In the present study, we have identified Daxx as a PML-interacting protein and characterized the functional interaction between Daxx and PML. We find a majority of Daxx in the nucleus of HeLa and HEp2 cells where it colocalizes with PML in the PODs. In the NB4 APL cell line, Daxx is distributed in the microparticulate structures that contain the PML-RARα oncoprotein (17
). The repressor function of Daxx is observed upon tethering it to a reporter gene via a heterologous DNA binding domain, as well as from a reporter containing a natural SF1-like promoter element. The mechanism by which Daxx represses basal transcription is found as involving histone deacetylation, for Daxx interacts with HDACs in vitro and in vivo and the histone deacetylase inhibitor, TSA, blocks the repressor activity. Coexpression of PML reverses the transcriptional repression by Daxx, which, in turn, correlates with the recruitment of Daxx to the PODs. In addition, we show that SUMO-1 modification of PML is required for both recruitment of Daxx to the PODs and efficient inhibition of Daxx-mediated repression. The physiological role of Daxx in transcriptional repression is further supported by the observation that Daxx associates with condensed chromatin in cells that lack PML. Together, these data establish novel roles for Daxx, as a transcriptional repressor, and for PML, as a protein that can potentially regulate the repressor function of Daxx.
Consistent with our findings, Daxx has recently been identified as an inhibitor of transcriptional activation by Pax3, a member of the homeodomain family of transcription factors (28
). Thus, Daxx not only is able to repress basal transcription, as suggested from our data, but can also inhibit transcriptional activation via interactions with DNA-binding transcription factors. While the exact mechanism of the inhibition of Pax3 transactivation by Daxx is unclear, our data elucidate the mechanism of Daxx-mediated repression of basal transcription as involving histone deacetylation. We observe Daxx localization to condensed chromatin in NT2 cells that lack detectable PML. Condensed chromatin is considered as a site of transcriptional repression that also includes transcriptionally silent centromeric heterochromatin. Other POD-associated proteins, such as SP100, have been demonstrated to interact with heterochromatin protein 1 (HP1) and also colocalize with centromeric chromatin (10
). Consistent with this idea, Daxx has been shown to interact with CENP-C in a yeast two-hybrid assay and partially to colocalize with interphase centromeres (50
). Also, Daxx has been shown to interact with DNA methyltransferase 1, which plays a role in gene silencing (43
Previous studies have implicated the PODs as sites of transcriptional activation. For example, PML has been demonstrated to interact with the transcription coactivator CBP and recruit CBP to the PODs (15
). Furthermore, PML can enhance the transactivation functions of both CBP and members of the nuclear receptor superfamily (15
). PML also induces genes of the major histocompatibility complex, while PML−/−
mice display reduced transactivation responses to atRA (64
). Finally, the transcriptional activator Sp140 (5
) and nascent RNA (35
) have been found in at least a subset of PODs. Our findings that Daxx represses basal transcription and PML inhibits such repressor activity suggest a new role for the POD structure in gene regulation. The POD may enhance transcription of target genes not only through recruitment of activators but also through the inactivation of repressors such as Daxx via recruitment by PML. Because other transcriptional repressors, such as PLZF, pRB, and Sp100, have also been found in the PODs, it will be interesting to determine if PML can regulate the repressor activities of these proteins as well.
Our observations that PML-RARα can interact with Daxx but not inhibit transcriptional repression by Daxx suggest a potential role for Daxx in acute promyelocytic leukemia. Support for this hypothesis is evident in our finding that Daxx, PML-RARα, and PML colocalize at diffusely distributed microparticulate structures in nucleus of the APL NB4 cells. The PML-RARα fusion protein disrupts the POD structure in these cells and, through its interaction with Daxx, may direct Daxx to the microparticulate structures, where it is capable of repressing gene expression. PML-RARα itself is a potent transcriptional repressor, which acts via the recruitment of the corepressors SMRT, N-CoR, and HDAC1 (40
). The POD structure is reorganized upon treatment of these cells with atRA or arsenic trioxide, leading to the degradation of the PML-RARα fusion protein and colocalization of Daxx and PML in the PODs (47
). Therefore, Daxx inactivation through localization to the PODs may be critical to the differentiation of normal hematopoietic cells. Since expression of the PML-RARα fusion protein disrupts the integrity of the PODs, Daxx may act as a constitutive repressor in the APL cells, which along with the repressor function of PML-RARα, may block expression of specific genes that are critical for cell differentiation and culminate in the subsequent APL pathology.
Daxx was initially identified as a Fas-binding protein that promoted Fas-mediated apoptosis via activation of the JNK kinase cascade pathway (12
). Interestingly, PML has also been found to be involved in apoptosis triggered by Fas, tumor necrosis factor alpha, and type I and II interferons, possibly by recruitment of the death effector Bax and cdk inhibitor p21 (37
). In contrast, expression of PML-RARα prevents apoptosis in response to these signals (51
). Our findings, together with these reports, suggest that the regulation of Daxx repressor function by PML may also be important in programmed cell death. Consistent with this possibility, several transcriptional repressors are known to play a role in apoptosis. For example, the adenovirus E1B and the cellular Bcl-2 oncoprotein block p53-mediated apoptosis by inhibiting transcriptional repression by p53, suggesting that p53 induces apoptosis via transcriptional repression (52
). In the case of Daxx, PML may recruit it to the PODs, where it is inactivated, thus allowing the expression of certain genes required for apoptosis. Conversely, PML-RARα might inhibit apoptosis in APL cells through disruption of the PODs, thereby promoting enhanced or constitutive repression of these target genes by Daxx and the PML-RARα fusion protein itself, which leads to the APL phenotype. Retinoic acid treatment would stimulate degradation of PML-RARα and restoration of the POD structure (17
). This would allow Daxx to be inactivated through sequestration to the PODs and allow apoptosis to proceed and would eventually lead to remission of the APL phenotype. Because PML can shuttle between the nucleus and cytoplasm (59
), it is possible that Daxx may be brought along with PML to regulate cytoplasmic events relevant to Fas-mediated apoptosis. However, a recent study reports that the loss of Daxx leads to extensive apoptosis in early mouse development (43
), a result seemingly opposite to other findings concerning the function of Daxx in apoptosis (12
). Therefore, the precise role of Daxx in apoptosis remains to be further elucidated.
Our data provide strong evidence for the roles of PML and the PODs in regulating the function of Daxx as a transcriptional repressor. Daxx and PML interact in vivo and colocalize in the PODs. Overexpression of PML recruits Daxx to the PODs, which correlates with a complete inhibition of transcriptional repression by Daxx. Although the detailed mechanism of this inhibition of Daxx by PML remains to be determined, our data provide several possibilities. First, PML might inactivate Daxx by transporting it to the PODs and separating it from HDAC and putative target genes. In response to certain stimuli such as interferon, PML levels increase in the PODs, which, via competition for Daxx binding or conformational change of Daxx upon PML binding, might result in the dissociation of Daxx from HDAC and recruitment of Daxx, but not HDAC, to the PODs. Confinement of Daxx in the PODs would thus block access to target genes, whose expression level would then increase to at least the basal level in the absence of Daxx repression. Our findings that PML overexpression results in increased Daxx levels in the PODs, while having no effect on HDAC1 distribution or repression by HDAC1, support this possibility. Alternatively, the increased PML levels may dissociate HDAC from Daxx and recruit both Daxx and its putative target genes, but not HDAC, to the PODs. Because Daxx requires HDAC and histone deacetylation for its repressor activity, the target genes may be expressed in the absence of HDAC. The presence of transcriptional activators in the PODs would facilitate transcription of target genes. With either possibility, it is evident that the POD is involved in maintaining the balance of Daxx function, depending on the PML level. At normal, physiological levels of PML, Daxx might repress transcription at areas of condensed chromatin. However, with increased PML expression, more Daxx is recruited to the PODs, thus reducing its overall repression activity. Although the precise mechanism of the inhibition of Daxx repression by the PODs awaits further investigation, our data clearly reveal a novel connection between Daxx and PML in regulating transcriptional repression that may play a critical role in acute promyelocytic leukemia and apoptosis.