Figure 1.
ZNF24 DNA binding domain is necessary for nuclear localization in HEK293 cells.
(A) Schematic of full-length and deletion ZNF24-GFP constructs used in this study (not to scale). The DNA binding domain is shown as four red boxes at the carboxyl terminus, with each box depicting a Krüppel-like zinc finger. All deletion constructs were fused to the N terminus of the GFP. (B) Protein localization of the ZNF24 constructs. HEK293 cells transfected with GFP control, pZNF24-GFP, p1-51-GFP, p1-200-GFP, p1-250-GFP, p161-250-GFP or p161-368-GFP. Digital images were taken by fluorescent microscopy. 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; C, exclusively cytoplasmic; 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 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).
Figure 2.
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).
Figure 3.
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).
Figure 4.
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).
Figure 5.
The lysine 286(K286) and arginine 290(R290) residues within the first and second ZF region are critical for the nuclear localization of ZNF24.
(A) Diagram of the ZNF24 mutants with point mutation of the lysine(K), argine(R), serines (S), threonines (T) or lysines (K) to alanines (A) within the first and second ZF region as indicated. (B) The protein localization was analyzed similarly as 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). (D) Conservation of the first and second ZF region of ZNF24 among the species.
Figure 6.
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).
Figure 7.
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.