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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 essential for its regulation of transcriptional targets.HeLa cells were co-transfected with ZNF24 or its mutant constructs along with β-Catenin promoter reporter, and luciferase activities were measured after 48 h. Shown was the mean + S.E. of at least three independent experiments. *P < 0.05 compared to Vector and **P < 0.05 compared to WT.
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pone-0079910-g007: The first and second zinc fingers are essential for its regulation of transcriptional targets.HeLa cells were co-transfected with ZNF24 or its mutant constructs along with β-Catenin promoter reporter, and luciferase activities were measured after 48 h. Shown was the mean + S.E. of at least three independent experiments. *P < 0.05 compared to Vector and **P < 0.05 compared to WT.

Mentions: We next examined the effect of ZNF24 nuclear localization on the transcriptional activity of ZNF24 toward the β-Catenin (CTNNB1) promoter. Using promoter luciferase reporter assays, we found that the wild-type ZNF24 enhanced the CTNNB1 promoter activity by ~2.7-fold (Figure 7, compare WT with Vector). In contrast, when the ZF region was missing, the ZNF24 mutants totally failed to activate the CTNNB1 promoter (Figure 7, compare dZF1-2 or p1-250 with WT). The K286A and R290A mutants showed no change in its transactivation function (Figure 7, compare K286A, R290A with WT). Taken together, these results suggest that the first and second ZFs that control the nuclear localization of ZNF24 are essential for ZNF24 regulation of its transcriptional target genes.


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 essential for its regulation of transcriptional targets.HeLa cells were co-transfected with ZNF24 or its mutant constructs along with β-Catenin promoter reporter, and luciferase activities were measured after 48 h. Shown was the mean + S.E. of at least three independent experiments. *P < 0.05 compared to Vector and **P < 0.05 compared to WT.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0079910-g007: The first and second zinc fingers are essential for its regulation of transcriptional targets.HeLa cells were co-transfected with ZNF24 or its mutant constructs along with β-Catenin promoter reporter, and luciferase activities were measured after 48 h. Shown was the mean + S.E. of at least three independent experiments. *P < 0.05 compared to Vector and **P < 0.05 compared to WT.
Mentions: We next examined the effect of ZNF24 nuclear localization on the transcriptional activity of ZNF24 toward the β-Catenin (CTNNB1) promoter. Using promoter luciferase reporter assays, we found that the wild-type ZNF24 enhanced the CTNNB1 promoter activity by ~2.7-fold (Figure 7, compare WT with Vector). In contrast, when the ZF region was missing, the ZNF24 mutants totally failed to activate the CTNNB1 promoter (Figure 7, compare dZF1-2 or p1-250 with WT). The K286A and R290A mutants showed no change in its transactivation function (Figure 7, compare K286A, R290A with WT). Taken together, these results suggest that the first and second ZFs that control the nuclear localization of ZNF24 are essential for ZNF24 regulation of its transcriptional target genes.

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