Function Of Daxx Research Articles

Age-dependent differential expression of death-associated protein 6 (Daxx) in various peripheral tissues and different brain regions of C57BL/6 male mice.

Death-associated protein 6 (DAXX) is a ubiquitous protein implicated in various cellular processes such as apoptosis, tumorigenesis, development and transcription. The role of DAXX is however ambiguous and many contradictory results regarding its function in apoptosis upon various cellular stresses are described in the literature. In order to have a better understanding of the role of DAXX throughout the entire organism under physiological stress conditions, we have characterized the mRNA levels, protein expression and the proteolytic processing of DAXX in the normal aging process in peripheral organs and brain regions in C57BL/6 male mice. Overall, Daxx mRNA expression decreases with aging in the liver, kidney, heart, cortex and cerebellum. In contrast, an increase is observed in the striatum. The protein expression of DAXX and of its proteolytic fragments increases with aging in the kidney, heart and cortex. In liver and spleen, no changes are observed while in the striatum and cerebellum, certain forms increase and others decrease with age, suggesting that the functions of DAXX may be cell type dependent. This study provides important details regarding the expression and post-translational modifications of DAXX in aging in the entire organism and provides reference data for the deregulation observed in age-associated diseases.

Abstract 2717: Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma

Abstract The unlimited proliferative capacity of cancer cells is closely linked to maintenance of their telomeres, which shorten with each cell division in normal cells. Cancer cells are able to maintain telomere length by multiple mechanisms, including activation of telomerase and the recombination based alternative lengthening of telomeres (ALT) pathway. ALT is prevalent in osteosarcoma, with approximately 50% of osteosarcoma cases using ALT for telomere maintenance. Mutations in the ATRX/DAXX chromatin remodeling complex and histone H3.3 correlate with activation of the ALT pathway in several tumor systems. While loss of ATRX is a frequent event in osteosarcoma tumors, alterations of DAXX have not been reported. We characterized the telomere maintenance mechanisms utilized by 11 osteosarcoma cell lines. Of these, 45% (5/11) were ALT positive and 45% (5/11) were telomerase positive. One cell line possessed features of both telomere maintenance mechanisms. Among ALT positive osteosarcoma cell lines, we observed frequent loss of ATRX expression (4/5) and a previously unreported translocation resulting in disruption of DAXX. The translocation abolishes recruitment of DAXX to nuclear PML bodies and prevents normal DAXX function. By reintroducing full length DAXX, we were able to suppress telomere maintenance by ALT as evidenced by multiple assays including loss of C-circles and ALT-associated PML bodies, thus demonstrating that continued DAXX deficiency is necessary for maintenance of the ALT mechanism. Suppression of ALT by DAXX reintroduction did not result in compensatory activation of telomerase. This first demonstration of ALT suppression by DAXX supports a mechanistic connection between loss of the ATRX/DAXX chromatin remodeling complex and telomere maintenance by ALT. Understanding this relationship may uncover vulnerabilities specific to ALT tumors that could potentially lead to the development of targeted therapies for diverse cancers that depend on the ALT pathway. Citation Format: Kathryn E. Driest, Joshua J. Waterfall, Robert L. Walker, Marbin A. Pineda, Ogan Abaan, Yuelin J. Zhu, Yonghong Wang, Corbin D. Ester, Sean R. Davis, Sven Bilke, Paul S. Meltzer. Reintroduction of DAXX suppresses alternative lengthening of telomeres in osteosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2717.

Daxx and TCF4 interaction links to oral squamous cell carcinoma growth by promoting cell cycle progression via induction of cyclin D1 expression.

ObjectivesDeath domain-associated protein (Daxx) has been recently implicated as a positive factor in ovarian cancer and prostate cancer, but the role of Daxx in oral squamous cell carcinoma (OSCC) has never been addressed. Herein, we investigate the expression and function of Daxx in OSCC.Materials and methodsRT-quantitative PCR, Western blotting, and immunohistochemistry were used to evaluation of the expression of Daxx in human OSCC cell lines and clinical surgical specimens. Short hairpin RNA targeting Daxx was transduced by lentivirus infection to knockdown the expression of Daxx in SAS and SCC25 cell lines, and the influence of this knockdown was evaluated by analyzing the growth and the cell cycle in transduced cells. Immunoprecipitation and sequential chromatin immunoprecipitation-quantitative PCR were used to analyze the associations between Daxx, TCF4, and cyclin D1 promoter. Xenograft tumor model was used to evaluate the in vivo tumorigenicity of Daxx in OSCC.ResultsDaxx mRNA and protein expression are elevated in several OSCC cell lines and human OSCC samples in comparison to those in normal tissue. We further find that depletion of Daxx decreases OSCC cell growth activity through G1 cell cycle arrest. Daxx silencing reduces cyclin D1 expression via a Daxx-TCF4 interaction, whereas the Daxx depletion-mediated G1 arrest can be relieved by ectopic expression of cyclin D1. Moreover, we show that in OSCC clinical samples, the expression of Daxx is significantly correlated with that of cyclin D1.ConclusionOur data demonstrate the importance of Daxx in regulation of cyclin D1 expression and provide the first evidence that Daxx exhibits tumor-promoting activity in OSCC.Clinical relevanceDaxx plays an important role in malignant transformation of OSCC and may serves as a target for cancer prevention and treatment.

Dengue virus disrupts Daxx and NF-κB interaction to induce CD137-mediated apoptosis

Dengue virus (DENV) is a positive-strand RNA virus of the Flavivirus family with 4 different serotypes. Clinical manifestations of DENV infection include dengue fever, dengue hemorrhagic fever, and dengue shock syndrome. Following DENV infection, apoptosis of hepatic cells is observed both in vitro and in vivo. However, the molecular mechanisms revealing how viral components affect cellular apoptosis remain unclear. In the present study, the role of death domain-associated protein 6 (Daxx) in DENV-mediated apoptosis was characterized by RNA interference and overexpression studies, and the anti-apoptotic function of Daxx during DENV infection was identified. Furthermore, the viral component, DENV capsid protein (DENV C), interacted with Daxx to disrupt interaction between Daxx and NF-κB. The liberated NF-κB activated the promoter of CD137, which is a member of the TNF family, and is previously shown to induce apoptosis during DENV infection. In summary, DENV C disrupts Daxx and NF-κB interaction to induce CD137-mediated apoptosis during DENV infection.

Non‐telomerase telomere maintenance in human cells (97.2)

Although it has recently been demonstrated that telomeres can be lengthened by a non‐telomerase mechanism (i.e., Alternative Lengthening of Telomeres; ALT) in normal mouse tissues, we have learnt most about ALT in mammalian cells from the study of in vitro‐immortalized and tumor‐derived human cell lines. Apart from its intrinsic biological interest, it is important to understand ALT because ~10% of human cancers utilize it to avoid senescence, and some of the tumor types where ALT is common, such as soft tissue sarcomas and astrocytic brain tumors, are currently difficult to treat. Loss of normal ATRX or DAXX function contributes to, but is insufficient for the upregulation of ALT in many cancers and cell lines. An increased non‐canonical telomeric repeat sequence content may also foster increased levels of ALT activity by decreasing shelterin binding sites. ALT involves synthesis of new telomeric DNA via recombination‐mediated copying of existing telomeric sequences. The template may be the DNA of another telomere or a more proximal region of the telomere that is being lengthened. There is a tight correlation between the level of C‐circles, i.e., extrachromosomal circles of partially double‐stranded telomeric DNA with an intact C‐rich strand and an incomplete G‐rich strand, and ALT activity. We are therefore using a C‐circle assay to identify genes/ proteins involved in the ALT mechanism.

Death Domain-associated Protein 6 (Daxx) Selectively Represses IL-6 Transcription through Histone Deacetylase 1 (HDAC1)-mediated Histone Deacetylation in Macrophages

As a multifunctional nuclear protein, death domain-associated protein 6 (Daxx) regulates a wide range of biological processes, including cell apoptosis and gene transcription. However, the function of Daxx in innate immunity remains unclear. In our study, we show that Daxx is highly expressed in macrophages and localized in nucleus of macrophages. The expression of Daxx is significantly up-regulated by stimulation with TLR ligands LPS and poly(I:C). Silence of Daxx selectively represses IL-6 expression at transcription level in LPS-activated macrophages. Upon stimulation of LPS, Daxx specifically binds to the promoter of IL-6 and inhibits histone acetylation at IL-6 promoter region. Further mechanism analyses show that histone deacetylase 1 (HDAC1) interacts with Daxx and binds to the promoter of IL-6. Daxx silencing decreases the association of HDAC1 to IL-6 promoter. Therefore, our data reveal that Daxx selectively represses IL-6 transcription through HDAC1-mediated histone deacetylation in LPS-induced macrophages, acting as a negative regulator of IL-6 during innate immunity and potentially preventing inflammatory response because of overproduction of IL-6.

Disease mutant analysis identifies a new function of DAXX in telomerase regulation and telomere maintenance.

Most human cancers depend on the telomerase to maintain telomeres; however, about 10% of cancers are telomerase negative and utilize the alternative lengthening of telomeres (ALT) mechanism. Mutations in the DAXX gene have been found frequently in both telomerase-positive and ALT cells, and how DAXX mutations contribute to cancers remains unclear. We report here that endogenous DAXX can localize to Cajal bodies, associate with the telomerase and regulate telomerase targeting to telomeres. Furthermore, disease mutations that are located in different regions of DAXX differentially impact on its ability to interact with its binding partners and its targeting to Cajal bodies and telomeres. In addition, DAXX knockdown by RNA interference led to reduced telomerase targeting to telomeres and telomere shortening. These findings collectively support a DAXX-centric pathway for telomere maintenance, where DAXX interaction with the telomerase regulates telomerase assembly in Cajal bodies and telomerase targeting to telomeres.

English

Death domain-associated protein (Daxx) was originally identii¬ed as a protein that specii¬cally binds to the death domain of the transmembrane death receptor Fas (also called CD95) in the cytoplasm and potentiates Fas-induced apoptosis. Over expression of Daxx enhances Fas-mediated apoptosis and activates the Jun N-terminal kinase (JNK) pathway. Daxx was found in the nuclears where it localizes to promyelocytic leukaemia (PML) oncogenic domains (PODs). As a highly conserved nuclear protein, Daxx plays an important role in proapoptosis, antiapoptosis and transcriptional regulation. This article reviews the latest advances in the study of the biological function of Daxx. Key words: Death domain-associated protein (Daxx), cell apoptosis, transcriptional regulation, biological function.

Phosphorylation Drives an Apoptotic Protein to Activate Antiapoptotic Genes: PARADIGM OF INFLUENZA A MATRIX 1 PROTEIN FUNCTION

During infection, viral proteins target cellular pathways that regulate cellular innate immune responses and cell death. We demonstrate that influenza A virus matrix 1 protein (M1), an established proapoptotic protein, activates nuclear factor-κB member RelB-mediated survival genes (cIAP1, cIAP2, and cFLIP), a function that is linked with its nuclear translocation during early infection. Death domain-associated protein 6 (Daxx) is a transcription co-repressor of the RelB-responsive gene promoters. During influenza virus infection M1 binds to and stabilizes Daxx protein by preventing its ubiquitination and proteasomal degradation. Binding of M1 with Daxx through its Daxx binding motif prevents binding of RelB and Daxx, resulting in up-regulation of survival genes. This interaction also prevents promoter recruitment of DNA methyltransferases (Dnmt1 and Dnmt3a) and lowers CpG methylation of the survival gene promoters, leading to the activation of these genes. Thus, M1 prevents repressional function of Daxx during infection, thereby exerting a survival role. In addition to its nuclear localization signal, translocation of M1 to the nucleus depends on cellular kinase-mediated phosphorylation as the protein kinase C inhibitor calphostin C effectively down-regulates virus replication. The study reconciles the ambiguity of dual antagonistic function of viral protein and potentiates a possible target to limit virus infection.

Tumor suppressor protein Pdcd4 interacts with Daxx and modulates the stability of Daxx and the Hipk2-dependent phosphorylation of p53 at serine 46

The tumor suppressor protein Pdcd4 is a nuclear/cytoplasmic shuttling protein that has been implicated in the development of several types of human cancer. In the nucleus, Pdcd4 affects the transcription of specific genes by modulating the activity of several transcription factors. We have identified the Daxx protein as a novel interaction partner of Pdcd4. Daxx is a scaffold protein with roles in diverse processes, including transcriptional regulation, DNA-damage signaling, apoptosis and chromatin remodeling. We show that the interaction of both proteins is mediated by the N-terminal domain of Pdcd4 and the central part of Daxx, and that binding to Pdcd4 stimulates the degradation of Daxx, presumably by disrupting the interaction of Daxx with the de-ubiquitinylating enzyme Hausp. Daxx has previously been shown to serve as a scaffold for protein kinase Hipk2 and tumor suppressor protein p53 and to stimulate the phosphorylation of p53 at serine 46 (Ser-46) in response to genotoxic stress. We show that Pdcd4 also disrupts the Daxx–Hipk2 interaction and inhibits the phosphorylation of p53. We also show that ultraviolet irradiation decreases the expression of Pdcd4. Taken together, our results support a model in which Pdcd4 serves to suppress the phosphorylation of p53 in the absence of DNA damage, while the suppressive effect of Pdcd4 is abrogated after DNA damage owing to the decrease of Pdcd4. Overall, our data demonstrate that Pdcd4 is a novel modulator of Daxx function and provide evidence for a role of Pdcd4 in restraining p53 activity in unstressed cells.

Death domain-associated protein（DAXX）による転写調節と細胞死

Death domain-associated protein (DAXX) is a multifunctional protein that modulates both cell death and transcription. DAXX predominantly localizes in the nucleus and regulates the transcription of genes. Several studies have indicated that DAXX is a mediator of lymphocyte death and/or growth suppression, although it is still unclear whether DAXX pro-cell death function is dependent on its transcriptional functions. We found that DAXX suppresses the gp130/STAT3-dependent cell growth and that DAXX interacts with STAT3 and inhibits its DNA-binding activity. Here we will discuss recent developments in our understanding of the nuclear functions of DAXX.

T cell proliferative and apoptotic responses in conditional Daxx knockout mice (163.16)

Abstract Apoptosis is essential for maintaining homeostasis in the immune system. Fas is a single transmembrane domain protein capable of inducing apoptotic death in lymphocytes. Mutations in the Fas receptor or it’s ligand, FasL, lead to autoimmune-lymphocproliferation diseases characterized by lymphadenopathy, splenomegaly and autoantibody production. The Fas-associated death domain (FADD) protein is an adaptor protein which binds to Fas, recruits and activates the initiator caspase 8. In addition to FADD, proteins including Daxx have also been indicated to bind to Fas. It has been proposed that Daxx mediates a parallel apoptotic pathway that is independent of FADD and Caspase 8, but is dependent on ASK1-medaited signaling. Deletion of FADD blocks Fas-induced apoptosis in T and B cells, which, however, does not lead to lymphoproliferation diseases as present in Fas mutant animals. Recent studies indicate that absence of FADD unleashes a potent necrosis-like death pathway. The in vivo function of Daxx in lymphocytes has not been properly analyzed as deletion of Daxx in germ cells lead to embryonic lethality. In this study, we used a conditional mutant mouse in which Daxx is deleted specifically in T cells. Our data revealed a novel function of Daxx in the regulation of proliferation and death in primary T cells.

Dualistic function of Daxx at centromeric and pericentromeric heterochromatin in normal and stress conditions

Nuclear structures ND10/PML NBs are linked to multiple processes, including the maintenance of intranuclear homeostasis by sequestering proteins into “nuclear depot.” This function presumes release of proteins from PML NBs and their redistribution to the alternative, supposedly “active” locations, in response to the external stress application. To further investigate this nuclear depot function, we focused on the intranuclear distribution of protein Daxx that in normal conditions is mainly accumulated at PML NBs, and has a minor association with centromeres and pericentromeres (CEN/periCEN). Here we report that application of physiological Heat Shock (HS) changes this balance forcing very robust and reversible accumulation of Daxx on CEN/periCEN heterochromatin. Heterochromatin architecture is essential for the proper orchestration of nuclear processes, while transcription from this part of genome is required for its maintenance. To understand functional consequences of Daxx deposition at CEN/periCEN, we tested for Daxx-dependency of heterochromatin transcription. Depletion of Daxx reduces accumulation of CEN RNA in normal conditions and periCEN RNA after HS application. Searching for the mechanism of Daxx-dependent regulation of heterochromatin transcription, we found that depletion of Daxx decreases incorporation of transcription-associated histone H3 variant, H3.3, into both CEN and periCEN. Surprisingly, HS-induced deposition of Daxx does not further elevate incorporation of H3.3 into CEN/periCEN that remained steady during stress and recovery. Instead, depletion of Daxx leads to HS-induced changes in the balance of epigenetic modifications at heterochromatin, most dramatically elevating levels of active H3K4Me2 modification at periCEN. We propose dualistic function of Daxx-containing complexes at CEN/periCEN: (1) regulation of H3.3 loading in normal conditions and (2) protection of epigenetic status upon stress-induced accumulation, thus collectively guarding epigenetic identity of CEN/periCEN heterochromatin.

Daxx mediates activation-induced cell death in microglia by triggering MST1 signalling

Microglia, the resident macrophages of the mammalian central nervous system, migrate to sites of tissue damage or infection and become activated. Although the persistent secretion of inflammatory mediators by the activated cells contributes to the pathogenesis of various neurological disorders, most activated microglia eventually undergo apoptosis through the process of activation-induced cell death (AICD). The molecular mechanism of AICD, however, has remained unclear. Here, we show that Daxx and mammalian Ste20-like kinase-1 (MST1) mediate apoptosis elicited by interferon-γ (IFN-γ) in microglia. IFN-γ upregulated the expression of Daxx, which in turn mediated the homodimerization, activation, and nuclear translocation of MST1 and apoptosis in microglial cells. Depletion of Daxx or MST1 by RNA interference also attenuated IFN-γ-induced cell death in primary rat microglia. Furthermore, the extent of IFN-γ-induced death of microglia in the brain of MST1-null mice was significantly reduced compared with that apparent in wild-type mice. Our results thus highlight new functions of Daxx and MST1 that they are the key mediators of microglial cell death initiated by the proinflammatory cytokine IFN-γ.

Structural and Functional Roles of Daxx SIM Phosphorylation in SUMO Paralog-Selective Binding and Apoptosis Modulation

Small ubiquitin-like modifier (SUMO) conjugation and interaction are increasingly associated with various cellular processes. However, little is known about the cellular signaling mechanisms that regulate proteins for distinct SUMO paralog conjugation and interactions. Using the transcriptional coregulator Daxx as a model, we show that SUMO paralog-selective binding and conjugation are regulated by phosphorylation of the Daxx SUMO-interacting motif (SIM). NMR structural studies show that Daxx (732)E-I-I-V-L-S-D-S-D(740) is a bona fide SIM that binds to SUMO-1 in a parallel orientation. Daxx-SIM is phosphorylated by CK2 kinase at residues S737 and S739. Phosphorylation promotes Daxx-SIM binding affinity toward SUMO-1 over SUMO-2/3, causing Daxx preference for SUMO-1 conjugation and interaction with SUMO-1-modified factors. Furthermore, Daxx-SIM phosphorylation enhances Daxx to sensitize stress-induced cell apoptosis via antiapoptotic gene repression. Our findings provide structural insights into the Daxx-SIM:SUMO-1 complex, a model of SIM phosphorylation-enhanced SUMO paralog-selective modification and interaction, and phosphorylation-regulated Daxx function in apoptosis.

Structural Characterization of the DAXX N-Terminal Helical Bundle Domain and Its Complex with Rassf1C

DAXX is a scaffold protein with diverse roles including transcription and cell cycle regulation. Using NMR spectroscopy, we demonstrate that the C-terminal half of DAXX is intrinsically disordered, whereas a folded domain is present near its N terminus. This domain forms a left-handed four-helix bundle (H1, H2, H4, H5). However, due to a crossover helix (H3), this topology differs from that of the Sin3 PAH domain, which to date has been used as a model for DAXX. The N-terminal residues of the tumor suppressor Rassf1C fold into an amphipathic α helix upon binding this DAXX domain via a shallow cleft along the flexible helices H2 and H5 (K(D) ∼60μM). Based on a proposed DAXX recognition motif as hydrophobic residues preceded by negatively charged groups, we found that peptide models of p53 and Mdm2 also bound the helical bundle. These data provide a structural foundation for understanding the diverse functions of DAXX.

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.

Regulation of c-met expression by transcription repressor Daxx

The protooncogene c-met encodes the tyrosine kinase receptor for the hepatocyte growth factor/scatter factor (HGF/SF). While overexpression of c-met is documented in many types of tumors, the mechanism of c-met regulation remains elusive. Here, we demonstrate Daxx as a repressor of c-met transcription. The expression of c-met is elevated in Daxx knockout mouse cells and is reversed by Daxx reconstitution. C-met promoter analysis of Daxx-/- cells reveled changes in chromatin acetylation, but not in DNA methylation. Daxx binds to the mouse c-met promoter and Daxx-binding region is sufficient for transcription repression, while HDAC2 is associated with c-met promoter mostly in Daxx+/+ cells, pointing to Daxx-dependent HDAC2 recruitment as a potential mechanism of c-met repression. HGF-induced cell mobility and invasion confirmed augmented activity of c-Met/HGF pathway in Daxx-/- cells. Finally, inverse correlation between Daxx and c-Met in cancer cell lines and in metastatic breast cancer specimens suggests potential function of Daxx as a c-met repressor during cancer progression.

Daxx Cooperates with the Axin/HIPK2/p53 Complex to Induce Cell Death

Daxx, a death domain-associated protein, has been implicated in proapoptosis, antiapoptosis, and transcriptional regulation. Many factors known to play critically important roles in controlling apoptosis and gene transcription have been shown to associate with Daxx, including the Ser/Thr protein kinase HIPK2, promyelocytic leukemia protein, histone deacetylases, and the chromatin remodeling protein ATRX. Although it is clear that Daxx may exert multiple functions, the underlying mechanisms remain far from clear. Here, we show that Axin, originally identified for its scaffolding role to control beta-catenin levels in Wnt signaling, strongly associates with Daxx at endogenous levels. The Daxx/Axin complex formation is enhanced by UV irradiation. Axin tethers Daxx to the tumor suppressor p53, and cooperates with Daxx, but not DaxxDeltaAxin, which is unable to interact with Axin, to stimulate HIPK2-mediated Ser(46) phosphorylation and transcriptional activity of p53. Interestingly, Axin and Daxx seem to selectively activate p53 target genes, with strong activation of PUMA, but not p21 or Bax. Daxx-stimulated p53 transcriptional activity was significantly diminished by small interfering RNA against Axin; Daxx fails to inhibit colony formation in Axin(-/-) cells. Moreover, UV-induced cell death was attenuated by the knockdown of Axin and Daxx. All these results show that Daxx cooperates with Axin to stimulate p53, and implicate a direct role for Axin, HIPK2, and p53 in the proapoptotic function of Daxx. We have hence unraveled a novel aspect of p53 activation and shed new light on the ultimate understanding of the Daxx protein, perhaps most pertinently, in relation to stress-induced cell death.

HGF/SF Regulates Expression of Apoptotic Genes in MCF‐10A Human Mammary Epithelial Cells

Hepatocyte growth factor/scatter factor (HGF/SF) induces scattering, morphogenesis, and survival of epithelial cells through activation of the MET tyrosine kinase receptor. HGF/SF and MET are involved in normal development and tumor progression of many tissues and organs, including the mammary gland. In order to find target genes of HGF/SF involved in its survival function, we used an oligonucleotide microarray representing 1,920 genes known to be involved in apoptosis, transcriptional regulation, and signal transduction. MCF-10A human mammary epithelial cells were grown in the absence of serum and treated or not with HGF/SF for 2 h. Total RNA was reverse-transcribed to cDNA in the presence of fluorescent Cy3-dUTP or Cy5-dUTP to generate fluorescently labeled cDNA probes. Microarrays were performed and the ratios of Cy5/Cy3 fluorescence were determined. The expression of three apoptotic genes was modified by HGF/SF, with A20 being upregulated, and DAXX and SMAC being downregulated. These changes of expression were confirmed by real-time quantitative PCR. According to current-knowledge, A20 is antiapoptotic and SMAC is proapoptotic, while a pro- or antiapoptotic function of DAXX is controversial. The fact that HGF/SF upregulates an antiapoptotic gene (A20) and downregulates a proapoptotic gene (SMAC) is in agreement with its survival effect in MCF-10A cells. This study identified novel apoptotic genes regulated by HGF/SF, which can contribute to its survival effect.