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Identification of a functional nuclear localization signal mediating nuclear import of the zinc finger transcription factor ZNF24.

Li JZ, Chen X, Gong XL, Hu HY, Shi D, Lu YM, Qiu L, Lu F, Hu ZL, Zhang JP - PLoS ONE (2013)

Bottom Line: Our results delimit the NLS to ZF1-2.ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization.Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacy, Second Military Medical University, Shanghai, China.

ABSTRACT
ZNF24 is a member of the SCAN domain family of Krüppel-like zinc finger (ZF) transcription factors, which plays a critical role in cell proliferation and differentiation. However, how ZNF24 enters the nucleus in order to exert its function remains unclear since its nuclear localization signal(s) (NLS) has not been identified. Here, we generated a series of GFP-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to ZF1-2. Deletion of ZF1-2 caused cytoplasmic accumulation of ZNF24. Fusion of the ZF1-2 to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the ZF1-2 is both necessary and sufficient for nuclear localization. ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. K286A and R290A mutation led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that ZNF24 interacted with importin-β and this interaction required the ZF motifs. The β-Catenin (CTNNB1) luciferase assays showed that the ZNF24 mutants defective in nuclear localization could not promote CTNNB1 promoter activation as the wild-type ZNF24 did. Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

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The ZF regions are essential for ZNF24 binding to importin-β1.GFP-tagged ZNF24 and mutants were transiently expressed and co-IPed with importin-β1 antibody and blotted with anti-importin-β1 or anti-GFP. Positions of the ZNF24 proteins are marked on the left side of the blots (H.C., IgG heavy chain).
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pone-0079910-g006: The ZF regions are essential for ZNF24 binding to importin-β1.GFP-tagged ZNF24 and mutants were transiently expressed and co-IPed with importin-β1 antibody and blotted with anti-importin-β1 or anti-GFP. Positions of the ZNF24 proteins are marked on the left side of the blots (H.C., IgG heavy chain).

Mentions: Transcription factors interact with the importin proteins (importin-β1 in most cases and importin-α/β1 complex in other cases) through their NLS(s) and are then shuttled into the nucleus[28]. To test if the K286, R290 and/or the ZF region are important for ZNF24 interaction with importin-β, indicated GFP-tagged ZNF24 proteins were transiently expressed in HEK293 cells and co-IPed with importin-β (Figure 6). Clearly, ZNF24 (WT) interacted with importin-β, whereas the negative control IgG did not. This interaction was completely abolished when the ZF region was deleted (p1-250-GFP), whereas mutation of K286 or/and R290 did not affect the association. These results suggest that the C-terminal ZF region is critical for ZNF24 interaction with importin-β during its nuclear translocation, and the K286 and R290 residues may use a different mechanism to regulate ZNF24 nuclear localization.


Identification of a functional nuclear localization signal mediating nuclear import of the zinc finger transcription factor ZNF24.

Li JZ, Chen X, Gong XL, Hu HY, Shi D, Lu YM, Qiu L, Lu F, Hu ZL, Zhang JP - PLoS ONE (2013)

The ZF regions are essential for ZNF24 binding to importin-β1.GFP-tagged ZNF24 and mutants were transiently expressed and co-IPed with importin-β1 antibody and blotted with anti-importin-β1 or anti-GFP. Positions of the ZNF24 proteins are marked on the left side of the blots (H.C., IgG heavy chain).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815127&req=5

pone-0079910-g006: The ZF regions are essential for ZNF24 binding to importin-β1.GFP-tagged ZNF24 and mutants were transiently expressed and co-IPed with importin-β1 antibody and blotted with anti-importin-β1 or anti-GFP. Positions of the ZNF24 proteins are marked on the left side of the blots (H.C., IgG heavy chain).
Mentions: Transcription factors interact with the importin proteins (importin-β1 in most cases and importin-α/β1 complex in other cases) through their NLS(s) and are then shuttled into the nucleus[28]. To test if the K286, R290 and/or the ZF region are important for ZNF24 interaction with importin-β, indicated GFP-tagged ZNF24 proteins were transiently expressed in HEK293 cells and co-IPed with importin-β (Figure 6). Clearly, ZNF24 (WT) interacted with importin-β, whereas the negative control IgG did not. This interaction was completely abolished when the ZF region was deleted (p1-250-GFP), whereas mutation of K286 or/and R290 did not affect the association. These results suggest that the C-terminal ZF region is critical for ZNF24 interaction with importin-β during its nuclear translocation, and the K286 and R290 residues may use a different mechanism to regulate ZNF24 nuclear localization.

Bottom Line: Our results delimit the NLS to ZF1-2.ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization.Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemical Pharmacy, Second Military Medical University, Shanghai, China.

ABSTRACT
ZNF24 is a member of the SCAN domain family of Krüppel-like zinc finger (ZF) transcription factors, which plays a critical role in cell proliferation and differentiation. However, how ZNF24 enters the nucleus in order to exert its function remains unclear since its nuclear localization signal(s) (NLS) has not been identified. Here, we generated a series of GFP-tagged deletion and point mutants and assessed their subcellular localization. Our results delimit the NLS to ZF1-2. Deletion of ZF1-2 caused cytoplasmic accumulation of ZNF24. Fusion of the ZF1-2 to green fluorescent protein (GFP) targeted GFP to the nucleus, demonstrating that the ZF1-2 is both necessary and sufficient for nuclear localization. ZNF24 containing histidine to leucine mutations that disrupt the structure of ZF1 or/and ZF2 retains appropriate nuclear localization, indicating that neither the tertiary structure of the zinc fingers nor specific DNA binding are necessary for nuclear localization. K286A and R290A mutation led to partial cytoplasmic accumulation. Co-immunoprecipitation demonstrated that ZNF24 interacted with importin-β and this interaction required the ZF motifs. The β-Catenin (CTNNB1) luciferase assays showed that the ZNF24 mutants defective in nuclear localization could not promote CTNNB1 promoter activation as the wild-type ZNF24 did. Taken together, these results suggest that consecutive ZF1-2 is critical for the regulation of ZNF24 nuclear localization and its transactivation function.

Show MeSH
Related in: MedlinePlus