Importin alpha1 is involved in the nuclear localization of Zac1 and the induction of p21WAF1/CIP1 by Zac1

Abstract

Zac1, a novel seven-zinc-finger transcription factor, preferentially binds GC-rich DNA elements and has intrinsic transactivation activity. To date, the NLS (nuclear localization signal) of Zac1 has not been empirically determined. We generated a series of EGFP (enhanced green fluorescence protein)-tagged deletion mutants of Zac1 and examined their subcellular localization, from which we defined two NLSs within the DNA-binding (or zinc-finger) domain. Fusion proteins consisting of the two EGFP-tagged zinc-finger clusters (zinc finger motifs 1-3 and 4-7) were located exclusively in the nucleus, demonstrating that each of the zinc-finger clusters is sufficient for nuclear localization. Physical interactions between these two zinc-finger clusters and importin alpha1 were demonstrated using an in vitro glutathione S-transferase pull-down assay. Finally, our results indicate that the association of Zac1 with importin alpha1 is also involved in regulating the transactivation activity of Zac1 on the p21WAF1/CIP1 gene and protein expression.

Figure 1. The NLS of Zac1 is identified in its N-terminal region

(a) The functional domains of mouse Zac1. (b and c) Schematic representations of various EGFP-tagged constructs used to transfect HeLa cells. Representative HeLa cells were transiently transfected with these constructs. Nuclei are identified by blue fluorescence after DAPI staining. The intracellular distribution of various EGFP-fusion proteins is shown underneath (c). Approx. 100 transfected cells were examined by fluorescence microscopy and classified by the presence of green fluorescence: exclusively in the nucleus (N), mainly in the nucleus (N>C) or evenly distributed throughout the nucleus and cytoplasm (N/C). Scale bar, 10 μm.

Figure 2. Sequence alignment of Zac1 (amino acids 1–90; zinc finger motifs 1–3) and PLAG1 (amino acids 1–120)

Sequence alignment is illustrated for identical amino acids by capital letters and for similar amino acids by plus signs (+) in between the two sequences. The highly conserved cysteine (C) and histidine (H) residues within the C₂H₂-zinc finger motif of Zac1 and PLAG1 are shown in bold capital letters. The KRKR sequence in the open box is the putative NLS of PLAG1. Two linkers (underlined) were swapped with alanine residues (indicated as A) by site-directed mutagenesis.

Figure 3. Two zinc-finger clusters function as nuclear import signals for Zac1

(a) The diagram shows seven C₂H₂-type zinc-finger motifs, within which the conserved cysteine (C) and histidine (H) residues are shown in upper case letters. ZF7* is a truncated ZF7 and its amino acid sequence is illustrated. (b–d) Representative fluorescence images are shown for the HeLa cells transiently transfected with various combinations of zinc-finger motifs compiling the Zac1–EGFP fusion proteins. Approx. 100 transfected cells were examined by fluorescence microscopy, and each of the EGFP-fusion proteins is classified by N, N>C and N/C as in Figure 1.

Figure 4. Zac1 physically interacts with importin α₁ through its zinc-finger motif in vitro

(a and b) Full-length importin α₁ was translated in vitro and incubated with bead-bound GST or various GST–Zac1 fusion proteins as indicated. Bound proteins were eluted, separated by SDS/PAGE and visualized by autoradiography. For comparison, the leftmost lane of each panel shows 10 or 15% of the input protein used in the binding assay reactions. M.W., molecular mass (kDa) (c) Location of the IBB domain and ten armadillo repeats. Various importin α₁ fragments were translated in vitro and incubated with bead-bound GST or GST–Zac1_ZF1−3 fusion protein. Results are representative of two independent experiments.

Figure 5. Importin α₁ co-localizes with Zac1 in the nucleus

HeLa cells were transfected with (a) 0.5 μg of DNA encoding DsRed conjugated to the indicated fragments of importin α₁ fusion proteins with various regions of importin α₁ as indicated, or (b) 0.5 μg of DNA encoding EGFP-tagged full-length Zac1 and DsRed conjugated to the indicated fragments of importin α₁. Approx. 100 transfected cells were examined by fluorescence microscopy.

Figure 6. Importin α₁ acts synergistically with Zac1 on the p21 promoter and protein induction in HeLa cells

(a and b) HeLa cells were transiently transfected with the p21-LUC reporter gene (0.4 μg) and pSG5HA.Zac1 (0.3 μg) and/or pSG5HA.importin α₁ (0.3 μg) reporter constructs. Luciferase activity in the transfected cell extracts is indicated in RLU. Numbers above the columns indicate the fold induction in RLU relative to that of the control cells in which only p21-LUC was transfected. (b) HeLa cell extracts were subjected to Western blot analysis probing with anti-p21, anti-p53, anti-HA and anti-α-tubulin antibodies. (c) HeLa cell extracts were used for ChIP assays followed by PCR analysis with two sets of primer for both: Sp1 A (−218/−87) and Sp1 B (−86/+142). Results (b and c) are representative of two independent experiments.

Figure 7. Importin α₁ associates with the Zac1–DNA-responsive element complex

(a) A modified DAPA carried out as described in the Materials and methods section is shown schematically. (b) HeLa nuclear extracts were subjected to analysis by the modified DAPA using the antibodies indicated and analysed by Western blot analysis. Results are representative of two independent experiments.