Identification of Daxx interacting with p73, one of the p53 family, and its regulation of p53 activity by competitive interaction with PML

Abstract

We performed a yeast two-hybrid screen using p73alpha, which is a member of the p53 family, as bait. We found that the p53 family members were functionally associated with Daxx, which was described originally as a cytoplasmic mediator of Fas signaling, but has been identified recently as a nuclear protein that co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription. Extensive yeast two-hybrid assays indicated a physical interaction between a region including the oligomerization domain (OD) of p73alpha (amino acids 345-380) or p53 (amino acids 319-360) and amino acids 161-311 and 667-740 (C-terminal S/P/T-rich domain) of hDaxx, which is the common binding region of Fas, ASK1 and PML. This interaction was further confirmed by in vitro GST pull-down and in vivo immunoprecipitation assays. Both Daxx and p73/p53 co-localized in nuclear dot-like structures, which are probably nuclear PML oncogenic domains (PODs) or the nuclear domain NB10. Transient co-expression of Daxx resulted in strong inhibition of p73- and p53-mediated transcriptional activation of the synthetic p53-responsive and p21WAF1 promoters. Consequently, Gal4-Daxx repressed basal transcription in a dose-dependent manner. Treatment with trichostatin A, which is an inhibitor of histone deacetylase, or PML over-expression relieved Daxx-mediated transcriptional repression of p53. The mechanism underlying PML-mediated derepression appears to be competitive binding between Daxx, p53 and PML. Taken together, these findings delineate a transcriptional regulatory network that is modulated by differential Daxx-p53-PML interactions in the nuclear PODs. Therefore, Daxx is implicated in the regulation of the cell cycle and apoptosis through transcriptional regulation of p53 and possibly its family members.

Figure 1

Structural characteristics of p53 family proteins and Daxx homologs. (A) p53 family (p53, p73α, p73β, p63α and p63γ). TAD, transcription activation domain; DBD, DNA-binding domain; OD, oligomerization domain; BD, basic regulatory domain; SAM, sterile α-motif. (B) Daxx homologs (mouse and human). PAH, paired amphipathic helix; D/E, aspartate- and glutamate-rich; S/P/T, serine/proline/threonine-rich. A partial portion of hDaxx was indicated as identified by yeast two-hybrid screening. The numbers indicate amino acid positions.

Figure 2

hDaxx interacts with the p53 family. (A) Identification of hDaxx by yeast two-hybrid screening using a human liver-derived cDNA library fused to the Gal4 activation domain (AD). (B) Interaction of the p53 family with hDaxx, analyzed by yeast two-hybrid and β-galactosidase assays using the LexA DBD-fused p53 family (in pBTM116) and the original Gal AD-fused hDaxx (in pGAD10) as described in detail in Materials and Methods.

Figure 3

Mapping of the specific region of the p53 family responsible for interaction with hDaxx. (A) p73. (B) p53. (C) p63. Yeast two-hybrid and β-galactosidase assays were performed using LexA-fused truncations of the p53 family (in pBTM116) and the original Gal AD-fused hDaxx (in pGAD10). LexA-fused mutants were almost evenly expressed in yeast as determined by western blotting analysis (data not shown).

Figure 3

Mapping of the specific region of the p53 family responsible for interaction with hDaxx. (A) p73. (B) p53. (C) p63. Yeast two-hybrid and β-galactosidase assays were performed using LexA-fused truncations of the p53 family (in pBTM116) and the original Gal AD-fused hDaxx (in pGAD10). LexA-fused mutants were almost evenly expressed in yeast as determined by western blotting analysis (data not shown).

Figure 4

Mapping of the hDaxx domain responsible for interaction with p73α (amino acids 49–636). Yeast two-hybrid and β-galactosidase assays were performed using LexA-fused p73α (amino acids 49–636) in pBTM116 and VP16-fused truncations of hDaxx in pASV3. VP16-fused mutants were almost evenly expressed in yeast (data not shown).

Figure 5

GST pull-down assays. In vitro translated ³⁵S-labeled products were incubated with GST, GST–Daxx (amino acids 607–740) or GST–p53 (amino acids 319–393) immobilized onto glutathione–Sepharose beads for 2 h. Beads were washed five times with buffer A [20 mM HEPES pH 7.5, 50 mM KCl, 2.5 mM MgCl₂, 10% glycerol, 1 mM dithiothreitol (DTT), 1% NP-40 and 200 mg/ml phenylmethylsulfonyl fluoride (PMSF)]. Bound proteins were eluted with SDS sample buffer and resolved by 10% SDS–PAGE for autoradiography.

Figure 6

In vivo interaction assay by immunoprecipitation. Transfection conditions in p53-null Saos2 cells were as follows: (A) GFP–Daxx and empty HA-pcDNA3 vector, HA-p73α or HA-p53; (B, left) p73α without tag (in pcDNA3) and empty GFP vector or GFP–Daxx; (B, right) Daxx and empty pcDNA3 or p73α; (C, left) p53 in pcDNA3 and empty GFP vector or GFP–Daxx; (C, right) Daxx and empty pcDNA3 or p53. After transfections, cells were lysed in RIPA buffer (250 mM NaCl, 50 mM HEPES pH 7.0, 0.1% NP40 and 5 mM EDTA) supplemented with protease inhibitors and pre-cleared by incubating with protein A–Sepharose for 1 h. Immuno precipitation was carried out with anti-HA, anti-Daxx or anti-GFP antibody. After resolution by 10% SDS–PAGE, western blotting was carried out using anti-GFP (A), anti-p73α (B) and anti-p53 (C) antibodies. A band in the control sample (C, right panel, Daxx alone) corresponds to the heavy chain of anti-Daxx antibody that can be detected by western blotting. The band is close to that of p53 because of running the gel for a short time.

Figure 7

Immunofluorescence assays using confocal microscopy. Transfected Saos2 cells with GFP–Daxx and HA-p73α (A) or HA-p53 (B) were replated on slides. Cells were fixed in 4% paraformaldehyde for 20 min at room temperature and were covered with blocking solution (5% goat serum in 3% bovine serum albumin in PBS) for 1 h. Cells were stained with anti-HA-Texas red-conjugated antibody for 1 h. Slides were then mounted in antifade mounting medium and analyzed by confocal microscopy.

Figure 8

Effect of Daxx on the transcriptional activity of the p53 family. Saos2 cells were transfected with a total of 2.2 µg of DNA comprising the p53 family, Daxx expression vectors, 0.2 µg of p53-responsive G5p53-CAT (A and C), 17m5-TATA-CAT (B) or 17m5-tk-CAT (D) reporter plasmid, and 0.2 µg of SV40-driven β-gal internal control plasmid. The amounts of each expression vector were as follows: (A) lane 2, Daxx 0.8 µg; lanes 3–6, p73α 0.2 µg, Daxx 0, 0.2, 0.4 and 0.8 µg; lanes 7–10, p53 0.2 µg, Daxx 0, 0.2, 0.4 and 0.8 µg. (B) lane 2, Daxx 0.8 µg; lanes 3–6, Gal4-p73α 0.2 µg, Daxx 0, 0.2, 0.4 and 0.8 µg; lanes 7–10, Gal4-p53 0.2 µg, Daxx 0, 0.2, 0.4 and 0.8 µg. (C) lanes 2–6, p73α 0.2 µg, Daxx 0.8 µg, TSA 0.1, 0.2 and 0.4 nM. (D) lane 1, Gal4 alone 0.8 µg; lanes 2–4, Gal4-Daxx (amino acids 607–740) 0.2, 0.4 and 0.8 µg; lanes 5–7, Gal4-Daxx (amino acids 607–740) 0.8 µg, TSA 0.1, 0.2 and 0.4 µM. Data represent the mean of at least three independent transfections. The relative amount of CAT was determined by CAT ELISA after normalization by β-galactosidase activity. The inset in (A) shows a representative western blot of p73α and p53 with each band corresponding with the bar immediately below.

Figure 9

PML3 relieves Daxx-mediated repression of p53 activated transcription possibly through the disruption of Daxx-p53 complex. (A) Transient transfection assays. Transfections were performed as described in Figure 8 except using p21WAF1-CAT as a reporter plasmid. The transfected amounts of Daxx, PML3 or PML-L were 0.2, 0.4 and 0.8 µg, respectively. The amount of p53 was 0.2 µg. Results represent the mean of three independent experiments. (B) Competition by PML3. For assays, 10 µl of in vitro translated p53 was mixed with purified GST–Daxx (amino acids 607–740) and further reacted with increasing volumes of PML3 (10, 20 and 40 µl). Afterwards, GST pull-down assays were performed as described in Figure 5. (C) Competition by PML-L. The same reactions were performed as (B) except using PML-L instead of PML3.