Abstract
The DEAD-box RNA helicase DDX3 plays divergent roles in tumorigenesis, however, its function in mitosis is unclear. Immunofluorescence indicated that DDX3 localized to centrosome throughout the cell cycle and colocalized with centrosome-associated p53 during mitosis in HCT116 and U2OS cells. DDX3 depletion promoted chromosome misalignment, segregation defects and multipolar mitosis, eventually leading to G2/M delay and cell death. DDX3 prevented multipolar mitosis by inactivation and coalescence of supernumerary centrosomes. DDX3 silencing suppressed Ser15 phosphorylation of p53 which is required for p53 centrosomal localization. Additionally, knockout of p53 dramatically diminished the association of DDX3 with centrosome, which was rescued by overexpression of the centrosomal targeting-defective p53 S15A mutant, indicating that centrosomal localization of DDX3 is p53 dependent but not through centrosomal location of p53. Furthermore, DDX3 knockdown suppressed p53 transcription through activation of DNA methyltransferases (DNMTs) along with hypermethylation of p53 promoter and promoting the binding of repressive histone marks to p53 promoter. Moreover, DDX3 modulated p53 mRNA translation. Taken together, our study suggests that DDX3 regulates epigenetic transcriptional and translational activation of p53 and colocalizes with p53 at centrosome during mitosis to ensure proper mitotic progression and genome stability, which supports the tumor-suppressive role of DDX3.
Highlights
Centrosome amplification and aneuploidy are hallmarks of cancer cells
In view of the centrosomal localization of p53 during mitosis[7, 8] and that DDX3 interacts with p5337, we examined if DDX3 colocalizes with p53 at centrosome by immunofluorescence with anti-p53 antibody
Knockdown of DDX3 diminished the binding of DDX3 and p53 to p53 promoter (Fig. 10a–c). These findings demonstrate that knockdown of DDX3 suppresses p53 promoter by enhancing DNA methyltransferases (DNMTs) and repressive histone marks binding to p53 promoter
Summary
Centrosome amplification and aneuploidy are hallmarks of cancer cells. In general, each cell has a single centrosome which duplicates once in S phase. Loss of p53 causes centrosome amplification which results in multiple mitotic spindle poles and aberrant chromosome segregation[10]. In cleavage failure and centrosome over-duplicated tetraploid cells, p53 abnormality impairs clustering of centrosomes and causes multipolar mitosis along with a high degree of aneuploidy[11,12,13]. Representative confocal images show the control mitotic cells with normal bipolar spindles (NB) and balanced chromosome segregation in metaphase (NB-M), anaphase (NB-A) and telophase (NB-T) while DDX3 knockdown promoted the abnormal mitosis such as chromosome misalignment (MisA) (arrow), chromosome segregation defect (arrowhead), lagging chromosome (Lag) and chromosome bridge (Bri) and multipolar mitosis (MuP-M, A, T). The proportions of sub-G1 phase population of the control and DDX3-knockdown cells are shown as the average value ± S.D. calculated from three independent experiments. The 3′UTR base pairs with the 5′UTR to form a steady RNA structure that is crucial for translational regulation of p53 mRNA22–24
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