RNAi reveals anti-apoptotic and transcriptionally repressive activities of DAXX

The function of DAXX, a highly conserved mammalian gene, has remained controversial; this is due, in part, to its identification in a variety of yeast two-hybrid screens. Targeted deletion in the mouse revealed that DAXX is essential for embryonic development. Furthermore, the increased levels of apoptosis observed in Daxx-knockout embryos and embryonic stem cell lines suggested that DAXX functions in an anti-apoptotic capacity. In contrast, overexpression studies showed that DAXX may promote apoptosis. Additional studies showed that, when overexpressed, DAXX could function as a transcriptional repressor. To clarify these matters, we have used RNAi to deplete endogenous DAXX and thereby assess DAXX function in cell lines previously tested in overexpression studies. Increased apoptosis was observed in DAXX-depleted cells, showing DAXX to be anti-apoptotic. The apoptosis induced by the absence of DAXX was rescued by Bcl-2 overexpression. In addition, transcriptional derepression was observed in RNAi-treated cells, indicating the ability of endogenous DAXX to repress gene expression and allowing for the identification of novel targets of DAXX repression, including nuclear factor κB (NF-κB)- and E2F1- regulated targets. Thus, depletion of DAXX by RNAi has verified the crucial role of endogenous DAXX as an anti-apoptotic regulator, and has allowed the identification of probable physiological targets of DAXX transcriptional repression.

interacting with the Fas receptor death domain, was shown to enhance Fas-mediated apoptosis when overexpressed (Yang et al., 1997). DAXX was proposed to function in a pathway independent of Fas-associated death domain and to mediate apoptosis through activation of the Jun N-terminal kinase (JNK) pathway via apoptosis signal-regulating kinase 1 (Chang et al., 1998;Chang et al., 1999;Ko et al., 2001;Yang et al., 1997). Similarly, a role for DAXX in transforming growth factor β (TGF-β)-induced apoptosis and associated JNK activation was shown in B-cell lymphomas and hepatocytes (Perlman et al., 2001). Interestingly, Fas-mediated apoptosis and JNK signaling was shown to be independent of DAXX in lymphoid cells (Villunger et al., 2000).
Other studies have proposed that the ability of DAXX to induce apoptosis relies not on the ability of DAXX to interact with Fas, but rather on a nuclear apoptotic pathway, consistent with DAXX localization studies. Furthermore, it was suggested that DAXX facilitates the induction of apoptosis in primary splenocytes and keratinocytes from within PODs and only in the presence of PML (Zhong et al., 2000). Similarly, Torii et al. report that the ability of DAXX to facilitate Fas-induced apoptosis requires DAXX localization to PODs (Torii et al., 1999).
The ability of DAXX to function as a transcriptional repressor has also been shown. DAXX was shown to bind to Pax3, a member of the paired box homeodomain family of transcription factors, and on overexpression DAXX repressed Pax3-mediated transcriptional activity (Hollenbach et al., The function of DAXX, a highly conserved mammalian gene, has remained controversial; this is due, in part, to its identification in a variety of yeast two-hybrid screens. Targeted deletion in the mouse revealed that DAXX is essential for embryonic development. Furthermore, the increased levels of apoptosis observed in Daxx-knockout embryos and embryonic stem cell lines suggested that DAXX functions in an anti-apoptotic capacity. In contrast, overexpression studies showed that DAXX may promote apoptosis. Additional studies showed that, when overexpressed, DAXX could function as a transcriptional repressor. To clarify these matters, we have used RNAi to deplete endogenous DAXX and thereby assess DAXX function in cell lines previously tested in overexpression studies. Increased apoptosis was observed in DAXX-depleted cells, showing DAXX to be anti-apoptotic. The apoptosis induced by the absence of DAXX was rescued by Bcl-2 overexpression. In addition, transcriptional derepression was observed in RNAi-treated cells, indicating the ability of endogenous DAXX to repress gene expression and allowing for the identification of novel targets of DAXX repression, including nuclear factor κB (NF-κB)-and E2F1-regulated targets. Thus, depletion of DAXX by RNAi has verified the crucial role of endogenous DAXX as an anti-apoptotic regulator, and has allowed the identification of probable physiological targets of DAXX transcriptional repression.
1999). Interaction of DAXX with the ETS1 transcription factor similarly caused repression of transcriptional activity (Li et al., 2000b). When tethered to a galactose 4 (Gal4)-DNA binding domain, DAXX could inhibit basal transcription, possibly through recruitment of histone deacetylases (Li et al., 2000a). In B cells, DAXX was shown to function as a co-repressor, and under certain circumstances as a co-activator, of Pax5 (BSAP) (Emelyanov et al., 2002). A recent report suggests that the ability of DAXX to interact with histone deacetylases, core histones and the chromatin-associated protein DEK, provides the mechanism by which DAXX represses transcription (Hollenbach et al., 2002).
We have used RNAi as a method to deplete cell lines of endogenous DAXX protein in an effort to determine conclusively the function of DAXX in apoptosis and transcriptional regulation. RNA interference (RNAi) is the process of gene-specific post-transcriptional silencing following the introduction of double-stranded RNA homologous to the gene of interest (reviewed by Hunter, 2000). The phenomenon, initially described in Caenorhabditis elegans, has recently been successfully used as a tool to inhibit gene expression in mammalian cells (Caplen et al., 2001;Elbashir et al., 2001). Specifically, transfection of 21-23-nucleotide double-stranded RNAs into human and mouse cell lines was shown to efficiently and specifically suppress the expression of target genes, either endogenous or overexpressed.
Our analysis of cell lines depleted of DAXX by RNAi has revealed increased levels of apoptosis, confirming the role of DAXX as an anti-apoptotic protein. Furthermore, transcriptional studies in DAXX-depleted cells have shown that endogenous DAXX represses transcriptional activity, and has allowed for the identification of probable physiological targets of DAXX repression.

Materials and Methods
Cell culture and transfections Cells were grown in DMEM media (Gibco) containing 10% bovine serum (Sigma) and 4 mM L-glutamine in the presence of antibiotics at 37°C with 5% CO2.
For DNA transfections, HeLa cells were transfected with 3 µg DNA per well of a six-well dish (or 8 µg DNA for a 10 cm plate) using Fugene6 reagent (Boehringer Mannheim) in medium containing serum. GenBank NM_007829 bp 666-686. RNA oligonucleotides were annealed as described previously .

DNA constructs
The Daxx expression vector contains full-length mouse Daxx with a C-terminal myc epitope tag expressed under the control of the chicken β-actin promoter in the pCAGGS vector (Niwa et al., 1991). pEGFP-C1 (Clontech) was used for expression of green fluorescent protein (GFP). The Bcl-2 expression vector contained full-length human Bcl-2 cloned into pCDNA3.1. cMet-luciferase (Met-luc) contains luciferase under control of the cMet promoter (Epstein et al., 1996). E2F1-luc and SP1-luc were kind gifts from P. Farnham (University of Wisconsin Medical School, Madison, WI) (Slansky et al., 1993). Other luciferase reporter constructs were obtained from Stratagene.

FACS analysis
Cells were collected 48, 72 or 96 hours following RNAi treatment, washed in PBS and fixed in 70% ethanol. Samples were incubated with RNase A (0.5 mg/ml) and propidium iodide (5 µg/ml), followed by analysis on a FACS Calibur flow cytometer (Becton-Dickinson). A minimum of 1.5×10 5 cells were analyzed for each FACS experiment sample. For GFP experiments, a minimum of 5×10 4 cells were analyzed.

Reporter assays
Lysates were prepared from six-well dishes 24 hours following DNA transfection in 250 µl Passive Lysis Buffer (Promega; Madison, WI), and a 50 µl sample was analyzed on an Automat LB953 luminometer (Berthold) with automatic injection of luciferase reagent (Fischer). All samples were co-transfected with 50-100 ng β-galactosidase reporter construct, pCMVβ (MacGregor and Caskey, 1989). Luciferase values were normalized for transfection efficiency by measuring βgalactosidase activity using the Galacto-star system (Tropix). All transfections were performed in triplicate.

Depletion of DAXX by RNAi
RNAi studies were used to deplete cell lines of endogenous DAXX protein. Two sets of 21-basepair double-stranded RNA oligonucleotides (hDx1 and hDx2) corresponding to human DAXX were tested by transfection into HeLa cells. Western blot analysis of cell lysates collected 72 hours after transfection revealed that hDx2 effected near complete depletion of endogenous DAXX protein, whereas hDx1 resulted in a modest decrease in DAXX (Fig. 1A). Note that background bands and β-actin control were unaffected by RNAi treatment, indicating the specificity of the RNAi effect and the equivalent amounts of protein loaded on the gel. Subsequent studies used exclusively hDx2 as an efficient means to deplete cells of endogenous DAXX. A time-course study revealed that the RNAi effect of hDx2 lasted 5 days post-transfection, with the effect largely dissipated by 9 days (Fig. 1B). The RNAi effect was evident as early as 48 hours post-transfection but was not apparent at 24 hours (data not shown).
The specificity of the hDx2 effect was confirmed using a control RNA oligonucleotide (mDx2), the mouse homolog of hDx2 which differs in sequence by three nucleotides plus an additional nucleotide. Indeed, mDx2 had no effect on DAXX protein levels (Fig. 1C). Transient transfection of mouse Daxx cDNA following RNAi treatment revealed that hDx2 had no effect on accumulation of overexpressed mouse DAXX, whereas mDx2 treatment prevented overexpression (Fig. 1D). These studies confirmed the species specificity of hDx2 and mDx2. As shown in Fig. 1E,F, hDx2 was also effective in U2OS and 293 cells, with the resulting depletion of DAXX protein being slightly more pronounced following a second round of transfection. Taken together, these studies show that treatment of human cell lines with hDx2 results in a specific and significant depletion of endogenous DAXX protein.

Increased levels of apoptosis upon depletion of DAXX by RNAi
Whether DAXX functions as a pro-and/or antiapoptotic molecule has been a matter of dispute in the literature (reviewed by Michaelson, 2000). Although results from knockout studies show that DAXX probably plays a role in preventing apoptosis in the early embryo and in ES cells, overexpression studies suggested that DAXX may function as a proapoptotic molecule in other cell types. Our ability to deplete DAXX using RNAi provided the opportunity to directly assess the function of DAXX in cell lines previously analyzed in overexpression studies (Chang et al., 1998;Chang et al., 1999;Ko et al., 2001;Torii et al., 1999;Yang et al., 1997).
The cell-cycle profile of HeLa cells transfected with hDx2 was analyzed by FACS. The sub-G1 peak, indicative of the apoptotic fraction due to fragmented DNA content, was modestly yet significantly increased in hDx2compared with mock-transfected cells ( Fig. 2A). The increased levels of apoptosis were evident as early as 48 hours posttransfection and were more pronounced 72 and 96 hours following RNAi transfection. The levels observed in the DAXX-depleted cells were similar to those observed in Daxxknockout ES cell lines (Michaelson et al., 1999). Elevated levels of apoptosis were similarly observed following transfection of hDx2 into 293 cells (data not shown). To verify that the increased apoptosis observed in HeLa cells was not a nonspecific RNAi effect, mDx2 was used in a similar experiment and showed no apoptotic effect (Fig. 2B).
As an additional method of assessing apoptosis, cleavage of the caspase target PARP was analyzed by western blot following RNAi treatment. In contrast to extracts prepared from mDx2-transfected HeLa cells, in which only full-length PARP (112 kDa) was evident, depletion of DAXX by hDx2 treatment resulted in the reproducible appearance of the cleaved version of PARP (85 kDa) (Fig. 2C), indicating activation of the caspase cascade.
It is well documented that Bcl-2, the founding member of a conserved family of proteins that regulate cell death, Samples in the upper panels are detected with anti-DAXX antibody, with the arrow indicating the DAXX band; samples in the lower panels are detected with anti-β-actin antibody as a loading control. Mock transfections were performed with buffer alone. hDx1, hDx2 and mDx2 refer to RNAi species, as described in Materials and Methods. Unless otherwise specified, cell lysates were prepared from HeLa cells, and were collected 72 hours posttransfection. In B, cells were harvested 3 days (3d), 5 days (5d) or 9 days (9d) following RNAi transfection. In D, cells were transfected with mouse Daxx cDNA (+mDaxx) 24 hours following RNAi treatment. Cell lysates were prepared from 293 (E) and U2OS (F) cells following one round (1X) or two rounds (2X) of transfection.
functions to inhibit apoptosis (reviewed by Adams and Cory, 2001). We tested whether overexpression of Bcl-2 could rescue the apoptosis induced by DAXX depletion. HeLa cells were transfected with Bcl-2 or vector control 4 hours post-RNAi treatment. Measurement by FACS analysis revealed a significant rescue of the apoptotic fraction following transfection with Bcl-2 (Fig. 3A). The rescue was not complete, as expected, given the limitation of transfection efficiency. To monitor transfected cells only, HeLa cells were co-transfected with GFP in addition to Bcl-2 or vector control following RNAi treatment. FACS analysis on GFP-positive staining cells revealed a complete rescue of apoptotic cells (Fig. 3B). Note that in addition to rescue of hDx2-induced apoptosis, Bcl-2 also rescued the apoptotic fraction generated presumably as a result of GFP toxicity (Liu et al., 1999), which was also evident in mocktransfected cells.

RNAi depletion of DAXX results in transcriptional derepression
Previous studies have shown that DAXX, when overexpressed, can mediate transcriptional repression (Emelyanov et al., 2002;Hollenbach et al., 2002;Hollenbach et al., 1999;Li et al., 2000a;Li et al., 2000b). We have assessed the ability of endogenous DAXX to mediate repression by measuring transcriptional activity of putative targets in cells depleted of DAXX by RNAi. Following RNAi treatment, HeLa cells were transfected with Met-luc (Epstein et al., 1996), a luciferase reporter gene driven by the Pax-3-regulated c-Met (hepatocyte growth factor) promoter. Cells treated with hDx2 RNAi revealed significantly increased levels of luciferase activity relative to mock-or mDx2-treated cells, indicating that depletion of endogenous DAXX causes de-repression of the c-Met promoter (Fig. 4A).
To show that the de-repression was solely a function of the loss of DAXX, an attempt was made to revert the de-repression by reconstituting cells with increasing levels of DAXX. Reconstitution was accomplished by transfection with mouse Journal of Cell Science 116 (2)

Fig. 3. Bcl-2 rescues apoptosis in cells depleted of DAXX by RNAi.
Mock-or hDx2-transfected HeLa cells were fixed and stained 72 hours post-RNAi treatment. DNA transfections were performed 4 hours after RNAi treatment. Percent apoptosis is calculated as the percentage of total cells comprising the sub-G1 peak as determined by FACS analysis cell-cycle profiling following propidium iodide staining. Averages and standard deviations were calculated from a minimum of two independent transfection experiments. In B, the sub-G1 peak of GFP-positive cells only is calculated. RNAi reveals DAXX-dependent activities Daxx, which is unaffected by hDx2 treatment (see Fig. 1D). In mock-treated cells, transfection of mouse Daxx cDNA resulted in decreased activity of Met-luc (Fig. 4B), indicative of repression of the c-Met promoter and consistent with previous studies showing the repressive effects of DAXX overexpression (Hollenbach et al., 1999;Li et al., 2000b). In hDx2-treated cells, where increased levels of c-Met activity were observed (Fig. 4B), transfection of increasing levels of Daxx resulted in an incremental decreases in luciferase activity (Fig. 4B). In the presence of a high concentration of transfected Daxx (2.0 µg), significant repression of Met-luc was observed. These results confirm that loss of DAXX is responsible for the de-repression observed in the RNAi experiments.
The ability of endogenous DAXX to mediate repression of several other luciferase reporter constructs was tested. Following depletion of DAXX by hDx2, significantly increased luciferase activity from reporters driven by NF-κB and E2F1 elements (NFκB-luc and E2F1-luc) was observed relative to mDx2-treated cells (Fig. 5A), indicating that NF-κB and E2F1 targets are probably repressed by endogenous DAXX. In contrast, a basal promoter element (TATA-luc) showed no difference in luciferase activity when treated with hDx2 as compared with mDx2 (Fig. 5A). Similarly, RNAi treatment had no significant effect on the transcriptional activity of several reporter constructs, including AP1-luc and others (Fig. 5A). Thus, it is likely that endogenous DAXX does not regulate this subset of promoters in the given cellular context.
The ability of DAXX to repress the panel of reporter constructs was also tested using DAXX overexpression studies. When DAXX was overexpressed, the Met-, NF-κB-and E2F1driven luciferase activities were accordingly repressed, but there was no significant effect on TATA-luc activity (Fig. 5B). These results are consistent with the effect of DAXX observed in the RNAi studies (Fig. 5A). However, in overexpression studies, DAXX was capable of moderately repressing AP1-luc, SP1-luc and cAMP-responsive element (CRE)-luc, reporters Fig. 4. Depletion of DAXX by RNAi results in transcriptional de-repression. HeLa cells were transfected with RNAi (or mock transfected), followed 24 hours later by DNA transfection with Met-Luciferase and β-gal constructs. Luciferase activity was measured after an additional 24 hours. In A, fold-de-repression was calculated by the fold increase of luciferase activity for hDx2-transfected relative to mock-transfected samples, with the mock-treated luciferase being set arbitrarily as 1. In B, DNA transfections in some cases included mouse Daxx cDNA (mDaxx), with the indicated microgram amount per well of six-well plate. All values represent averages of three independent transfections with standard deviation. All experiments were normalized by co-transfection with β-gal. HeLa cells were transfected with hDx2 or mDx2 followed 24 hours later by DNA transfection with reporter plasmids. Fold de-repression was calculated by the fold increase of luciferase activity for hDx2-transfected relative to mDx2transfected samples, with the mDx2-treated luciferase value for each reporter being set arbitrarily as 1. (B) HeLa cells were transfected with reporter plasmids, with or without mouse Daxx cDNA. Fold repression is calculated by computing the fold difference between cells transfected without DAXX (mock transfected) relative to cells transfected with Daxx. All values represent averages of three independent transfections with standard deviation. All experiments were normalized by co-transfection with β-gal. that were not accordingly de-repressed following depletion of endogenous DAXX (Fig. 5A). Taken together, these studies probably identify a set of physiological targets of DAXX, which include the Pax-3 regulated c-Met, as well as NF-κB and E2F1 targets.

Discussion
In this report, we have used RNAi technology to assess the function of endogenous DAXX. Our results show that DAXX protects cells from apoptosis, thus confirming an anti-apoptotic role for DAXX. Furthermore, these studies illustrate the ability of endogenous DAXX to repress transcriptional targets.
We have taken advantage of the power of RNAi to evaluate the function of DAXX. Only recently has RNAi been shown to be effective in inhibiting gene expression in mammalian systems (Caplen et al., 2001;Elbashir et al., 2001). As a technical application, our studies exploit RNAi as a tool to evaluate mammalian gene function, namely apoptosis and transcriptional regulation. Recently, a few examples of successful use of RNAi in assessing cell growth and division were reported (Du et al., 2001;Harborth et al., 2001;Ohta et al., 2002). Our studies have further taken advantage of the specificity of RNAi to enable human cells depleted of DAXX to be reconstituted with mouse DAXX. By reintroducing DAXX into RNAi-depleted cells, we could show the specificity of the RNAi effect in mediating transcriptional de-repression. This type of reconstitution experiment is a powerful and effective method that will undoubtedly prove useful in validating phenotypes observed following RNAi treatment.
The ability to effect nearly complete depletion of endogenous protein was crucial for these studies. The observations that Daxx heterozygous mice develop normally and that Daxx heterozygous embryos as well as ES cells show no increase in apoptosis, despite the reduced levels of DAXX protein (Michaelson et al., 1999), suggest that modest levels of DAXX within the cell are probably sufficient for normal gene function. Therefore, it is likely that a relatively complete reduction in protein levels is required to achieve functional deletion of DAXX. Previous studies have attempted to use antisense technology to assess DAXX function. For example, in a study by Gongora and colleagues (Gongora et al., 2001), the significant levels of residual DAXX evident following antisense treatment make it difficult to interpret the effect observed on interferon-induced apoptosis in pro-B cells. Antisense studies were also employed to assess TGF-β-induced apoptosis in murine hepatocytes; although protection from apoptosis was observed, the extent to which depletion of DAXX was achieved was not reported (Perlman et al., 2001). The RNAi studies reported here show nearly complete depletion of DAXX and thus enable a more meaningful interpretation of resulting phenotypes.
RNAi studies have allowed us to evaluate the function of endogenous DAXX, a matter that until now has been somewhat controversial. Previously, results from the Daxx-knockout studies revealed that embryos and ES cell lines lacking DAXX have increased levels of apoptosis (Michaelson et al., 1999). In contrast to the knockout findings, several studies suggested that when overexpressed, DAXX could enhance Fas-mediated apoptosis (Chang et al., 1998;Chang et al., 1999;Ko et al., 2001;Yang et al., 1997), although not in lymphoid cells (Villunger et al., 2000). Other studies argued for DAXX mediating apoptosis from within the nucleus (Torii et al., 1999;Zhong et al., 2000). Taken together, the accumulating data were consistent with the notion that DAXX might have a dual function with respect to apoptosis, depending on the cellular context. Although DAXX may function in an anti-apoptotic capacity in development and possibly in the lymphoid system, it might be pro-apoptotic in fibroblasts and other cell types. The RNAi data presented here, however, suggest that DAXX also has an anti-apoptotic effect in cell lines such as HeLa and 293, in which overexpression studies previously showed the opposite effect. We deduce that DAXX may be anti-apoptotic in a variety of contexts, given that many of the studies arguing for a pro-apoptotic role used overexpression methodology. Nevertheless, the possibility that DAXX may be pro-apoptotic under certain circumstances cannot be ruled out.
Indeed, the levels of apoptosis that we observed here are consistent with the percentage of apoptotic cells measured in the Daxx-knockout ES cell lines (Michaelson et al., 1999). Although a truncated DAXX transcript and polypeptide observed in the knockout was of potential concern, we have recently generated a true DAXX null and the phenotype mimics that of the original knockout (J.S.M. and P.L., unpublished observations). Nevertheless, although our results suggest that the absence of DAXX results in a consistent level of apoptosis in several cellular contexts, the levels of apoptosis observed both in the RNAi studies and in the knockout ES lines are relatively modest. It is possible that cells lacking DAXX are only susceptible to apoptosis at a particular phase of the cell cycle. Alternatively, it is conceivable that cells with an exceedingly low level of DAXX can escape apoptosis. Finally, as discussed below, apoptosis may be secondary to the transcriptional effects of DAXX, in which case the apoptotic outcome may be dictated by fine differences in the transcriptional status of a given cell.
Significantly, we have found that Bcl-2 can rescue cells from the apoptosis induced by the absence of DAXX. This observation confirms that the death induced by DAXX depletion is indeed apoptotic. Furthermore, this finding indicates that cells lacking DAXX are subject to an apoptotic pathway that is Bcl-2 dependent. This would imply that the absence of DAXX cannot directly induce effector caspase activation. The caspase cascade is, nevertheless, probably involved in the apoptotic effect, given our observation of PARP cleavage in RNAi-treated cells. The ability of Bcl-2 to rescue the apoptotic effect of DAXX depletion is not surprising, given the ability of Bcl-2 to rescue many apoptotic responses, including that presumably induced by GFP overexpression (Liu et al., 1999) as observed in this study (Fig. 3B).
The RNAi studies also show the role of endogenous DAXX in mediating transcriptional repression. It was previously observed that overexpression of DAXX resulted in repression of targets of various transcription factors, such as ETS and Pax family members (Hollenbach et al., 1999;Li et al., 2000a;Li et al., 2000b). Our RNAi data show that endogenous DAXX can indeed mediate transcriptional repression of a variety of targets, including the c-Met promoter regulated by Pax 3. It is likely, however, that in our experimental system, regulation of the c-Met promoter is through other factors, given that Pax-3 is not expressed in HeLa cells. In cases where we observed transcriptional de-repression following RNAi treatment, such as Met-luc, NF-κB-luc and E2F1-luc, we detected corresponding levels of repression when DAXX was overexpressed. These probably represent true targets of DAXX. In contrast, several targets showed no change with RNAi treatment, indicating the specificity of DAXX repression and arguing against DAXX being a general repressor. Interestingly, modest repression of AP1-luc, SP1-luc and CREluc was observed with DAXX overexpression. We consider it likely that these are not physiological targets of DAXX, but rather that the repression observed is an artifact of overexpression. Alternatively, they may be potential targets of DAXX repression under certain circumstances. We believe that RNAi thus provides an advantageous method to identify physiological targets of transcriptional regulators.
Our results suggest that AP1 transcriptional regulation is probably not a physiological target of DAXX. Previous studies had suggested that DAXX mediated JNK activation (Chang et al., 1998;Perlman et al., 2001;Yang et al., 1997), a process which results in increased transcriptional activity of AP-1 (reviewed by Davis, 2000). However, in our RNAi studies we observed no effect on AP-1 and, upon overexpression of DAXX, we actually detected modest repression of AP-1. Our findings thus do not support the claim that DAXX induces JNK activation. JNK activation by DAXX has similarly been challenged by several other studies in both lymphoid (Villunger et al., 2000) and fibroblast cell lines (Charette et al., 2000;Hofmann et al., 2001;Torii et al., 1999).
The question remains regarding the link between the role of DAXX in protecting from apoptosis and its function in transcriptional repression. It is possible that these represent independent activities of DAXX. However, our data provide a potential explanation for how the transcriptional effects of DAXX may dictate an apoptotic outcome. For example, the strong repression of E2F1 targets by DAXX and its derepression in the absence of DAXX correlate with the positive role for E2F1 in inducing apoptosis (reviewed by Black and Azizkhan-Clifford, 1999). Furthermore, the repression of NF-κB target genes by DAXX may reduce the expression of proapoptotic genes, which, in the absence of DAXX, are then upregulated to induce apoptosis. Alternatively, it is conceivable that the activation of NF-κB in the absence of DAXX, as observed in our transcriptional studies, may represent a response to apoptotic signals. Such a mechanism would suggest that the transcriptional activities of DAXX are secondary to its apoptotic function. Future experimentation will be required to dissect the interplay between the role of DAXX in apoptosis and its function in transcriptional repression.