CK2 kinase activity but not its binding to CK2 promoter regions is implicated in the regulation of CK2α and CK2β gene expressions

Protein kinase CK2, a ubiquitous serine/threonine kinase in control of a variety of crucial cellular functions, is composed of catalytic a- and a0-subunits and non-catalytic b-subunits which form holoenzymes such as CK2(ab)2, CK2aa\u27b2, or CK2(a\u27b)2. In addition, there is sample evidence for the occurrence of the individual subunits beside the holoenzyme. While the CK2 subunits are well analyzed on the protein level, only little is known about the regulation of their transcription. The existence of multiple forms of CK2 subunits raised the question about a mutual regulation of their expression. Here we defined two 50-upstream regions of the CK2alpha and the CK2beta genes, respectively, as sequences with promoter activities. We found that CK2alpah and CK2alpha\u27 stimulated the expression of the reporter constructs whereas, CK2beta was inactive. Using chromatin immunoprecipitation assays, we were unable to detect binding of endogenous CK2 subunits to these promoter sequences in vivo. However, it turned out that inhibition of the kinase activity of CK2 attenuated the promoter activity indicating that CK2alpha and CK2alpha\u27 might regulate their gene expression indirectly by phosphorylation reactions. Thus, we have shown here (i) that under normal physiological conditions CK2 does not bind to CK2 promoter regions and (ii) that the CK2 kinase activity is implicated in the regulation of its own expression

p53 and cell cycle dependent transcription of kinesin family member 23 (KIF23) is controlled via a CHR promoter element bound by DREAM and MMB complexes

The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21WAF1/CIP1 was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth

The CHR element mediates cell cycle-dependent expression of <i>KIF23</i>.

<p>Human foreskin fibroblasts (HFF) were arrested in G₀ by serum starvation and re-entered the cell cycle after serum addition. Cells were harvested at 0 (G₀), 12 (G₁), 22 (S) or 26 (G₂/M) hours after restimulation. (A) mRNA levels of <i>KIF23</i> splice variants were measured separately or with primers detecting both splice variants by real-time RT-PCR. (B) KIF23 protein levels were detected by western blot with β-actin serving as loading control. (C) Cell cycle distribution of HFF cells was confirmed by FACS analysis. (D) Dual luciferase reporter gene assays were carried out in serum-starved (G₀) and 24 h restimulated (G₂/M) NIH3T3 cells. Activities of wild-type and CHR mutant <i>KIF23</i> promoter constructs as well as the vector control are displayed as relative light units (RLU). (E) DNA content of serum-starved and 24 h restimulated NIH3T3 cells was analyzed by FACS.</p

KIF23 protein levels are diminished after p53 activation and elevated following <i>p53</i> knockdown.

<p>KIF23, p53 and p21 protein levels were detected by western blot. GAPDH or β-actin served as loading control. (A) In D53wt or D53mut cells the expression of transgenes was induced for 9 h. (B+C) Wild-type HCT116 cells (HCT116<i>+/+</i>) or HCT116 cells lacking p53 (HCT116 <i>p53−/−</i>) or p21 (HCT116 <i>p21−/−</i>) were treated with (B) doxorubicin for 48 and 72 h or with (C) nutlin-3 for 24 h. DMSO was used as solvent control. (D) Human foreskin fibroblasts (HFF) were mock transfected or transfected with siRNA targeting p53 and control siRNA, respectively.</p

The DREAM and MMB complexes bind to the CHR in the <i>KIF23</i> promoter.

<p>(A) <i>In vitro</i> analysis of DREAM and MMB protein binding to the <i>KIF23</i> wild-type (wt) or the CHR mutant promoter. Nuclear extracts of density-arrested or asynchronously growing NIH3T3 cells were employed for DNA-affinity purification using biotinylated human <i>KIF23</i> promoter probes. Binding was tested by western blot analysis using antibodies against p130, E2f4, Lin37 and B-myb. As a protein binding to all <i>KIF23</i> probes, Nfya was detected. As a negative control, a fragment of the <i>Gapdhs</i> promoter was used. (B-D) T98G cells were synchronized by serum starvation and restimulated to re-enter the cell cycle for 22 h. (B) FACS analysis confirmed the cell cycle distribution. (C) Nuclear extracts were prepared and chromatin immunoprecipitation analysis of DREAM and MMB binding to the human <i>KIF23</i> promoter <i>in vivo</i> was performed with antibodies targeting E2F4, LIN9 and B-Myb. As a negative control, a non-targeting rabbit antibody (IgG) was used. All signals are given relative to the input (**p≤0.01; ***p≤0.001; n = 4). (D) The <i>GAPDHS</i> promoter served as negative control. (E-G) ChIP experiments were performed in untreated HCT116 cells or HCT116 cells treated with doxorubicin for 48 h. (E) Binding of p53, p130, E2F4, LIN9 and B-Myb to the <i>KIF23</i> promoter was analyzed (*p≤0.05; **p≤0.01; n.s. not significant; n ≥ 2) using (F) <i>p21</i> and (G) <i>GAPDHS</i> promoters as controls.</p