Functional interplay between the RK motif and linker segment dictates Oct4-DNA recognition.
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.
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
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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.
Affiliation: Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.