Identification of Daxx interacting with p73, one of the p53 family, and its regulation of p53 activity by competitive interaction with PML.
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.
Kim
Eun-Joo
EJ
Department of Bioscience and Biotechnology/Institute of Bioscience, Sejong University, 98 Kunja-dong, Kwangjin-gu, Seoul 143-747, Korea.
Park
Jong-Sup
JS
Um
Soo-Jong
SJ
D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites / Q000235:genetics; D001667:Binding, Competitive / Q000187:drug effects; D015153:Blotting, Western; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D056970:Co-Repressor Proteins; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016147:Genes, Tumor Suppressor; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D056572:Histone Deacetylase Inhibitors; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D018832:Molecular Chaperones; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D000071156:Promyelocytic Leukemia Protein; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D056970:Co-Repressor Proteins; C106985:DAXX protein, human; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D018832:Molecular Chaperones; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D000071156:Promyelocytic Leukemia Protein; D011993:Recombinant Fusion Proteins; C000606460:TP73 protein, human; D014157:Transcription Factors; D000072160:Tumor Protein p73; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C070576:PML protein, human; D049452:Green Fluorescent Proteins; C012589:trichostatin A; D015500:Chloramphenicol O-Acetyltransferase; D005982:Glutathione Transferase