The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition.

After DNA viruses enter the nucleus, they initiate a transcriptional cascade which is followed by replication. We investigated whether these processes take place at specific nuclear sites or, as suggested by the mode of entry, randomly throughout the nucleus. Three distinct nuclear domains, nuclear factor-1 sites, coiled bodies, and nuclear domain 10 (ND10), were used as markers to investigate the relative position of DNA virus replication sites. We found that all three nuclear domains had a very high spatial correlation with each other in uninfected cells. After adenoviral infection, nuclear factor 1 and coiled bodies were found associated with some viral replication domains. Simian virus 40 begins replication adjacent to ND10 but adenovirus 5 and herpes simplex type 1 modified ND10s before replication. Adenovirus E4orf 3 gene deletion mutants retain ND10 and begin replication at the peripheries of ND10. The same was found for the herpes simplex virus type 1 immediate early gene 1 mutants. That the deposition and replication of adenovirus 5 and herpesvirus type 1 at ND10 was not a mutant phenotype was confirmed by finding the input wild-type virus juxtaposed to ND10. The transport of viral genomes to ND10 does not require viral gene expression. Thus, the peripheries of ND10 represent preferred sites where early steps of transcription and replication of at least three DNA virus families take place, suggesting a new set of functional properties for this large nuclear domain.

Human cytomegalovirus immediate early interaction with host nuclear structures: definition of an immediate transcript environment.

The development of an induced transcript environment was investigated at the supramolecular level through comparative localization of the human cytomegalovirus immediate early (IE) transcripts and specific nuclear domains shortly after infection. Compact aggregates of IE transcripts form only adjacent to nuclear domain 10 (ND10), and the viral protein IE86 accumulates exclusively juxtaposed to the subpopulation of ND10 with transcripts. The stream of transcripts is funneled from ND10 into the spliceosome assembly factor SC35 domain through the accumulation of IE86 protein, which recruits some components of the basal transcription machinery. Concomitantly the IE72 protein binds to ND10 and later disperses them. The domain containing the zinc finger region of IE72 is essential for this dispersal. Positional analysis of proteins IE86 and IE72, IE transcripts, ND10, the spliceosome assembly factor SC35, and basal transcription factors defines spatially and temporally an immediate transcript environment, the basic components of which exist in the cell before viral infection, providing the structural environment for the virus to usurp.

PML is critical for ND10 formation and recruits the PML-interacting protein daxx to this nuclear structure when modified by SUMO-1.

Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML-/- cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1-modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozygous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

[Cloning universal probes for detecting mycoplasma contamination of cell cultures]. / Kolnirovanie universal'nykh zondov dlia vyiavleniia mikoplazmennogo zarazheniia kletochnykh kul'tur.

To obtain the universal polynucleotide hybridization probes for testing mycoplasmal contaminations in cell cultures, we have cloned several DNA fragments from the srRNA gene of Acholeplasma laidlawii. Before cloning, in order to exclude cross-hybridization of these probes with eukaryotic rRNA, the thermodynamic parameters of duplex formation between DNA complementary to mycoplasmal rRNA and eukaryotic rRNA had been studied. Using a set of computer methods, the region which forms weak heteroduplexes with eukaryotic srRNA was revealed. This region occupies positions 250 to 550 position of the mycoplasmal srRNA. Three different DNA fragments which include the region were generated in PCR, cloned in pUC18, and their hybridization characteristics were evaluated. In appropriate hybridization conditions the probes hybridize with all mycoplasmal RNAs studied without cross-hybridization with eukaryotic ribosomal RNA and DNA, and allow one to detect virtually any mycoplasmas (or any prokaryote) in cell cultures. Blot-hybridization of universal probes with mycoplasmal DNA digested by BsuRI allows one to identify the different species of mycoplasmas.

[Monoclonal antibodies against protein Daxx and its localization in nuclear domains 10]. / Monoklonal'nye antitela k belku Daxx i ego lokalizatsiia v iadernykh domenakh 10.

Nuclear domains 10 (ND10) were first detected occasionally using antibodies to an antigen of unknown nature (Ascoli, Maul, 1991). Further on it was shown that ND10 were sites of locality of the number of proteins (PML, Sp 100, pRB) (Sterndorf et al., 1992; Kamitani et al., 1998), the majority of which are modified with ubiquitin-like small proteins-modifiers (SUMO) (Ishov, Maul, 1996). In addition, it was shown that ND10 were sites of primary localization, transcription and replication of some DNA-viruses (SV40, virus of simple herpes 1, adenovirus 5) (Ishov, Maul, 1996; Ishov et al., 1997). Except for SV40, these viruses produce proteins able to modify ND10, or leading to degradation of ND10-associated proteins (Maul et al., 1993; Maul, Everett, 1994). This degradation is accompanied with protein desumofication and, later, with hydrolysis on the ubiquitin-proteosomal way (Everett et al., 1998, 1999). Cell incubation with interferon leads to augmentation of the number and dimension of ND10 owing to increased expression of Sp100 and PML (Lavau et al., 1995; Grotzinger et al., 1996). In all, these data make it possible to put forward a hypothesis that ND10 may represent a peculiar cell storage ("dépôt") of proteins regulated according to the "accumulation-drop" principle (Ishov et al., 1997; Maul, 1998). However, this hypothesis requires further factual grounds.

USP7 and Daxx regulate mitosis progression and taxane sensitivity by affecting stability of Aurora-A kinase.

A large number of patients are resistant to taxane-based chemotherapy. Functional mitotic checkpoints are essential for taxane sensitivity. Thus, mitotic regulators are potential markers for therapy response and could be targeted for anticancer therapy. In this study, we identified a novel function of ubiquitin (Ub)-specific processing protease-7 (USP7) that interacts and cooperates with protein death domain-associated protein (Daxx) in the regulation of mitosis and taxane resistance. Depletion of USP7 impairs mitotic progression, stabilizes cyclin B and reduces stability of the mitotic E3 Ub ligase, checkpoint with forkhead and Ring-finger (CHFR). Consequently, cells with depleted USP7 accumulate Aurora-A kinase, a CHFR substrate, thus elevating multipolar mitoses. We further show that these effects are independent of the USP7 substrate p53. Thus, USP7 and Daxx are necessary to regulate proper execution of mitosis, partially via regulation of CHFR and Aurora-A kinase stability. Results from colony formation assay, in silico analysis across the NCI60 platform and in breast cancer patients suggest that USP7 levels inversely correlate with response to taxanes, pointing at the USP7 protein as a potential predictive factor for taxane response in cancer patients. In addition, we demonstrated that inhibition of Aurora-A attenuates USP7-mediated taxane resistance, suggesting that combinatorial drug regimens of Taxol and Aurora-A inhibitors may improve the outcome of chemotherapy response in cancer patients resistant to taxane treatment. Finally, our study offers novel insights on USP7 inhibition as cancer therapy.

Regulation of mitosis and taxane response by Daxx and Rassf1.

Current theories suggest that mitotic checkpoint proteins are essential for proper cellular response to taxanes, a widely used family of chemotherapeutic compounds. We recently showed that absence or depletion of protein Daxx increases cellular taxol (paclitaxel) resistance-a common trait of patients diagnosed with several malignancies, including breast cancer. Further investigation of Daxx-mediated taxol response revealed that Daxx is important for the proper timing of mitosis progression and cyclin B stability. Daxx interacts with mitotic checkpoint protein RAS-association domain family protein 1 (Rassf1) and partially colocalizes with this protein during mitosis. Rassf1/Daxx depletion or expression of Daxx-binding domain of Rassf1 elevates cyclin B stability and increases taxol resistance in cells and mouse xenograft models. In breast cancer patients, we observed the inverse correlation between Daxx and clinical response to taxane-based chemotherapy. These data suggest that Daxx and Rassf1 define a mitotic stress checkpoint that enables cells to exit mitosis as micronucleated cells (and eventually die) when encountered with specific mitotic stress stimuli, including taxol. Surprisingly, depletion of Daxx or Rassf1 does not change the activity of E3 ubiquitin ligase anaphase promotion complex/C in in vitro settings, suggesting the necessity of mitotic cellular environment for proper activation of this checkpoint. Daxx and Rassf1 may become useful predictive markers for the proper selection of patients for taxane chemotherapy.

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.

Evidence for separate ND10-binding and homo-oligomerization domains of Sp100.

Nuclear domains called ND10 or PML nuclear bodies consist of an aggregation of several proteins, most notably PML and Sp100. PML is essential in the nucleation and formation of ND10 as well as in the recruitment of other ND10-associated proteins such as Daxx, pRb, BLM and Sp100. In cells induced to overexpress Sp100, ND10 binding of Sp100 was saturable and excess Sp100 formed new aggregation sites devoid of other ND10-associated proteins, suggesting that homo-oligomerization is the basis for aggregation. To determine whether Sp100 binds to ND10 through hetero- or oligomerization, Sp100 deletion variants fused with GFP were transfected into cells with and without endogenous Sp100, and the localization of the GFP-labeled fragments was determined relative to ND10. Amino acids 29-152 were sufficient for deposition of the GFP-labeled fragments at ND10 in the absence of endogenous Sp100 (heterologous binding) and for self-aggregation (formation of new Sp100 deposits). None of the shorter fragments was deposited at ND10 or self-aggregated. The 29-152 amino acid fragment and some larger fragments, but not the full-size Sp100, induced elongation of ND10, which at their ends contain only Sp100, probably due to self-aggregation. By fusing a peptide consisting of the p53-binding domain from hMDM2 to the Sp100(29-152) fragment, this self-aggregation could be blocked while retaining the limited ND10 binding capacity, indicating that the Sp100 self-aggregation domain and the ND10 binding domain are separate entities. This fusion peptide was used to demonstrate the potential of ND10 to recruit p53 as a protein not usually present at this site. Such deposited p53 was protected from turnover. The capacity of ND10 to recruit Sp100 may serve primarily to reduce its availability.

Nuclear domain 10 as preexisting potential replication start sites of herpes simplex virus type-1.

The herpes simplex virus type 1 (HSV-1) nuclear replication cycle begins at localized sites, but it has remained unclear whether these sites are associated with any defined nuclear structure. We have previously shown that during infection, the HSV-1 immediate-early protein ICP0 dispersed proteins associated with ND10, nuclear sites that contain high concentrations of PML and other potentially regulatory proteins and correspond to the ultrastructurally defined nuclear bodies. Using in situ hybridization and immunohistochemical techniques, we found that ICP0 mutants of HSV-1 replicate in the close proximity with ND10, but increasing replication sites develop away from these nuclear structures. Input wild-type HSV-1 DNA was found preferentially adjacent to ND10 before ICP0 modified these nuclear structures and did not colocalize with ICP8 containing so-called prereplication sites. The sites where HSV-1 can begin replication then need to be redefined as preexisting potential replication sites. Viral RNA was also found associated with ND10 before early protein synthesis (ICP8), suggesting that input virus genomes are deposited at ND10 before they start replication. The deposition of input viral DNA at ND10 is virus gene expression independent, probably indicating cell regulation of this process. Taken together, these data demonstrate that some very early processes of the nuclear viral replication cycle happen in close proximity or at the periphery of ND10. The localization of input HSV-1 to ND10 represents a new host-virus interaction and provides an unexpected functional property for this large nuclear site.

Review: properties and assembly mechanisms of ND10, PML bodies, or PODs.

Nuclear domain 10 (ND10), also referred to as PML bodies or PODs, are discrete interchromosomal accumulations of several proteins including PML and Sp100. We describe here developments in the visualization of ND10 and the mechanism of ND10 assembly made possible by the identification of proteins that are essential for this process using cell lines that lack individual ND10-associated proteins. PML is critical for the proper localization of all other ND10-associated proteins under physiological conditions. Introducing PML into a PML -/- cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10, attesting to its essential nature in ND10 formation. This recruitment includes Daxx, a protein that can bind PML and is highly enriched in condensed chromatin in the absence of PML. The segregation of Daxx from condensed chromatin to ND10 by increased accumulation of Sentrin/SUMO-1 modified PML suggests the presence of a variable equilibrium between these two nuclear sites. These findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML. Additional adapter proteins are suggested to exist by the behavior of Sp100, and Sp100 will provide the basis for their identification. Further information about the dynamic balance of proteins between ND10 and the actual site of functional activity of these proteins will establish whether ND10 function as homeostatic regulators or only in storage of excess proteins destined for turnover.

Nuclear redistribution of BRCA1 during viral infection.

The functions and the intracellular localization of the breast/ovarian susceptibility gene product, BRCA1, has been controversial. To arrive at a clear understanding of its localization and relative position to other nuclear structures, a new monoclonal antibody was produced and characterized by immunohistochemical techniques with other BRCA1 antibodies. Each of the antibodies specifically detected BRCA1 as localized to specific nuclear domains and did so in a variety of cells and in a cell cycle-dependent manner. However, all antibodies also cross-reacted with the centrosomal domain, suggesting that BRCA1 is also localized to this important mitotic component. We found that the BRCA1-containing nuclear domains are different than any of the well-defined nuclear domains. However, a cell cycle-related partial overlap was found for HP1alpha, a chromo-domain-containing protein involved in heterochromatin maintenance. Cellular stimuli, such as heat shock and herpes virus infection, dispersed BRCA1 from its domains. In contrast, infection with adenovirus 5 recruited BRCA1 to regions of viral transcription and replication. These disparate distributions of BRCA1 may provide clues to its function.

Non-apoptotic chromosome condensation induced by stress: delineation of interchromosomal spaces.

Chromosomes are known to occupy distinct territories, suggesting the existence of definite borders. Visualization of these borders requires chromatin condensation like that seen in prophase cells. We developed a novel method to induce chromosome condensation in all cells regardless of cell cycle stage using a complex set of stresses. The cells were not apoptotic, as indicated by the absence of DNA damage, maintenance of the intact lamina and scaffold attachment factor A, and by the continuation of metabolic processes as well as proliferative capacity. That the appearance of chromosome condensation did not represent a premature mitotic event was shown by the absence of fibrillarin and Ki67 envelopment of chromosomes, continued protein synthesis and the reversibility of chromosome condensation. That chromosome condensation was achieved was demonstrated by the removal of chromatin from the nuclear envelope and chromosome painting. Specific genetic sites known to be at the surface of chromosomes retained their positions as shown by in situ hybridization. Stress-induced chromosome condensation was used to prove that specific nuclear domains such as ND10 are interchromosomally located and that green fluorescent protein-tagged ND10-associated proteins are useful markers for chromosomal boundaries after adenovirus 5 track formation in vivo. From these observations we conclude that chromosomal territories appear to have boundaries that exclude developing macromolecular aggregates.

Nuclear domain 10 (ND10) associated proteins are also present in nuclear bodies and redistribute to hundreds of nuclear sites after stress.

The promyelocytic leukemia protein fused to the retinoic acid receptor alpha in t(15;17) acute promyelocytic leukemia, the primary biliary cirrhosis autoantigen, Sp100, as well as the incompletely characterized protein NDP55, are co-localized in specific immunohistochemically defined nuclear domains (ND10), which are potential equivalents of ultrastructurally defined nuclear bodies. We investigated whether the distribution of these proteins depends on environmental conditions and whether ND10 correlate with nuclear bodies. Certain nuclear bodies and ND10 react in a similar way and share antigens. Interferon exposure doubled the number of ND10 and increased the frequency of nuclear bodies, whereas herpes simplex virus infection or heat shock modify both. Redistribution of ND10-associated proteins to hundreds of small sites throughout the chromatin was inducible by stress in the form of heat shock and exposure to Cd++ ions. The change of distribution was rapid and independent of protein synthesis, and thus not part of the classical heat shock response. The very rapid redistribution of these proteins after heat shock, together with the development of ND10 upon interferon activation, raises the possibility that ND10 represent storage sites of certain matrix proteins readily accessible throughout the chromatin in response to stress or other effectors that induce global nuclear changes.

Adenovirus replication is coupled with the dynamic properties of the PML nuclear structure.

Wild-type PML and at least four other novel proteins are localized within discrete nuclear structures known as PODs. We demonstrate here that during adenovirus infection, immediate early viral proteins from the E1 and E4 transcription units associate with the POD, which in turn undergoes a dramatic morphological change. During this process, the auto-antigen Sp-100 and NDP55 but not PML, relocate from the POD to the viral inclusion bodies, the sites of adenovirus DNA replication and late RNA transcription. The E4-ORF3 11-kD protein alone will induce this reorganization and reciprocally, viruses carrying mutations in the E4-domain fail to do so. These same viral mutants are defective in viral replication as well as the accumulation of late viral mRNAs and host cell transcription shutoff. We show that interferon (INF) treatment enhances the expression of PML, reduces or blocks PODs reorganization, and inhibits BrdU incorporation into viral inclusion bodies. In addition, cell lines engineered to overexpress PML prevent PODs from viral-induced reorganization and block or severely delay adenovirus replication. These results suggest that viral replication relies on components of the POD and that the structure is a target of early viral proteins.