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 first and second zinc fingers are necessary for efficient nuclear localization.(A) Schematic of different ZNF24-GFP deletions constructs. The shaded boxes represent features described in Figure 1. (B) Subcellular localization of the different constructs. The indicated constructs were transfected into HEK293 cells was conducted as described in Figure 1. DAPI stain is shown to delineate nuclear boundaries (blue). Patterns of localization are summarized on the right by observing 100 transfected cells from 10 to 15 independent fields. N, exclusively nuclear; N=C, most cells show both nuclear and cytoplasmic localization with similar staining intensity in the nucleus and cytoplasm; N>C, most cells show both nuclear and cytoplasmic localization with brighter staining intensity in the nucleus. (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-g002: The first and second zinc fingers are necessary for efficient nuclear localization.(A) Schematic of different ZNF24-GFP deletions constructs. The shaded boxes represent features described in Figure 1. (B) Subcellular localization of the different constructs. The indicated constructs were transfected into HEK293 cells was conducted as described in Figure 1. DAPI stain is shown to delineate nuclear boundaries (blue). Patterns of localization are summarized on the right by observing 100 transfected cells from 10 to 15 independent fields. N, exclusively nuclear; N=C, most cells show both nuclear and cytoplasmic localization with similar staining intensity in the nucleus and cytoplasm; N>C, most cells show both nuclear and cytoplasmic localization with brighter staining intensity in the nucleus. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).

Mentions: We next carried out experiments to further delimit the NLS within the ZF region. This region consists of four Krüppel zinc finger motifs that are highly homologous to each other. We evaluated the contribution of each zinc finger motif to nuclear localization. A series of mutants were generated that deleted one to four zinc fingers. Figure 2A illustrates a schematic of these deletions. Deletion of the ZF4 (p1-329-GFP) or the ZF3-4 (p1-301-GFP) had no effect on the nuclear localization of ZNF24 (Figure 2B). However, deletion of the ZF2-4(p1-273-GFP) led to both nuclear and cytoplasmic localization with brighter staining intensity in the nucleus. Deletion of whole ZF region (p1-250-GFP) led to diffusively present throughout the cell with similar staining intensity in the nucleus and cytoplasm (Figure 2B). These data suggest that ZF1-2 (aa 251 to 301) is necessary for efficient nuclear localization of ZNF24.


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 first and second zinc fingers are necessary for efficient nuclear localization.(A) Schematic of different ZNF24-GFP deletions constructs. The shaded boxes represent features described in Figure 1. (B) Subcellular localization of the different constructs. The indicated constructs were transfected into HEK293 cells was conducted as described in Figure 1. DAPI stain is shown to delineate nuclear boundaries (blue). Patterns of localization are summarized on the right by observing 100 transfected cells from 10 to 15 independent fields. N, exclusively nuclear; N=C, most cells show both nuclear and cytoplasmic localization with similar staining intensity in the nucleus and cytoplasm; N>C, most cells show both nuclear and cytoplasmic localization with brighter staining intensity in the nucleus. (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-g002: The first and second zinc fingers are necessary for efficient nuclear localization.(A) Schematic of different ZNF24-GFP deletions constructs. The shaded boxes represent features described in Figure 1. (B) Subcellular localization of the different constructs. The indicated constructs were transfected into HEK293 cells was conducted as described in Figure 1. DAPI stain is shown to delineate nuclear boundaries (blue). Patterns of localization are summarized on the right by observing 100 transfected cells from 10 to 15 independent fields. N, exclusively nuclear; N=C, most cells show both nuclear and cytoplasmic localization with similar staining intensity in the nucleus and cytoplasm; N>C, most cells show both nuclear and cytoplasmic localization with brighter staining intensity in the nucleus. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).
Mentions: We next carried out experiments to further delimit the NLS within the ZF region. This region consists of four Krüppel zinc finger motifs that are highly homologous to each other. We evaluated the contribution of each zinc finger motif to nuclear localization. A series of mutants were generated that deleted one to four zinc fingers. Figure 2A illustrates a schematic of these deletions. Deletion of the ZF4 (p1-329-GFP) or the ZF3-4 (p1-301-GFP) had no effect on the nuclear localization of ZNF24 (Figure 2B). However, deletion of the ZF2-4(p1-273-GFP) led to both nuclear and cytoplasmic localization with brighter staining intensity in the nucleus. Deletion of whole ZF region (p1-250-GFP) led to diffusively present throughout the cell with similar staining intensity in the nucleus and cytoplasm (Figure 2B). These data suggest that ZF1-2 (aa 251 to 301) is necessary for efficient nuclear localization of ZNF24.

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