the role of the RK motif in modulating the transcription activity of Oct4, a known NLS was fused to the C-terminus of each Oct4 mutant to restore their nuclear localization (Figure 3A). The exogenous NLS indeed re-directed the RK-motif mutant proteins exclusively to the nuclei (Figure 3A–C), as previously observed (45). However, the restoration of nuclear localization did not rescue the abolished transactivation and reprogramming functions, and virtually no improvement in transcriptional activity was obtained for the Oct4 mutants with an exogenous NLS (Figure 3D and E). Notably, similar expression levels of the mutant Oct4 proteins were observed in these assays (Supplementary Figure S4). Therefore, our data establish that the transcriptional regulatory function of the RK-motif is independent of its nuclear localization activity.

Bottom Line: Intriguingly, computational simulations reveal that the function of the RK motif may be finely tuned by H-bond interactions with the partially disordered linker segment and that breaking these interactions significantly enhances the DNA binding and reprogramming activities of Oct4.These findings uncover a self-regulatory mechanism for specific Oct4-DNA recognition and provide insights into the functional crosstalk at the molecular level that may illuminate mechanistic studies of the Oct protein family and possibly transcription factors in the POU family.Our gain-of-function Oct4 mutants might also be useful tools for use in reprogramming and regenerative medicine.

View Article: PubMed Central - PubMed

Affiliation: Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.

Show MeSH
Related in: MedlinePlus