Limits...
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.

Show MeSH

Related in: MedlinePlus

The binding to zinc ions by ZF1 or/and ZF2 is not required for ZNF24 nuclear localization.(A) Schematic of different ZNF24-GFP mutant constructs and are denoted by an asterisk. The zinc-chelating histidines of ZF1 or/and ZF2 are mutated to leucine, respectively. The shaded boxes represent the same features described in Figure 1. (B) Subcellular localization of mutant ZNF24-GFPs. Indirect immunofluoresence was conducted on the indicated constructs and analyzed as described in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3815127&req=5

pone-0079910-g004: The binding to zinc ions by ZF1 or/and ZF2 is not required for ZNF24 nuclear localization.(A) Schematic of different ZNF24-GFP mutant constructs and are denoted by an asterisk. The zinc-chelating histidines of ZF1 or/and ZF2 are mutated to leucine, respectively. The shaded boxes represent the same features described in Figure 1. (B) Subcellular localization of mutant ZNF24-GFPs. Indirect immunofluoresence was conducted on the indicated constructs and analyzed as described in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).

Mentions: The major biochemical function of the ZFs in the nucleus is to contact the target gene promoter DNA in a Zn2+-binding-dependent manner[30,31]. The ZF motif of ZNF24 contains two conserved Cys and two conserved His residues. To determine whether these bindings are required for the nuclear localization of ZNF24, we individually disrupted the Zn2+-binding motifs (H269/273L, H297/301L and H269/273/297/301L) in ZF1 or/and ZF2 in the full-length context according for the reference[28], and examined the localization of the mutants in the cells (Figure 4). Surprisingly, none of these mutations affected the nuclear localization of ZNF24. Figure 4C quantifies the Fn/c ratios of these mutants (H269/273L、H297/301L and H269/273/297/301L) and confirms the visual phenotypes seen in Figure 4B. These results suggest that the ZFs regulate ZNF24 nuclear localization through a mechanism other than the Zn2+-dependent DNA binding or the tertiary zinc finger structure.


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 binding to zinc ions by ZF1 or/and ZF2 is not required for ZNF24 nuclear localization.(A) Schematic of different ZNF24-GFP mutant constructs and are denoted by an asterisk. The zinc-chelating histidines of ZF1 or/and ZF2 are mutated to leucine, respectively. The shaded boxes represent the same features described in Figure 1. (B) Subcellular localization of mutant ZNF24-GFPs. Indirect immunofluoresence was conducted on the indicated constructs and analyzed as described in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079910-g004: The binding to zinc ions by ZF1 or/and ZF2 is not required for ZNF24 nuclear localization.(A) Schematic of different ZNF24-GFP mutant constructs and are denoted by an asterisk. The zinc-chelating histidines of ZF1 or/and ZF2 are mutated to leucine, respectively. The shaded boxes represent the same features described in Figure 1. (B) Subcellular localization of mutant ZNF24-GFPs. Indirect immunofluoresence was conducted on the indicated constructs and analyzed as described in Figure 1. Patterns of localization are summarized on the left. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).
Mentions: The major biochemical function of the ZFs in the nucleus is to contact the target gene promoter DNA in a Zn2+-binding-dependent manner[30,31]. The ZF motif of ZNF24 contains two conserved Cys and two conserved His residues. To determine whether these bindings are required for the nuclear localization of ZNF24, we individually disrupted the Zn2+-binding motifs (H269/273L, H297/301L and H269/273/297/301L) in ZF1 or/and ZF2 in the full-length context according for the reference[28], and examined the localization of the mutants in the cells (Figure 4). Surprisingly, none of these mutations affected the nuclear localization of ZNF24. Figure 4C quantifies the Fn/c ratios of these mutants (H269/273L、H297/301L and H269/273/297/301L) and confirms the visual phenotypes seen in Figure 4B. These results suggest that the ZFs regulate ZNF24 nuclear localization through a mechanism other than the Zn2+-dependent DNA binding or the tertiary zinc finger structure.

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