Open AccessTumor suppressor protein p53-mediated repression of human mitotic centromere-associated kinesin gene expression is exerted via down-regulation of Sp1 level
Author(s) -
Do Youn Jun,
Ji Young Lee,
Hae Sun Park,
Yun Han Lee,
Young Ho Kim
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0189698
Subject(s) - psychological repression , kinesin , centromere , suppressor , mitosis , microbiology and biotechnology , biology , regulation of gene expression , kinetochore , gene expression , gene , genetics , microtubule , chromosome
The repressive role of p53 on the human mitotic centromere-associated kinesin ( MCAK ) core promoter from ‒266 to +54, relative to the transcription start site, has been determined. The MCAK mRNA and protein levels were 2.1- and 3.0-fold higher, respectively, in HCT116 (p53 ‒/‒ ) than in HCT116 (p53 +/+ ) cells. Enforced down-regulation of p53 levels either in HCT116 (p53 +/+ ) cells by p53 RNAi treatment or in MCF-7 cells using shRNA for p53 (shp53) resulted in a remarkable increase in the MCAK protein level. Site-directed mutagenesis and ChIP analyses showed that p53-mediated repression of the MCAK core promoter activity was not directly exerted by p53-binding to putative p53-response elements (p53-RE1 at −173/−166 and p53-RE2 at −245/−238), but indirectly by attenuating Sp1 binding to GC-motifs (GC1 at −93/−84 and GC2 at −119/−110). Treatment of HEK-293 cells bearing the MCAK core promoter-reporter (pGL2-320-Luc) with mithramycin A, which down-regulates Sp1 gene expression, reduced the promoter activity as well as endogenous MCAK levels. Exposure of HCT116 (p53 +/+ ) cells to nutlin-3a, a validated activator of p53, caused a simultaneous reduction in Sp1 and MCAK protein levels, but not in HCT116 (p53 −/− ) cells. In contrast to wild-type (wt)-p53, tumor-derived p53 mutants (p53 V143A , p53 R248W , and p53 R273H ) failed to repress the Sp1-dependent activation of the MCAK promoter and to down-regulate endogenous levels of Sp1 and MCAK proteins. Collectively, these findings demonstrate that p53 can repress MCAK promoter activity indirectly via down-regulation of Sp1 expression level, and suggest that MCAK elevation in human tumor cells might be due to p53 mutation.