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First published online April 22, 2009
doi: 10.1242/10.1242/jcs.041749

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Characterization of Snail nuclear import pathways as representatives of C₂H₂ zinc finger transcription factors

¹ Instituto de Neurociencias, CSIC-UMH, Avda. Ramón y Cajal s/n, San Juan de Alicante, Spain
² Instituto de Biología Molecular y Celular, UMH, Elche, Spain

View larger version (47K): [in this window] [in a new window]   Fig. 1. Several importins mediate Snail1 nuclear import. (A) Identification of importins that bind to immobilized Snail1 in pull-down assays using a cytosolic HeLa extract as the source of importins. The assay was performed in the presence or absence of RanGTP (5 µM) as indicated. Bound importins were identified by western blotting. (B-I) Nuclear import assays with digitonin-permabilised HeLa cells. (C-G) GFP-Snail1 was efficiently imported by the importins that strongly bound Snail1: Impβ, alone or in combination with Imp, transportin and Imp7, but not by the inactive adapter Imp alone. (H,I). Imp9 and Imp13 could not efficiently import Snail1 in to the nucleus.

View larger version (50K): [in this window] [in a new window]   Fig. 2. Identification of the Snail1 NLS. (A,D,E) Pull-down assays were performed with the indicated Snail1 protein fragments in the absence of RanGTP as described in Fig. 1. Bound importins (Imp, Impβ, Imp7 and transportin) were identified by western blotting. (A) Assays carried out with either the full length (FL) or the N-terminal (NH; 1-151) or C-terminal (CH; 152-264) halves. (B) Three-dimensional model of the four zinc fingers of Snail1 showing the position of all the mutated residues. (C) List of the mutated residues in all the Snail1 mutants (M1-M9) used in the pull-down assays shown in D and E. (F) Three-dimensional model showing the position of the residues that when mutated, impair Snail1 nuclear import.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Snail1 NLS is functional in cell lines. Constructs driving the expression of GFP (A) or the indicated GFP- (B-L) or GFP-GST- (M-P) Snail1 fusion proteins were transiently transfected into MCF7 cells. The cells were fixed 24 hours after transfection and the subcellular localization of the proteins was analysed by confocal microscopy. (Q) Pull-down assays with the indicated Snail proteins were performed as described in Fig. 2. (R-U) Subcellular localization of the indicated GFP-GST-Snail1 mutants in transiently transfected MCF7 cells. Single mutations in zinc fingers (Sm ZF) were as follows: ZF1: K161E; ZF2: K187E and ZF3: R220E.

View larger version (25K): [in this window] [in a new window]   Fig. 4. The Snail1 NLS residues are necessary for DNA binding but not for the conformation of the binding domain. (A) EMSA showing that Snail1 mutants M2 (K161E, K170E), M3 (K187E, R191E) and M9 (R220E, R224E) have lost their ability to form retardation complexes (RCI and RCII) with a Snail1-specific DNA probe, unlike the M8 mutant (Q239E) that forms complexes comparable with those of the wild type (wt). Upper panel: Snail proteins input used in the assay. FP: free probe. (B) Luciferase reporter assay showing that the Snail1 mutant versions M2, M3 and M9 are unable to repress the activity of the E-cadherin promoter (-1000 bp). Relative luciferase activity is related to the activity detected in cells cotransfected with the luciferase reporter construct and an empty vector (V0). (C) The CD spectra of the indicated wild-type and mutant Snail1 proteins do not display significant differences. (D) Thermal denaturation of the indicated Snail1 proteins monitored by far-UV CD reveal two main transitions at around 63 and 83°C in all cases.

View larger version (44K): [in this window] [in a new window]   Fig. 5. The proper folding of the four zinc fingers is necessary for Snail1 binding to the importins and subsequent nuclear localization (A) Pull-down assays with the indicated Snail1 proteins were performed in the absence of RanGTP as described in Fig. 1. (B-G) Subcellular localization of GFP-GST or the indicated GFP-GST-Snail1 fusion proteins in transiently transfected MCF7 cells.

View larger version (74K): [in this window] [in a new window]   Fig. 6. Snail1 nuclear import pathways are conserved in Snail proteins. (A) Pull-down assays were performed with immobilized Snail-like or Snail2, and the bound importins were identified by western blotting. Note that the same importins that bind to Snail1 (Fig. 1A) also bind to Snail-like and Snail2. (B-D) Pull-down assays performed with the indicated immobilized proteins in the absence of RanGTP. (B) Only the C-terminal half of Snail-like and Snail2 can bind to importins. NH, N-terminal half (Snail-like: 1-145; Snail2: 1-156); CH, C-terminal half (Snail-like: 146-258; Snail2: 157-268). (C) Mutations in equivalent residues to those identified in Snail1 also impair the binding of Snail-like or Snail2 to importins. (D) The import pathways are conserved in amphioxus Snail [C-BfSnail, C-terminal half (141-253)].

View larger version (52K): [in this window] [in a new window]   Fig. 7. Conservation of the NLS in C2H2 zinc finger transcription factors. (A) Alignment of zinc fingers (ZF) 2-4 of representative members of the Snail superfamily (Snail and Scratch proteins). These fingers correspond to number 1-3 in human Snail1 and Snail1L. The three pairs of basic residues necessary for nuclear import (red) are conserved in all the Snail proteins. Scratch proteins have lost the second K in finger 2. Note that only five out of the six residues are required for importin binding and nuclear localization. (B) Alignment of three consecutive zinc fingers of representative human members of five additional families of C2H2 transcription factors, which also contained the identified NLS. The basic residues are shown in red and the zinc coordinating cysteines and histidines are shown in blue. Zinc finger residues are shaded in red. (C) Schematic representation of the relative positions occupied by the NLS described. Exposed basic residues that conform the basic interface for importin binding are shown in red. In some cases, these basic residues can be found in adjacent positions (shown in pink) in other transcription factors. Linker regions (green);hydrophobic residues required for zinc finger conformation (Knight and Shimeld, 2001) (blue). Accession numbers of the aligned proteins: Hs-Snail1 (NP005976), Hs-SnailL (EAW70471), Hs-Snail2 (NP003059), Hs-Snail3 (NP840101), Dm-Snail (NP476732), Dm-Worniu (AAF12733), Dm-Escargot (AAA28513), Bf-Snail (AAC35351), Hs-Scratch1 (NP112599), Hs-Scratch2, (NP149120), Dm-Scratch (AAA91035), Dm-ScratchL1 (NP647845), Dm-ScratchL2 (NP612040), Bf-Scratch (Bf Scaffold 229), Hs-Egr1 (P18146), HsEKLF (NP006554), Hs-RFLAT1 (NP057079), Hs-SP1 (P08047) and Hs-FEZ1 (NP001019784). Hs, Homo sapiens; Dm, Drosophila melanogaster; Bf, Branchyostoma floridae.

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