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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 first and second zinc fingers are sufficient for nuclear localization.(A) The ZF1-2- GFP construct is a fusion of the first and second zinc fingers to GFP. ZF1-GFP is a fusion of the first zinc finger to GFP. ZF2-GFP is a fusion of the second zinc finger to GFP. HEK293 cells were transfected with the indicated constructs, and direct immunofluorescence was conducted as described in Figure 1. (B) The subcellular localization of each mutant is summarized on the right. Representative cells from each construct are shown. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).
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pone-0079910-g003: The first and second zinc fingers are sufficient for nuclear localization.(A) The ZF1-2- GFP construct is a fusion of the first and second zinc fingers to GFP. ZF1-GFP is a fusion of the first zinc finger to GFP. ZF2-GFP is a fusion of the second zinc finger to GFP. HEK293 cells were transfected with the indicated constructs, and direct immunofluorescence was conducted as described in Figure 1. (B) The subcellular localization of each mutant is summarized on the right. Representative cells from each construct are shown. (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 determined if the ZF1-2 was sufficient for nuclear localization. The ZF1-2 (aa 251-301) was fused to GFP (ZF1-2-GFP see Figure 3A) and examined for subcellular distribution. In contrast to GFP, which was distributed almost equally between the nucleus and cytoplasm, ZF1-2-GFP fusion proteins were predominantly localized to the nucleus in both HEK293 (Figure 3B) and HeLa cells (data not shown). Surprisingly, ZF1-GFP or ZF2-GFP fusion proteins were localized to the nucleus and cytoplasm with brighter staining intensity in the nucleus (Figure 3B). Figure 3C quantifies the Fn/c ratios of ZF1-2-GFP、ZF1-GFP and ZF2-GFP and confirms the visual phenotypes seen in Figure 3B. Taken together, these results demonstrate that the ZF1-2 of ZNF24 encode an NLS that is both necessary and sufficient for 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 first and second zinc fingers are sufficient for nuclear localization.(A) The ZF1-2- GFP construct is a fusion of the first and second zinc fingers to GFP. ZF1-GFP is a fusion of the first zinc finger to GFP. ZF2-GFP is a fusion of the second zinc finger to GFP. HEK293 cells were transfected with the indicated constructs, and direct immunofluorescence was conducted as described in Figure 1. (B) The subcellular localization of each mutant is summarized on the right. Representative cells from each construct are shown. (C) Results (mean±S.E.M., n=100) for quantitative analysis of images to determine the nuclear to cytoplasmic fluorescence ratio (Fn/c).
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Related In: Results  -  Collection

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

pone-0079910-g003: The first and second zinc fingers are sufficient for nuclear localization.(A) The ZF1-2- GFP construct is a fusion of the first and second zinc fingers to GFP. ZF1-GFP is a fusion of the first zinc finger to GFP. ZF2-GFP is a fusion of the second zinc finger to GFP. HEK293 cells were transfected with the indicated constructs, and direct immunofluorescence was conducted as described in Figure 1. (B) The subcellular localization of each mutant is summarized on the right. Representative cells from each construct are shown. (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 determined if the ZF1-2 was sufficient for nuclear localization. The ZF1-2 (aa 251-301) was fused to GFP (ZF1-2-GFP see Figure 3A) and examined for subcellular distribution. In contrast to GFP, which was distributed almost equally between the nucleus and cytoplasm, ZF1-2-GFP fusion proteins were predominantly localized to the nucleus in both HEK293 (Figure 3B) and HeLa cells (data not shown). Surprisingly, ZF1-GFP or ZF2-GFP fusion proteins were localized to the nucleus and cytoplasm with brighter staining intensity in the nucleus (Figure 3B). Figure 3C quantifies the Fn/c ratios of ZF1-2-GFP、ZF1-GFP and ZF2-GFP and confirms the visual phenotypes seen in Figure 3B. Taken together, these results demonstrate that the ZF1-2 of ZNF24 encode an NLS that is both necessary and sufficient for 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