DAXX drives de novo lipogenesis and contributes to tumorigenesis.

Cancer cells exhibit elevated lipid synthesis. In breast and other cancer types, genes involved in lipid production are highly upregulated, but the mechanisms that control their expression remain poorly understood. Using integrated transcriptomic, lipidomic, and molecular studies, here we report that DAXX is a regulator of oncogenic lipogenesis. DAXX depletion attenuates, while its overexpression enhances, lipogenic gene expression, lipogenesis, and tumor growth. Mechanistically, DAXX interacts with SREBP1 and SREBP2 and activates SREBP-mediated transcription. DAXX associates with lipogenic gene promoters through SREBPs. Underscoring the critical roles for the DAXX-SREBP interaction for lipogenesis, SREBP2 knockdown attenuates tumor growth in cells with DAXX overexpression, and DAXX mutants unable to bind SREBP1/2 have weakened activity in promoting lipogenesis and tumor growth. Remarkably, a DAXX mutant deficient of SUMO-binding fails to activate SREBP1/2 and lipogenesis due to impaired SREBP binding and chromatin recruitment and is defective of stimulating tumorigenesis. Hence, DAXX's SUMO-binding activity is critical to oncogenic lipogenesis. Notably, a peptide corresponding to DAXX's C-terminal SUMO-interacting motif (SIM2) is cell-membrane permeable, disrupts the DAXX-SREBP1/2 interactions, and inhibits lipogenesis and tumor growth. These results establish DAXX as a regulator of lipogenesis and a potential therapeutic target for cancer therapy.

Discovery of histone deacetylase 3 (HDAC3)-specific PROTACs.

Histone deacetylases (HDACs) are validated drug targets for cancer treatment. Increased HDAC isozyme selectivity and novel strategies to inhibit HDAC activity could lead to safer and more effective drug candidates. Nonetheless, it is quite challenging to develop isozyme-specific HDACi due to the highly conserved catalytic domain. We discovered XZ9002, a first-in-class HDAC3-specific PROTAC that potently degraded HDAC3. Importantly, XZ9002 is more effective to inhibit cancer cell proliferation than its proteolysis-inactive counterpart, suggesting HDAC3 degradation is a novel and promising anticancer approach.

Inhibition of androgen receptor transactivation function by adenovirus type 12 E1A undermines prostate cancer cell survival.

BACKGROUND: Mutations or truncation of the ligand-binding domain (LBD) of androgen receptor (AR) underlie treatment resistance for prostate cancer (PCa). Thus, targeting the AR N-terminal domain (NTD) could overcome such resistance. METHODS: Luciferase reporter assays after transient transfection of various DNA constructs were used to assess effects of E1A proteins on AR-mediated transcription. Immunofluorescence microscopy and subcellular fractionation were applied to assess intracellular protein localization. Immunoprecipitation and mammalian two-hybrid assays were used to detect protein-protein interactions. qRT-PCR was employed to determine RNA levels. Western blotting was used to detect protein expression in cells. Effects of adenoviruses on prostate cancer cell survival were evaluated with CellTiter-Glo assays. RESULTS: Adenovirus 12 E1A (E1A12) binds specifically to the AR. Interestingly, the full-length E1A12 (266 aa) preferentially binds to full-length AR, while the small E1A12 variant (235 aa) interacts more strongly with AR-V7. E1A12 promotes AR nuclear translocation, likely through mediating intramolecular AR NTD-LBD interactions. In the nucleus, AR and E1A12 co-expression in AR-null PCa cells results in E1A12 redistribution from nuclear foci containing CBX4 (also known as Pc2), suggesting a preferential AR-E1A12 interaction over other E1A12 interactors. E1A12 represses AR-mediated transcription in reporter gene assays and endogenous AR target genes such as ATAD2 and MYC in AR-expressing PCa cells. AR-expressing PCa cells are more sensitive to death induced by a recombinant adenovirus expressing E1A12 (Ad-E1A12) than AR-deficient PCa cells, which could be attributed to the increased viral replication promoted by androgen stimulation. Targeting the AR by E1A12 promotes apoptosis in PCa cells that express the full-length AR or C-terminally truncated AR variants. Importantly, inhibition of mTOR signaling that blocks the expression of anti-apoptotic proteins markedly augments Ad-E1A12-induced apoptosis of AR-expressing cells. Mechanistically, Ad-E1A12 infection triggers apoptotic response while activating the PI3K-AKT-mTOR signaling axis; thus, mTOR inhibition enhances apoptosis in AR-expressing PCa cells infected by Ad-E1A12. CONCLUSION: Ad12 E1A inhibits AR-mediated transcription and suppresses PCa cell survival, suggesting that targeting the AR by E1A12 might have therapeutic potential for treating advanced PCa with heightened AR signaling.

The subgingival microbiome in patients with established rheumatoid arthritis.

OBJECTIVES: To profile and compare the subgingival microbiome of RA patients with OA controls. METHODS: RA (n = 260) and OA (n = 296) patients underwent full-mouth examination and subgingival samples were collected. Bacterial DNA was profiled using 16 S rRNA Illumina sequencing. Following data filtering and normalization, hierarchical clustering analysis was used to group samples. Multivariable regression was used to examine associations of patient factors with membership in the two largest clusters. Differential abundance between RA and OA was examined using voom method and linear modelling with empirical Bayes moderation (Linear Models for Microarray Analysis, limma), accounting for the effects of periodontitis, race, marital status and smoking. RESULTS: Alpha diversity indices were similar in RA and OA after accounting for periodontitis. After filtering, 286 taxa were available for analysis. Samples grouped into one of seven clusters with membership sizes of 324, 223, 3, 2, 2, 1 and 1 patients, respectively. RA-OA status was not associated with cluster membership. Factors associated with cluster 1 (vs 2) membership included periodontitis, smoking, marital status and Caucasian race. Accounting for periodontitis, 10 taxa (3.5% of those examined) were in lower abundance in RA than OA. There were no associations between lower abundance taxa or other select taxa examined with RA autoantibody concentrations. CONCLUSION: Leveraging data from a large case-control study and accounting for multiple factors known to influence oral health status, results from this study failed to identify a subgingival microbial fingerprint that could reliably discriminate RA from OA patients.

Intrinsic cellular signaling mechanisms determine the sensitivity of cancer cells to virus-induced apoptosis.

Cancer cells of epithelial and mesenchymal phenotypes exhibit different sensitivities to apoptosis stimuli, but the mechanisms underlying this phenomenon remain partly understood. We constructed a novel recombinant adenovirus expressing Ad12 E1A (Ad-E1A12) that can strongly induce apoptosis. Ad-E1A12 infection of epithelial cancer cells displayed dramatic detachment and apoptosis, whereas cancer cells of mesenchymal phenotypes with metastatic propensity were markedly more resistant to this virus. Notably, forced detachment of epithelial cells did not further sensitize them to Ad-E1A12-induced apoptosis, suggesting that cell detachment is a consequence rather than the cause of Ad-E1A12-induced apoptosis. Ad-E1A12 increased phosphorylation of AKT1 and ribosomal protein S6 through independent mechanisms in different cell types. Ad-E1A12-induced AKT1 phosphorylation was PI3K-dependent in epithelial cancer cells, and mTOR-dependent in mesenchymal cancer cells. Epithelial cancer cells upon Ad-E1A12-induced detachment could not sustain AKT activation due to AKT1 degradation, but AKT1 activation was maintained in mesenchymal cancer cells. Expression of epithelial cell-restricted miR-200 family in mesenchymal cells limited mTOR signaling and sensitized them to Ad-E1A12-induced cell killing. Thus, epithelial cancer cells rely on the canonical PI3K-AKT signaling pathway for survival, while mesenchymal cancer cells deploy the PI3K-independent mTORC2-AKT axis in response to strong death stimuli.

Identification of histone deacetylase inhibitors with benzoylhydrazide scaffold that selectively inhibit class I histone deacetylases.

Inhibitors of histone deacetylases (HDACi) hold considerable therapeutic promise as clinical anticancer therapies. However, currently known HDACi exhibit limited isoform specificity, off-target activity, and undesirable pharmaceutical properties. Thus, HDACi with new chemotypes are needed to overcome these limitations. Here, we identify a class of HDACi with a previously undescribed benzoylhydrazide scaffold that is selective for the class I HDACs. These compounds are competitive inhibitors with a fast-on/slow-off HDAC-binding mechanism. We show that the lead compound, UF010, inhibits cancer cell proliferation via class I HDAC inhibition. This causes global changes in protein acetylation and gene expression, resulting in activation of tumor suppressor pathways and concurrent inhibition of several oncogenic pathways. The isotype selectivity coupled with interesting biological activities in suppressing tumor cell proliferation support further preclinical development of the UF010 class of compounds for potential therapeutic applications.

p53 SUMOylation promotes its nuclear export by facilitating its release from the nuclear export receptor CRM1.

Chromosomal region maintenance 1 (CRM1) mediates p53 nuclear export. Although p53 SUMOylation promotes its nuclear export, the underlying mechanism is unclear. Here we show that tethering of a small, ubiquitin-like modifier (SUMO) moiety to p53 markedly increases its cytoplasmic localization. SUMO attachment to p53 does not affect its oligomerization, suggesting that subunit dissociation required for exposing p53's nuclear export signal (NES) is unnecessary for p53 nuclear export. Surprisingly, SUMO-mediated p53 nuclear export depends on the SUMO-interacting motif (SIM)-binding pocket of SUMO-1. The CRM1 C-terminal domain lacking the NES-binding groove interacts with tetrameric p53, and the proper folding of the p53 core domain, rather than the presence of the N- or C-terminal tails, appears to be important for p53-CRM1 interaction. The CRM1 Huntington, EF3, a subunit of PP2A, and TOR1 9 (HEAT9) loop, which regulates GTP-binding nuclear protein Ran binding and cargo release, contains a prototypical SIM. Remarkably, disruption of this SIM in conjunction with a mutated SIM-binding groove of SUMO-1 markedly enhances the binding of CRM1 to p53-SUMO-1 and their accumulation in the nuclear pore complexes (NPCs), as well as their persistent association in the cytoplasm. We propose that SUMOylation of a CRM1 cargo such as p53 at the NPCs unlocks the HEAT9 loop of CRM1 to facilitate the disassembly of the transporting complex and cargo release to the cytoplasm.

Small-molecule inhibitors of acetyltransferase p300 identified by high-throughput screening are potent anticancer agents.

Acetyltransferase p300 (KAT3B) plays key roles in signaling cascades that support cancer cell survival and sustained proliferation. Thus, p300 represents a potential anticancer therapeutic target. To discover novel anticancer agents that target p300, we conducted a high-throughput screening campaign. A library of 622,079 compounds was assayed for cytotoxicity to the triple-negative breast cancer (TNBC) cell line MDA-MB-231 but not to the human mammary epithelial cells. The resulting compounds were tested in a biochemical assay for inhibiting the enzymatic activity of p300. One compound (L002, NSC764414) displayed an IC50 of 1.98 µmol/L against p300 in vitro, inhibited acetylation of histones and p53, and suppressed STAT3 activation in cell-based assays. L002 could be docked to the active site of the p300 catalytic domain. Biochemical tests of a series of related compounds revealed functional groups that may impact inhibitory potency of L002 against p300. Interestingly, these analogs showed inhibitory activities against the cellular paralog of p300 (CBP), p300/CBP-associated factor, and GCN5, but not to other acetyltransferases (KAT5, KAT6B, and KAT7), histone deacetylases, and histone methyltransferases. Among the NCI-60 panel of cancer cell lines, leukemia and lymphoma cell lines were extremely sensitive to L002, whereas it is toxic to only a limited number of cell lines derived from solid tumors. Notably, breast cancer cell lines, especially those derived from TNBC, were highly susceptible to L002. In vivo, it potently suppressed tumor growth and histone acetylation of MDA-MB-468 xenografts. Thus, these new acetyltransferase inhibitors are potential anticancer therapeutics.

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection.

Successful viral replication entails elimination or bypass of host antiviral mechanisms. Here, we show that shRNA-mediated knockdown of murine double minute (Mdm2) and its paralog Mdm4 enhanced the expression of early and late viral gene products during adenovirus (HAdV) infection. Remarkably, whereas the expression of HAdV genes was low in p53-deficient mouse embryonic fibroblasts (p53KO MEFs), the HAdV early gene products were efficiently expressed in Mdm2/p53 double-knockout (DKO) and Mdm4/p53 DKO MEFs, and viral capsid proteins were produced in Mdm2/p53 DKO MEFs. Thus, Mdm2 and Mdm4 seem to have potent antiviral property. In cells infected with wt HAdV or a mutant virus lacking the E1B-55K gene (dl 1520), both Mdm2 and Mdm4 were rapidly depleted, whereas replication-deficient mutant viruses (Ad-GFP) or ΔpTP with deletions within the coding sequence of preterminal binding protein failed to induce their downregulation. Reduced expression of Mdm2 and Mdm4 was not due to general shutoff of host protein synthesis. Additionally, expression of a dominant-negative mutant of Cul5 did not affect Mdm2/Mdm4 downregulation. Thus, viral replication but not the presence of E1B-55K is required for Mdm2/Mdm4 degradation. Surprisingly, treatment of HAdV-infected cells with proteasome inhibitor MG132 only partially restored the protein levels of Mdm2 and Mdm4, suggesting that they may also be downregulated through an additional mechanism independent of proteasome. Interestingly, cyclin D1 and p21 appear to be downregulated similarly during HAdV infection. Collectively, our work provides the first biochemical evidence for antiviral function of Mdm2 and Mdm4 and that viruses employ efficient countermeasure to ensure viral replication.

Inhibition of p53 by adenovirus type 12 E1B-55K deregulates cell cycle control and sensitizes tumor cells to genotoxic agents.

Adenovirus E1B-55K represses p53-mediated transcription. However, the phenotypic consequence of p53 inhibition by E1B-55K for cell cycle regulation and drug sensitivity in tumor cells has not been examined. In HCT116 cells with constitutive E1B-55K expression, the activation of p53 target genes such as the p21, Mdm2, and Puma genes was attenuated, despite markedly elevated p53 protein levels. HCT116 cells with E1B-55K expression displayed a cell cycle profile similar to that of the isogenic HCT116p53(-/-) cells, including unhindered S-phase entry despite DNA damage. Surprisingly, E1B-55K-expressing cells were more sensitive to drug treatment than parental cells. Compared to HCT116 cells, HCT116p53(-/-) cells were more susceptible to both doxorubicin and etoposide, and E1B-55K expression had no effects on drug treatment. E1B-55K expression increased the rate of cell proliferation in HCT116 but not in HCT116p53(-/-) cells. Thus, deregulation of p53-mediated cell cycle control by E1B-55K probably underlies sensitization of HCT116 cells to anticancer drugs. Consistently, E1B-55K expression in A549, A172, and HepG2 cells, all containing wild-type (wt) p53, also enhanced etoposide-induced cytotoxicity, whereas in p53-null H1299 cells, E1B-55K had no effects. We generated several E1B-55K mutants with mutations at positions occupied by the conserved Phe/Trp/His residues. Most of these mutants showed no or reduced binding to p53, although some of them could still stabilize p53, suggesting that binding might not be essential for E1B-55K-induced p53 stabilization. Despite heightened p53 protein levels in cells expressing certain E1B-55K mutants, p53 activity was largely suppressed. Furthermore, most of these E1B-55K mutants could sensitize HCT116 cells to etoposide and doxorubicin. These results indicate that E1B-55K might have utility for enhancing chemotherapy.

Identification of two independent SUMO-interacting motifs in Daxx: evolutionary conservation from Drosophila to humans and their biochemical functions.

Daxx is essential for embryonic development and implicated in apoptosis and transcriptional regulation. It is found only in the animal kingdom and appears to arise first in insects. In the Drosophila genus, the Daxx orthologs are much larger than those in other species. Here we show that in addition to a conserved core of approximately 200 residues, Daxx possesses several conserved domains and two essentially invariable short SUMO-interacting motifs (SIMs), each located at one or the other terminus of the protein. Both can independently interact with SUMO. The Daxx I7/733K double mutant with one mutation in each of the two SIMs no longer interacts with SUMO. Daxx interacts with Ubc9 and this interaction strictly requires at least one SIM. Interestingly, the Ubc9 H20D mutation that abolishes non-covalent Ubc9-SUMO interaction also interrupts Daxx-Ubc9 interaction. Thus, SUMO serves as the intermediate for Daxx-Ubc9 interaction. Surprisingly, Daxx I7/733K double mutant could still colocalize with PML. Furthermore, wt Daxx also strongly colocalizes with PMLDeltaS mutant, in which all three sumoylation sites are mutated, whereas PMLDeltaS only weakly colocalizes with Daxx I7/733K mutant, suggesting that SIM-SUMO interaction is not essential for but enhances PML-Daxx interaction. Remarkably, Daxx strongly stimulates c-Jun-mediated transcription and both SIMs are required for this stimulation. PML also activates c-Jun, which requires all three sumoylation sites. Coexpression of Daxx and PML revealed that they independently regulate c-Jun, with Daxx exerting a dominant role. These results suggest that the conserved SIMs are involved in mediating protein-protein interactions that underlie Daxx's diverse cellular functions.

Repression of p53-mediated transcription by adenovirus E1B 55-kDa does not require corepressor mSin3A and histone deacetylases.

The Ad E1B 55-kDa protein (E1B) is a potent transcriptional repressor. In vitro biochemical studies revealed that direct p53-E1B interaction is essential for E1B to block p53-activated transcription and a corepressor may be involved. To understand how E1B represses p53-mediated transcription in vivo, we expressed E1B in several tumor cell lines that express wild type p53. Here we show that E1B strongly suppresses the expression of p53 target genes such as p21 and Puma-alpha in normal growth conditions or after cells were treated with p53-activating chemotherapeutic agents, suggesting that E1B-mediated gene repression is dominant and cannot be reversed via p53 activation. Interestingly, we found that E1B binds to corepressor mSin3A. Mutagenesis analysis indicated that the sequence motif "LHLLA" near the NH(2) terminus of E1B is responsible for mSin3A binding, and this motif is conserved among E1B proteins from different Ad serotypes. The conserved paired amphipathic helix domain 1 of mSin3A is critical for mSin3A-E1B interaction. Surprisingly, E1B mutants that cannot bind to mSin3A can still repress p53 target genes, indicating that it is not the corepressor required for E1B-mediated gene repression. In support of this notion, repression of p53 target genes by E1B is insensitive to HDAC inhibitor trichostatin A. We further show that both the NH(2)- and COOH-terminal domains of E1B are required for the repression function. Therefore, E1B employs a unique repression mechanism to block p53-mediated transcription.

An EBF3-mediated transcriptional program that induces cell cycle arrest and apoptosis.

In a genome-wide screen for putative tumor suppressor genes, the EBF3 locus on the human chromosome 10q26.3 was found to be deleted or methylated in 73% of the examined cases of brain tumors. EBF3 is expressed in normal brain but is silenced in brain tumors. Therefore, it is suggested that EBF3 is a tumor suppressor. However, it remains unknown whether inactivation of EBF3 locus also occurs in other types of tumors and what functions of EBF3 underlie EBF3-mediated tumor suppression. We show here that expression of EBF3 resulted in cell cycle arrest and apoptosis. The expression of cyclin-dependent kinase inhibitors was profoundly affected with early activation and then repression of p21(cip1/waf1) and persistent activation of both p27(kip1) and p57(kip2), whereas genes involved in cell survival and proliferation were suppressed. EBF3 bound directly to p21(cip1/waf1) promoter and regulated transcription from both p21(cip1/waf1) and p27(kip1) promoters in reporter assays. Apoptosis occurred 48 hours after EBF3 expression with caspase-3 activation. Silencing of the EBF3 locus was observed in brain, colorectal, breast, liver, and bone tumor cell lines and its reactivation was achieved on treatment with 5-aza-2'-deoxycytidine and trichostatin A in a significant portion of these tumor cells. Therefore, EBF3 regulates a transcriptional program underlying a putative tumor suppression pathway.

Negative regulation of p53 functions by Daxx and the involvement of MDM2.

In normal cells p53 activity is tightly controlled and MDM2 is a known negative regulator. Here we show that via its acidic domain, Daxx binds to the COOH-terminal domain of p53, whose positive charges are critical for this interaction, as Lys to Arg mutations preserved, but Lys to Ala or Ser to Glu mutations abolished Daxx-p53 interaction. These results thus implicate acetylation and phosphorylation of p53 in regulating its binding to Daxx. Interestingly, whereas Daxx did not bind to p53 in cells as assessed by immunoprecipitation, MDM2 expression restored p53-Daxx interaction, and this correlated with deacetylation of p53. In p53/MDM2-null mouse embryonic fibroblasts (DKO MEF), Daxx repressed p53 target promoters whose p53-binding elements were required for the repression. Coexpression of Daxx and MDM2 led to further repression. p53 expression in DKO MEF induced apoptosis and Daxx expression relieved this effect. Similarly, in HCT116 cells, Daxx conferred striking resistance to 5-fluorouracil-induced apoptosis. As p53 is required for 5-fluorouracil-induced cell death, our data show that Daxx can suppress cell death induced by p53 overexpression and p53-dependent stress response. Collectively, our data reveal Daxx as a novel negative regulator of p53. Importantly, posttranslational modifications of p53 inhibit Daxx-p53 interaction, thereby relieving negative regulation of p53 by Daxx.

Sequestration of p53 in the cytoplasm by adenovirus type 12 E1B 55-kilodalton oncoprotein is required for inhibition of p53-mediated apoptosis.

The adenovirus E1B 55-kDa protein is a potent inhibitor of p53-mediated transactivation and apoptosis. The proposed mechanisms include tethering the E1B repression domain to p53-responsive promoters via direct E1B-p53 interaction. Cytoplasmic sequestration of p53 by the 55-kDa protein would impose additional inhibition on p53-mediated effects. To investigate further the role of cytoplasmic sequestration of p53 in its inhibition by the E1B 55-kDa protein we systematically examined domains in both the Ad12 55-kDa protein and p53 that underpin their colocalization in the cytoplasmic body and show that the N-terminal transactivation domain (TAD) of p53 is essential for retaining p53 in the cytoplasmic body. Deletion of amino acids 11 to 27 or even point mutation L22Q/W23S abolished the localization of p53 to the cytoplasmic body, whereas other parts of TAD and the C-terminal domain of p53 are dispensable. This cytoplasmic body is distinct from aggresome associated with overexpression of some proteins, since it neither altered vimentin intermediate filaments nor associated with centrosome or ubiquitin. Formation of this structure is sensitive to mutation of the Ad12 55-kDa protein. Strikingly, mutation S476/477A near the C terminus of the Ad12 55-kDa protein eliminated the formation of the cytoplasmic body. The equivalent residues in the Ad5 55-kDa protein were shown to be critical for its ability to inhibit p53. Indeed, Ad12 55-kDa mutants that cannot form a cytoplasmic body can no longer inhibit p53-mediated effects. Conversely, the Ad12 55-kDa protein does not suppress p53 mutant L22Q/W23S-mediated apoptosis. Finally, we show that E1B can still sequester p53 that contains the mitochondrial import sequence, thereby potentially preventing the localization of p53 to mitochondria. Thus, cytoplasmic sequestration of p53 by the E1B 55-kDa protein plays an important role in restricting p53 activities.

PCAF is a coactivator for p73-mediated transactivation.

The tumor suppressor p53-related p73 shares significant amino-acid sequence identity with p53. Like p53, p73 recognizes canonical p53 DNA-binding sites and activates p53-responsive target genes and induces apoptosis. Moreover, transcription coactivator p300/CBP binds to and coactivates with both p53 and p73 in stimulating the expression of their target genes. Here, we report that coactivator PCAF binds to p73. The N-terminal transactivation domain (TAD) and the conserved oligomerization domain (OD) of p73 are both required for its interaction with PCAF. Conversely, PCAF's HAT-domain is required for and both the N-terminal region and Bromo domain enhance binding of PCAF to p73. Significantly, PCAF stimulates p73-mediated transactivation, and binding of PCAF to p73 is necessary for p73's transactivation activity. PCAF-specific siRNA dramatically reduces p73-mediated transactivation. Stimulation of p73-mediated transactivation by PCAF requires the HAT domain of PCAF and the p53-binding site within the p21 promoter. In vivo, coexpression of wild-type, but not HAT-deficient PCAF with p73beta markedly increases p21 expression. Furthermore, cotransfection of PCAF and p73 leads to increased apoptosis and reduced colony formation. Collectively, these data suggest that p73 recruit PCAF to specific promoters to activate the transcription of p73 target genes.

Adenovirus E1B 55-kilodalton oncoprotein binds to Daxx and eliminates enhancement of p53-dependent transcription by Daxx.

The adenovirus E1B 55-kDa protein impairs the p53 pathway and enhances transformation, although the underlying mechanisms remain to be defined. We found that Daxx binds to the E1B 55-kDa protein in a yeast two-hybrid screen. The two proteins interact through their C termini. Mutation of three potential phosphorylation sites (S489/490 and T494 to alanine) within the E1B 55-kDa protein did not affect its interaction with Daxx, although such mutations were previously shown to inhibit E1B's ability to repress p53-dependent transcription and to enhance transformation. In addition to their coimmunoprecipitation in 293 extracts, purified Daxx interacted with the E1B 55-kDa protein in vitro, indicating their direct interaction. In 293 cells, Daxx colocalized with the E1B 55-kDa protein within discrete nuclear dots, where p53 was also found. Such structures were distinct from PML (promyelocytic leukemia protein) bodies, and it appeared that Daxx was displaced from PML bodies. Thus, the Daxx concentration was diminished in dots with a prominent presence of PML and vice versa. Indeed, PML overexpression led to dramatic redistribution of Daxx from p53-E1B 55-kDa protein complexes to PML bodies. Additionally, expression of the E1B 55-kDa protein in Saos2 osteosarcoma cells reduced the number of PML bodies. Our data suggest that E1B and PML compete for available Daxx in the cell. Surprisingly, Daxx significantly augmented p53-mediated transcription and the E1B 55-kDa protein eliminated this effect. Thus, it is likely that the E1B 55-kDa protein sequesters Daxx and p53 in specific nuclear locations, where p53 cannot activate transcription. One consequence of the Daxx-E1B interaction might be an alteration of normal interactions of Daxx, PML, and p53, which may contribute to cell transformation.