Sequestration and inhibition of Daxx-mediated transcriptional repression by PML

Acute promyelocytic leukemia (APL) arises as a result of chromosomal translocation involving the retinoic acid (RA) receptor α (RARα) gene on chromosome 17 fused with either the promyelocytic leukemia gene (PML) on chromosome 15, the promyelocytic leukemia zinc finger gene (PLZF) on chromosome 11, the nucleophosmin/B23 (NPM) gene on chromosome 5, or the nuclear mitotic apparatus gene (NuMA) on chromosome 11 (30, 39). The t(15;17) translocation between PML and RARα accounts for nearly all APL cases. This translocation creates an oncogenic fusion protein, PML-RARα, which contains both the DNA-binding domain (DBD) and ligand-binding domains of RARα and the N terminus of PML. Transgenic mice that overexpress PML-RARα or PLZF-RARα developed an APL-like phenotype (9, 21, 26), suggesting that these fusion proteins are directly involved in APL pathogenesis. Recent studies have focused on analyzing the functional properties of PML-RARα and PLZF-RARα (20, 22, 25, 40) in order to understand the molecular basis of leukemogenesis. Both fusion proteins form homodimers that bind to RA response elements and interact with the nuclear receptor corepressors SMRT (silencing mediator for retinoid and thyroid hormone action) and N-CoR (nuclear receptor corepressor), which in turn recruit a histone deacetylase complex (1, 27, 40, 46). Pharmacological concentrations of all-trans-RA (atRA) induce dissociation of the corepressors from PML-RARα, but not PLZF-RARα, due to the presence of an additional, RA-insensitive corepressor-interacting surface on PLZF. This differential degree of dissociation of corepressors induced by atRA correlates with the ability of histone deacetylase inhibitors and atRA to induce terminal differentiation of these two subtypes of APL cells. These findings indicate that abnormalities in transcriptional repression by the oncogenic fusion proteins may be involved in leukemogenesis. PML belongs to a family of proteins characterized by the presence of a RING finger domain (8). RING finger proteins are implicated in transcriptional regulation, and some members of the RING family are associated directly with chromatin (53). Ablation and overexpression experiments suggest an important role of PML in the regulation of cell growth, hematopoietic cell differentiation, tumorigenesis, apoptosis, and RA signaling (44, 63). In normal cells, PML is concentrated within 10 to 20 nuclear structures known as nuclear domains 10 (ND10), Kruppel bodies, nuclear bodies, or PML-oncogenic domains (PODs) (2, 17, 33, 59, 65). The POD structure is disrupted in the t(15;17) translocated APL cells (17, 33, 65), presumably through interaction of wild-type PML with PML-RARα. Interestingly, the POD structure reorganizes upon treatment with atRA or arsenic trioxide (As2O3), a process that correlates with differentiation of APL cells, indicating that the POD structure might affect promyelocyte differentiation. In addition to PML, the POD contains several other proteins, including the 100-kDa nuclear protein antigen (Sp100) (2), the small ubiquitin-related modifier (SUMO-1 [41], also known as PML-interacting clone 1 [PIC1] [7], ubiquitin-like 1 [UBL1] [57], or sentrin [48]), and the 140-kDa protein (Sp140) (6). Sp100 is a nuclear antigen recognized by autoantibodies from patients with primary biliary cirrhosis (62). Expression of both PML and Sp100 are upregulated by interferon (23). SUMO-1 was recently identified as a ubiquitin-like protein that forms covalent conjugates with PML and Sp100 (7, 58). In addition, the CREB-binding protein (CBP) and the retinoblastoma tumor suppressor (pRB) have been found in the PODs (35, 61). Also, the PODs are targets of several viral proteins, which alter POD structure (11, 14, 18). Although there is evidence for POD's role in transcriptional activation (15, 35), DNA replication (19), apoptosis (51, 64), and viral infection (14, 42), the precise function of PODs in these processes remains unclear. We have sought to understand the function of PODs through identification of PML-interacting proteins that also localize in the PODs. By using the yeast two-hybrid system, we identified SUMO-1 and the Fas-binding protein Daxx (68) (J. D. Chen and R. M. Evans, unpublished data). Daxx has been shown to promote Fas-mediated apoptosis through activation of the Jun NH2-terminal kinase (JNK) and JNK kinase kinase ASK1 (apoptosis signal-regulating kinase 1) (12). Recent data suggest that Daxx is not sufficient for Fas-mediated apoptosis, since a Fas mutant that selectively binds to Daxx but not the Fas-adaptor death domain-containing protein (FADD/MORT1) failed to induce apoptosis (13). Other evidence suggests that Daxx may interact with the centromeric protein-c (CENP-C) and may bind to a steroidogenic factor 1 (SF-1)-like DNA element (32, 50). Therefore, the exact mechanism by which Daxx regulates Fas-mediated apoptosis may involve nuclear processes. In the present study, we have characterized both biochemical and functional interactions between Daxx and PML. Daxx resides primarily in the cell nucleus, where it forms a complex with PML. Confocal immunofluorescence data demonstrate that Daxx colocalizes with PML in the PODs, and such colocalization persists in NB4 APL cells (36) before and after treatment with atRA and As2O3. Daxx possesses strong transcriptional repressor activity and appears to interact directly with histone deacetylases. Intriguingly, overexpression of PML inhibits Daxx-mediated transcriptional repression and, in cells that lack PML, Daxx is preferentially associated with condensed chromatin. Our data reveal a new role for Daxx in transcriptional repression and suggest a novel function of PML and the POD structure in the suppression of transcriptional repression.