Importin α1 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.

Modulation of the Cyclin-Dependent Kinase Inhibitor p21WAF1/Cip1 Gene by Zac1 through the Antagonistic Regulators p53 and Histone Deacetylase 1 in HeLa Cells

“…Induction of p53 in response to DNA-damaging agents could result in the formation of p53 and Sp1 complexes and, simultaneously, dissociate HDAC1 from the COOH terminus of Sp1 (20,28,33,45). Therefore, our findings on the physical interaction between Zac1 and HDAC1 suggest that Zac1 might act on the p21 promoter not only through the p53-dependent pathway, as a p53 coactivator or by competition of HDAC1 from HDAC1-p53 complex (35), but also through the Sp1-dependent pathway (13), as an Sp1 coactivator or in competition with HDAC1 from the HDAC1-Sp1 complex (data not shown).…”

“…Functionally, Zac1 not only exerts an antiproliferative effect but also acts as a transcriptional cofactor for p53 and a number of nuclear receptors (NR; refs. [13][14][15]. The NR coregulating activity of Zac1 may result from its ability to bind directly to NRs and to interact with two classes of NR coactivators: the p160 and the CBP/p300 family of coactivators.…”

Grail as a molecular determinant for the functions of the tumor suppressor p53 in tumorigenesis

“…Reporters for pG13-, p21-, Mdm2-, PUMA-, and Bax-LUC have been described previously. 26,30 Physical and functional interactions between Grail and p53 Y-C Chen et al…”

“…H1299 nuclear extracts were incubated with DAPA binding buffer (10 mM Tris-HCl, pH 7.5, 50 mM KCl, and 1 mM DTT) as described previously. 30 Specific protein-DNA-agarose complexes were washed, analyzed by SDS-PAGE, and detected by immunoblotting with an anti-p53 antibody.…”

Nuclear retention of importin α coordinates cell fate through changes in gene expression

“…All tested stress-responsive or apoptosis-related genes exhibited reduced mRNA levels in cells transfected with either full-length or C-mutant isoform of importin a2. On the other hand, p21, which previously exhibited little or no change in human importin a1-expressing HeLa cells without Zac1 (Huang et al, 2007), replication-dependent histones were listed as downregulated genes. Since the histone genes are known to be clustered together in the genome (Marzluff et al, 2002), we assumed that nuclear importin a2 may access specific gene clusters.…”