Distinct roles of DMAP1 in mouse development

DMAP1 (DNMT1-associated protein 1) is a member of the TIP60-p400 complex that maintains embryonic stem (ES) cell pluripotency and a complex containing the somatic form of DNA methyltransferase 1 (DNMT1s). DMAP1 interacts with DNMT1s through a domain that is absent in Dnmt1(V)(/)(V) mice expressing just the oocyte form (DNMT1o). A Dmap1-null allele was generated to study the role of DMAP1 in development. Consistent with the phenotypes of loss of other members of the TIP60-p400 complex, Dmap1(-/-) mice died during preimplantation in both Dnmt1(+/+) and Dnmt1(V)(/)(V) backgrounds. Unexpectedly, in the Dnmt1(V)(/)(V) background, Dmap1(+/-) parents produced mainly Dmap1(+/-) mice. Most Dmap1(+/+) progeny died during midgestation, with loss of DNA methylation on imprinted genes, suggesting that DMAP1 influences maintenance methylation mediated by DNMT1o. In this regard, a DMAP1-DNMT1o complex was detected in ES cells when DNMT1o was stably expressed but not when transiently expressed, indicating a novel interaction between DMAP1 and DNMT1o. These results suggest that DMAP1-DNMT1s and DMAP1-DNMT1o interactions are essential for normal development and that DMAP1-DNMT1o complexes are not readily formed in the embryo. Therefore, DMAP1 mediates distinct preimplantation epigenetic reprogramming processes: TIP60-p400 nucleosome remodeling and DNMT1 maintenance methylation.     

Sumoylation of Daxx regulates IFN-induced growth suppression of B lymphocytes and the hormone receptor-mediated transactivation

Daxx has been shown to play an essential role in type I IFN-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the molecular mechanisms of how Daxx acts on growth suppression of B lymphocytes, we examined functions of a sumoylation-defective Daxx KA mutant (Daxx K630/631A), which substituted Lys 630 and Lys 631 to Ala. Importantly, Daxx KA localized in the cytoplasm, whereas wild-type Daxx localized in the nucleus. Murine pro-B cell line Ba/F3 expressing Daxx KA revealed a resistance to the IFN-induced growth suppression. It is noteworthy that treatment with an exportin inhibitor, leptomycin B, resulted in nuclear localization of Daxx KA and recovery of the IFN-induced growth suppression in Ba/F3 cells. Moreover, Daxx KA decreased the binding potential to promyelocytic leukemia protein (PML), and overexpression of PML recruited Daxx KA into PML oncogenic domains. Notably, a Daxx-small ubiquitin-related modifier fusion protein exhibited increased nuclear localization and ability to suppress cell growth in Ba/F3 cells. These results demonstrate that the IFN-induced growth suppression of B lymphocytes requires nuclear localization of Daxx through its sumoylation and proper interactions with PML.     

Role of the ASK1-SEK1-JNK1-HIPK1 signal in Daxx trafficking and ASK1 oligomerization

Overexpression of JNK binding domain inhibited glucose deprivation-induced JNK1 activation, relocalization of Daxx from the nucleus to the cytoplasm, and apoptosis signal-regulating kinase 1 (ASK1) oligomerization in human prostate adenocarcinoma DU-145 cells. However, SB203580, a p38 inhibitor, did not prevent relocalization of Daxx and oligomerization of ASK1 during glucose deprivation. Studies from in vivo labeling and immune complex kinase assay demonstrated that phosphorylation of Daxx occurred during glucose deprivation, and its phosphorylation was mediated through the ASK1-SEK1-JNK1-HIPK1 signal transduction pathway. Data from immunofluorescence staining and protein interaction assay suggest that phosphorylated Daxx may be translocated to the cytoplasm, bind to ASK1, and subsequently lead to ASK1 oligomerization. Mutation of Daxx Ser667 to Ala results in suppression of Daxx relocalization during glucose deprivation, suggesting that Ser667 residue plays an important role in the relocalization of Daxx. Unlike wild-type Daxx, a Daxx deletion mutant (amino acids 501-625) mainly localized to the cytoplasm, where it associated with ASK1, activated JNK1, and induced ASK1 oligomerization without glucose deprivation. Taken together, these results show that glucose deprivation activates the ASK1-SEK1-JNK1-HIPK1 pathway, and the activated HIPK1 is probably involved in the relocalization of Daxx from the nucleus to the cytoplasm. The relocalized Daxx may play an important role in glucose deprivation-induced ASK1 oligomerization.     

Release of RASSF1C from the nucleus by Daxx degradation links DNA damage and SAPK/JNK activation

Stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) responds to a variety of stress stimuli and controls cell fates such as cell cycle entrance, apoptosis and senescence. Stimuli such as ultraviolet irradiation and chemical reagents that damage genomic DNA induce the activation of the SAPK/JNK signaling pathway. However, it is unclear how the signal arising in the nucleus owing to DNA damage is transmitted to SAPK/JNK in the cytoplasm. Here, we report that the nuclear components Daxx and Ras-association domain family 1C (RASSF1C) link DNA damage to SAPK/JNK activation in HeLa cells. In response to DNA damage, Daxx localized in promyelocytic leukaemia-nuclear bodies (PML-NBs) undergoes ubiquitination and degradation. RASSF1C, a tumor suppressor and newly identified binding partner of Daxx, is constitutively anchored by Daxx in PML-NBs but is released from the nucleus when Daxx is degraded. This released RASSF1C translocates to cytoplasmic microtubules and participates in the activation of SAPK/JNK. Our data define a novel mechanism by which the Daxx-RASSF1C complex in PML-NBs couples nuclear DNA damage to the cytoplasmic SAPK/JNK signaling pathway.     

Physical interactions and functional coupling between Daxx and sodium hydrogen exchanger 1 in ischemic cell death

Daxx, a death domain-associated protein, is implicated in ischemic cell death. To clarify the mechanism of cell death mediated by Daxx, a yeast two-hybrid assay was performed. Sodium hydrogen exchanger isoform 1 (NHE1) was identified as a Daxx-interacting protein. During ischemic stress, Daxx translocates from the nucleus to the cytoplasm, where it colocalizes with NHE1. Daxx binds to the ezrin/radixin/moesin-interacting domain of NHE1, in competition with ezrin. Consistent with this finding, transfection of the constitutively cytoplasmic mutant, Daxx(W621A), inhibited ezrin-mediated Akt-1 activation. Moreover, transfection of Daxx(W621A), but not the Daxx(S667A) mutant that is confined to the nucleus, accelerated pH(i) recovery from an acid load, indicating that the cytoplasmic protein activates NHE1. Based on the results, we propose that ischemic insult triggers the nucleocytoplasmic translocation of Daxx, following which cytoplasmic Daxx stimulates the NHE1 transporter activity and suppresses activation of the NHE1-ezrin-Akt-1 pathway. Our data support a novel molecular function of Daxx as an upstream regulator of NHE1 in ischemic cell death.     

A kinase-independent function of Ask1 in caspase-independent cell death

Ask1 (apoptosis signal-regulating kinase 1) is activated as a consequence of cell exposure to a variety of stresses and can then initiate apoptosis. A known pathway of apoptosis downstream of Ask1 involves the activation of the stress-activated protein kinases, the release of cytochrome c from mitochondria, the activation of caspases, and the fragmentation of nuclei. Here, we characterized a novel mechanism of Ask1-mediated cell killing that is triggered by the interaction with Daxx. Co-transfection of Ask1 and Daxx induced a caspase-independent cell-death process characterized at the morphological level by distinctive crumpled nuclei easily distinguishable from the condensed and fragmented nuclei seen during classical caspase-dependent apoptosis. The kinase activity of Ask1 was not involved in this process, because mutants lacking kinase activity were as efficient as wild type Ask1 in mediating Daxx-induced cell death. Ask1N, a deletant that lacks the C-terminal half including the kinase domain of Ask1, was constitutively active in producing crumpled nuclei. In contrast, Ask1DeltaN, the reciprocal deletant that possesses constitutive kinase activity, produced fragmented nuclei typical of caspase-dependent death processes. We conclude that in addition to a caspase-dependent pro-apoptotic function that depends on its kinase activity, Ask1 possesses a caspase-independent killing function that is independent on its kinase activity and is activable by interaction with Daxx. In the physiological situation, such an activity is induced as a consequence of the translocation of Daxx from the nucleus to the cytoplasm, a condition that occurs following activation of the death receptor Fas.     

Interaction of Daxx, a Fas binding protein, with sentrin and Ubc9

Sentrin is a ubiquitin-like protein that can covalently modify cellular proteins, and is a Fas binding protein that protects cells against anti-Fas induced cell death. However, the mechanism by which sentrin exerts its effect upon Fas-mediated apoptosis is not well known. Thus, this study examined the interaction of sentrin with Daxx. Sentrin interacted with Daxx but not with FADD when analyzed by yeast two-hybrid assay. In vitro translated Daxx bound to GST-sentrin fusion protein. FLAG-sentrin fusion protein was also coimmunoprecipitated with Daxx in BOSC23 cells. Also, Daxx interacted with Ubc9, an essential protein as a key conjugating enzyme. Amino acids 625-740 of Daxx, known as Fas binding region, was also mapped as sentrin and Ubc9 binding region. Colocalization of Fas, sentrin, and Ubc9 binding regions suggests the importance of that region upon the regulation of Daxx. Our data also demonstrated that sentrin could homooligomerize by protein-protein interaction.     

DNA Methyltransferase 1-associated Protein (DMAP1) Is a Co-repressor That Stimulates DNA Methylation Globally and Locally at Sites of Double Strand Break Repair

Correction of double strand DNA breaks proceeds in an error-free pathway of homologous recombination (HR), which can result in gene silencing of half of the DNA molecules caused by action by DNA methyltransferase 1 (DNMT1) (Cuozzo, C., Porcellini, A., Angrisano, T., Morano, A., Lee, B., Di Pardo, A., Messina, S., Iuliano, R., Fusco, A., Santillo, M. R., Muller, M. T., Chiariotti, L., Gottesman, M. E., and Avvedimento, E. V. (2007) PLoS Genet. 3, e110). To explore the mechanism that leads to HR-induced silencing, a genetic screen was carried out based on the silencing of a GFP reporter to identify potential partners. DMAP1, a DNMT1 interacting protein, was identified as a mediator of this process. DMAP1 is a potent activator of DNMT1 methylation in vitro, suggesting that DMAP1 is a co-repressor that supports the maintenance and de novo action of DNMT1. To examine critical roles for DMAP1 in vivo, lentiviral shRNA was used to conditionally reduce cellular DMAP1 levels. The shRNA transduced cells grew poorly and eventually ceased their growth. Analysis of the tumor suppressor gene p16 methylation status revealed a clear reduction in methylated CpGs in the shRNA cells, suggesting that reactivation of a tumor suppressor gene pathway caused the slow growth phenotype. Analysis of HR, using a fluorescence-based reporter, revealed that knocking down DMAP1 also caused hypomethylation of the DNA repair products following gene conversion. DMAP1 was selectively enriched in recombinant GFP chromatin based on chromatin immunoprecipitation analysis. The picture that emerges is that DMAP1 activates DNMT1 preferentially at sites of HR repair. Because DMAP1 depleted cells display enhanced HR, we conclude that it has additional roles in genomic stability.     

Inhibition of Daxx-mediated apoptosis by heat shock protein 27

Heat shock protein 27 (HSP27) confers cellular protection against a variety of cytotoxic stresses and also against physiological stresses associated with growth arrest or receptor-mediated apoptosis. Phosphorylation modulates the activity of HSP27 by causing a major change in the supramolecular organization of the protein, which shifts from oligomers to dimers. Here we show that phosphorylated dimers of HSP27 interact with Daxx, a mediator of Fas-induced apoptosis, preventing the interaction of Daxx with both Ask1 and Fas and blocking Daxx-mediated apoptosis. No such inhibition was observed with an HSP27 phosphorylation mutant that is only expressed as oligomers or when apoptosis was induced by transfection of a Daxx mutant lacking its HSP27 binding domain. HSP27 expression had no effect on Fas-induced FADD- and caspase-dependent apoptosis. However, HSP27 blocked Fas-induced translocation of Daxx from the nucleus to the cytoplasm and Fas-induced Daxx- and Ask1-dependent apoptosis. The observations revealed a new level of regulation of the Fas pathway and suggest a mechanism for the phosphorylation-dependent protective function of HSP27 during stress and differentiation.     

Daxx enhances Fas-mediated apoptosis in a murine pro-B cell line,BAF3

Daxx has been shown to play an essential in type I interferon (IFN-/β)-mediated suppression of B cell development and apoptosis. Recently, we demonstrated that Tyk2 is directly involved in IFN signaling for the induction and nuclear translocation of Daxx, which may result in growth arrest and/or apoptosis of B lymphocyte progenitors. To clarify the mechanism of Daxx-mediated apoptosis signaling in B lymphocyte progenitors, here we introduced an efficient suicide switch in a murine pro-B cell line, BAF3, by expressing FK506-binding protein-fused Fas intracellular domain (FKBP-Fas) and Daxx. It allows us to monitor Fas/Daxx-mediated signal by induction of Fas dimerization with the dimerizer drug AP20187. AP20187-mediated Fas dimerization induced not only apoptosis but also Jun N-terminal kinase (JNK) activation. However, AP20187 had no effect on cells expressing either Fas or Daxx only. Furthermore, expression of a JNK inhibitor, the JNK-binding domain of JIP-1, resulted in resistance to AP20187-mediated apoptosis in cells expressing FKBP-Fas and Daxx. These results imply that our novel suicide switch system may provide a powerful tool to delineate or identify the signaling molecules for Daxx-mediated apoptotic machinery in B lymphocyte progenitors through JNK activation.