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The ADP ribosyl transferase [poly(ADP-ribose) polymerase] ARTD3(PARP3) is a newly characterized member of the ARTD(PARP) family that catalyzes the reaction of ADP ribosylation, a key posttranslational modification of proteins involved in different signaling pathways from DNA damage to energy metabolism and organismal memory. This enzyme shares high structural similarities with the DNA repair enzymes PARP1 and PARP2 and accordingly has been found to catalyse poly(ADP ribose) synthesis. However, relatively little is known about its in vivo cellular properties. By combining biochemical studies with the generation and characterization of loss-of-function human and mouse models, we describe PARP3 as a newcomer in genome integrity and mitotic progression. We report a particular role of PARP3 in cellular response to double-strand breaks, most likely in concert with PARP1. We identify PARP3 as a critical player in the stabilization of the mitotic spindle and in telomere integrity notably by associating and regulating the mitotic components NuMA and tankyrase 1. Both functions open stimulating prospects for specifically targeting PARP3 in cancer therapy.

International audience; The nerve growth factor (NGF)-tyrosine kinase receptor TrkA plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation, and proliferation. In breast cancer cells, TrkA stimulation results in the activation of cellular growth, but downstream signaling largely remains to be described. Here we used a proteomics-based approach to identify partners involved in TrkA signaling in breast cancer cells. Wild type and modified TrkA chimeric constructs with green fluorescent protein were transfected in MCF-7 cells, and co-immunoprecipitated proteins were separated by SDS-PAGE before nano-LC-MS/MS analysis. Several TrkA putative signaling partners were identified among which was the DNA repair protein Ku70, which is increasingly reported for its role in cell survival and carcinogenesis. Physiological interaction of Ku70 with endogenous TrkA was induced upon NGF stimulation in non-transfected cells, and co-localization was observed with confocal microscopy. Mass spectrometry analysis and Western blotting of phosphotyrosine immunoprecipitates demonstrated the induction of Ku70 tyrosine phosphorylation upon NGF stimulation. Interestingly no interaction between TrkA and Ku70 was detected in PC12 cells in the absence or presence of NGF, suggesting that it is not involved in the initiation of neuronal differentiation. In breast cancer cells, RNA interference indicated that whereas Ku70 depletion had no direct effect on cell survival, it induced a strong potentiation of apoptosis in TrkA-overexpressing cells. In conclusion, TrkA signaling appears to be proapoptotic in the absence of Ku70, and this protein might therefore play a role in the long time reported ambivalence of tyrosine kinase receptors that can exhibit both anti- and eventually proapoptotic activities.

Parp-1 and Parp-2 are activated by DNA breaks and have been implicated in the repair of DNA single-strand breaks (SSB). Their involvement in double-strand break (DSB) repair mediated by homologous recombination (HR) or nonhomologous end joining (NHEJ) remains unclear. We addressed this question using chicken DT40 cells, which have the advantage of carrying only a PARP-1 gene but not a PARP-2 gene. We found that PARP-1(-/-) DT40 mutants show reduced levels of HR and are sensitive to various DSB-inducing genotoxic agents. Surprisingly, this phenotype was strictly dependent on the presence of Ku, a DSB-binding factor that mediates NHEJ. PARP-1/KU70 double mutants were proficient in the execution of HR and displayed elevated resistance to DSB-inducing drugs. Moreover, we found deletion of Ligase IV, another NHEJ gene, suppressed the camptothecin of PARP-1(-/-) cells. Our results suggest a new critical function for Parp in minimizing the suppressive effects of Ku and the NHEJ pathway on HR.

International audience; DNA double-stranded breaks (DSBs) can be repaired by several mechanisms, including classical NHEJ (c-NHEJ) and a poorly defined, error-prone process termed alternative NHEJ (a-NHEJ). How cells choose between these alternatives to join physiologic DSBs remains unknown. Here, we show that deletion of RAG2's C-terminus allows a-NHEJ to repair RAG-mediated DSBs in developing lymphocytes from both c-NHEJ-proficient and c-NHEJ-deficient mice, demonstrating that the V(D)J recombinase influences repair pathway choice in vivo. Analysis of V(D)J junctions revealed that, contrary to expectation, junctional characteristics alone do not reliably distinguish between a-NHEJ and c-NHEJ. These data suggest that a-NHEJ is not necessarily mutagenic, and may be more prevalent than previously appreciated. Whole genome sequencing of a lymphoma arising in a p53(-/-) mouse bearing a C-terminal RAG2 truncation reveals evidence of a-NHEJ and also of aberrant recognition of DNA sequences resembling RAG recognition sites.

International audience; Recent genome-wide RNAi screens have identified >842 human genes that affect the human immunodeficiency virus (HIV) cycle. The list of genes implicated in infection differs between screens, and there is minimal overlap. A reason for this variance is the interdependence of HIV infection and host cell function, producing a multitude of indirect or pleiotropic cellular effects affecting the viral infection during RNAi screening. To overcome this, the authors devised a 15-dimensional phenotypic profile to define the viral infection block induced by CD4 silencing in HeLa cells. They demonstrate that this phenotypic profile excludes nonspecific, RNAi-based side effects and viral replication defects mediated by silencing of housekeeping genes. To achieve statistical robustness, the authors used automatically annotated RNAi arrays for seven independent genome-wide RNAi screens. This identified 56 host genes, which reliably reproduced CD4-like phenotypes upon HIV infection. The factors include 11 known HIV interactors and 45 factors previously not associated with HIV infection. As proof of concept, the authors confirmed that silencing of PAK1, Ku70, and RNAseH2A impaired HIV replication in Jurkat cells. In summary, multidimensional, visual profiling can identify genes required for HIV infection.

International audience; The H/ACA RNAs represent an abundant, evolutionarily conserved and functionally diverse class of non-coding RNAs. Many H/ACA RNAs direct pseudouridylation of rRNAs and snRNAs, while members of the rapidly growing group of 'orphan' H/ACA RNAs participate in pre-rRNA processing, telomere synthesis and probably, in other nuclear processes. The yeast snR30 'orphan' H/ACA snoRNA has long been known to function in the nucleolytic processing of 18S rRNA, but its molecular role remained unknown. Here, we provide biochemical and genetic evidence demonstrating that during pre-rRNA processing, two evolutionarily conserved sequence elements in the 3'-hairpin of snR30 base-pair with short pre-rRNA sequences located in the eukaryote-specific internal region of 18S rRNA. The newly discovered snR30-18S base-pairing interactions are essential for 18S rRNA production and they constitute a complex snoRNA target RNA transient structure that is novel to H/ACA RNAs. We also demonstrate that besides the 18S recognition motifs, the distal part of the 3'-hairpin of snR30 contains an additional snoRNA element that is essential for 18S rRNA processing and that functions most likely as a snoRNP protein-binding site.

International audience; The assembly of functional immune receptor genes via V(D)J recombination in developing lymphocytes generates DNA double-stranded breaks intermediates that are repaired by non-homologous end joining (NHEJ). This repair pathway requires the sequential recruitment and activation onto coding and signal DNA ends of several proteins, including the DNA-dependent protein kinase and the nuclease Artemis. Artemis activity, triggered by the DNA-dependent protein kinase, is necessary to process the genes hairpin-sealed coding ends but appears dispensable for the ligation of the reciprocal phosphorylated, blunt-ended signal ends into a signal joint. The DNA-dependent protein kinase is however present on signal ends and could potentially recruit and activate Artemis during signal joint formation. To determine whether Artemis plays a role during the resolution of signal ends during V(D)J recombination, we analyzed the structure of signal joints generated in developing thymocytes during the rearrangement of T cell receptor genes in wild type mice and mice mutated for NHEJ factors. These joints exhibit junctional diversity resulting from N nucleotide polymerization by the terminal nucleotidyl transferase and nucleotide loss from one or both of the signal ends before they are ligated. Our results show that Artemis participates in the repair of signal ends in vivo. Furthermore, our results also show that while the DNA-dependent protein kinase complex protects signal ends from processing, including deletions, Artemis seems on the opposite to promote their accessibility to modifying enzymes. In addition, these data suggest that Artemis might be the nuclease responsible for nucleotide loss from signal ends during the repair process.

International audience; Little information is available on the ability of terminally differentiated cells to efficiently repair DNA double strand breaks (DSBs), and one might reasonably speculate that efficient DNA repair of these threatening DNA lesions, is needed in cells of long life span with no or limited regeneration from precursor. Few tissues are available besides neurons that allow the study of DNA DSBs repair activity in very long-lived cells. Adipocytes represent a suitable model since it is generally admitted that there is a very slow turnover of adipocytes in adult. Using both Pulse Field Gel Electrophoresis (PFGE) and the disappearance of the phosphorylated form of the histone variant H2AX, we demonstrated that the ability to repair DSBs is increased during adipocyte differentiation using the murine pre-adipocyte cell line, 3T3F442A. In mammalian cells, DSBs are mainly repaired by the non-homologous end-joining pathway (NHEJ) that relies on the DNA dependent protein kinase (DNA-PK) activity. During the first 24 h following the commitment into adipogenesis, we show an increase in the expression and activity of the catalytic sub-unit of the DNA-PK complex, DNA-PKcs. The increased in DNA DSBs repair activity observed in adipocytes was due to the increase in DNA-PK activity as shown by the use of DNA-PK inhibitor or sub-clones of 3T3F442A deficient in DNA-PKcs using long term RNA interference. Interestingly, the up-regulation of DNA-PK does not regulate the differentiation program itself. Finally, similar positive regulation of DNA-PKcs expression and activity was observed during differentiation of primary culture of pre-adipocytes isolated from human sub-cutaneous adipose tissue.Our results show that DNA DSBs repair activity is up regulated during the early commitment into adipogenesis due to an up-regulation of DNA-PK expression and activity. In opposition to the general view that DNA DSBs repair is decreased during differentiation, our results demonstrate that an up-regulation of this process might be observed in post-mitotic long-lived cells.

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.

Free in PubMed Central : http://www.pubmedcentral.nih.gov/picrender.fcgi?tool=pmcentrez&artid=1951289&blobtype=pdf; The heterodimer Ku70/80 Ku is the DNA-binding component of the DNA-PK complex required for the nonhomologous end-joining pathway. It participates in numerous nuclear processes, including telomere and chromatin structure maintenance, replication, and transcription. Ku interacts with retroviral preintegration complexes and is thought to interfere with the retroviral replication cycle, in particular the formation of 2-long terminal repeat (LTR) viral DNA circles, viral DNA integration, and transcription. We describe here the effect of Ku80 on both provirus integration and the resulting transgene expression in cells transduced with retroviral vectors. We found that transgene expression was systematically higher in Ku80-deficient xrs6 cells than in Ku80-expressing CHO cells. This higher expression was observed irrespective of the presence of the viral LTR and was also not related to the nature of the promoter. Real-time PCR monitoring of the early viral replicative steps demonstrated that the absence of Ku80 does not affect the efficiency of transduction. We analyzed the transgene distributions localization in nucleus by applying a three-dimensional reconstruction model to two-dimensional fluorescence in situ hybridization images. This indicated that the presence of Ku80 resulted in a bias toward the transgenes being located at the periphery of the nucleus associated with their being repressed; in the absence of this factor the transgenes tend to be randomly distributed and actively expressed. Therefore, although not strictly required for retroviral integration, Ku may be involved in targeting retroviral elements to chromatin domains prone to gene silencing.

Poly(ADP-ribose) polymerase 3 (PARP-3) is a novel member of the PARP family of enzymes that synthesize poly(ADP-ribose) on themselves and other acceptor proteins. Very little is known about this PARP, which is closely related to PARP-1 and PARP-2. By sequence analysis, we find that PARP-3 may be expressed in two isoforms which we studied in more detail to gain insight into their possible functions. We find that both PARP-3 isoforms, transiently expressed as GFP or FLAG fusions, are nuclear. Detection of endogenous PARP-3 with a specific antibody also shows a widespread nuclear distribution, appearing in numerous small foci and a small number of larger foci. Through co-localization experiments and immunoprecipitations, the larger nuclear foci were identified as Polycomb group bodies (PcG bodies) and we found that PARP-3 is part of Polycomb group protein complexes. Furthermore, using a proteomics approach, we determined that both PARP-3 isoforms are part of complexes comprising DNA-PKcs, PARP-1, DNA ligase III, DNA ligase IV, Ku70, and Ku80. Our findings suggest that PARP-3 is a nuclear protein involved in transcriptional silencing and in the cellular response to DNA damage. J. Cell. Biochem. 100: 385–401, 2007. © 2006 Wiley-Liss, Inc.

In Agrobacterium -mediated genetic transformation of plant cells, the bacterium exports a well defined transferred DNA (T-DNA) fragment and a series of virulence proteins into the host cell. Following its nuclear import, the single-stranded T-DNA is stripped of its escorting proteins, most likely converts to a double-stranded (ds) form, and integrates into the host genome. Little is known about the precise mechanism of T-DNA integration in plants, and no plant proteins specifically associated to T-DNA have been identified. Here we report the direct involvement of KU80, a protein that binds dsT-DNA intermediates. We show that ku80 -mutant Arabidopsis plants are defective in T-DNA integration in somatic cells, whereas KU80-overexpressing plants exhibit increased susceptibility to Agrobacterium infection and increased resistance to DNA-damaging agents. The direct interaction between dsT-DNA molecules and KU80 in planta was confirmed by immunoprecipitation of KU80 dsT-DNA complexes from Agrobacterium -infected plants. Transformation of KU80-overexpressing plants with two separate T-DNA molecules resulted in an increased rate of extrachromosomal T-DNA to T-DNA recombination, indicating that KU80 bridges between dsT-DNAs and double-strand breaks. This last result further supports the notion that integration of T-DNA molecules occurs through ds intermediates and requires active participation of the host's nonhomologous end-joining repair machinery.

International audience; In this study, we investigated the subcellular and molecular mechanisms underlying promyelocytic leukemia (PML)-induced premature senescence. We demonstrate that intact PML nuclear bodies are not required for the induction of senescence. We have determined further that of seven known PML isoforms, only PML IV is capable of causing premature senescence, providing the first evidence for functional differences among these isoforms. Of interest is the fact that in contrast to PML(+/+) fibroblasts, PML(-/-) cells are resistant to PML IV-induced senescence. This suggests that although PML IV is necessary for this process to occur, it is not sufficient and requires other components for activity. Finally, we provide evidence that PML IV-induced senescence involves stabilization and activation of p53 through phosphorylation at Ser46 and acetylation at Lys382, and that it occurs independently of telomerase and differs from that elicited by oncogenic Ras. Taken together, our data assign a specific pro-senescent activity to an individual PML isoform that involves p53 activation and is independent from PML nuclear bodies.

International audience; Posttranslational modification with SUMO1 regulates protein/protein interactions, localization, and stability. SUMOylation requires the E1 enzyme Aos1/Uba2 and the E2 enzyme Ubc9. A family of E3-like factors, PIAS proteins, was discovered recently. Here we show that the nucleoporin RanBP2/Nup358 also has SUMO1 E3-like activity. RanBP2 directly interacts with the E2 enzyme Ubc9 and strongly enhances SUMO1-transfer from Ubc9 to the SUMO1 target Sp100. The E3-like activity is contained within a 33 kDa domain of RanBP2 that lacks RING finger motifs and does not resemble PIAS family proteins. Our findings place SUMOylation at the cytoplasmic filaments of the NPC and suggest that, at least for some substrates, modification and nuclear import are linked events.

Article en ligne : http://www.jbc.org/cgi/content/full/277/7/4918; International audience; Ku has been implicated in nuclear processes, including DNA break repair, transcription, V(D)J recombination, and telomere maintenance. Its mode of action involves two distinct mechanisms: one in which a nonspecific binding occurs to DNA ends and a second that involves a specific binding to negative regulatory elements involved in transcription repression. Such elements were identified in mouse mammary tumor virus and human T cell leukemia virus retroviruses. The purpose of this study was to investigate a role for Ku in the regulation of human immunodeficiency virus (HIV)-1 transcription. First, HIV-1 LTR activity was studied in CHO-K1 cells and in CH0-derived xrs-6 cells, which are devoid of Ku80. LTR-driven expression of a reporter gene was significantly increased in xrs-6 cells. This enhancement was suppressed after re-expression of Ku80. Second, transcription of HIV-1 was followed in U1 human cells that were depleted in Ku by using a Ku80 antisense RNA. Ku depletion led to a increase of both HIV-1 mRNA synthesis and viral production compared with the parent cells. These results demonstrate that Ku acts as a transcriptional repressor of HIV-1 expression. Finally, a putative Ku-specific binding site was identified within the negative regulatory region of the HIV-1 long terminal repeat, which may account for this repression of transcription.

International audience; SUMO belongs to a growing number of ubiquitin-like proteins that covalently modify their target proteins. Although some evidence supports a role of SUMO modification in regulating protein stability, most studied examples support a model by which SUMO alters the interaction properties of its targets, often affecting their subcellular localization behavior. Examination of the PML nuclear bodies, whose principal components are SUMO-modified, has revealed this modification to be essential for their structural and functional integrity. This and other examples thus support the view that SUMO regulates the stability not of individual proteins, but rather that of entire multiprotein complexes.

International audience; The SP100 protein, together with PML, represents a major constituent of the PML-SP100 nuclear bodies (NBs). The function of these ubiquitous subnuclear structures, whose integrity is compromised in pathological situations such as acute promyelocytic leukemia (APL) or DNA virus infection, remains poorly understood. There is little evidence for the occurrence of actual physiological processes within NBs. The two NB proteins PML and SP100 are covalently modified by the ubiquitin-related SUMO-1 modifier, and recent work indicates that this modification is critical for the regulation of NB dynamics. In exploring the functional relationships between NBs and chromatin, we have shown previously that SP100 interacts with members of the HP1 family of nonhistone chromosomal proteins and that a variant SP100 cDNA encodes a high-mobility group (HMG1/2) protein. Here we report the isolation of a further cDNA, encoding the SP100C protein, that contains the PHD-bromodomain motif characteristic of chromatin proteins. We further show that TIF1α, a chromatin-associated factor with homology to both PML and SP100C, is also modified by SUMO-1. Finally, in vitro experiments indicate that SUMO modification of SP100 enhances the stability of SP100-HP1 complexes. Taken together, our results suggest an association of SP100 and its variants with the chromatin compartment and, further, indicate that SUMO modification may play a regulatory role in the functional interplay between the nuclear bodies and chromatin.

Background The relationship between the population doubling time and the quantity of silver-stained nucleolar organizer region (AgNOR) interphase proteins was studied in cell culture at three different temperatures used to modulate the cell cycle duration. Methods After MIB 1 and AgNOR combined staining, the quantity of AgNOR proteins was measured in cycling cells by image cytometry. Results Among the several parameters calculated, the AgNOR relative area showed a strong correlation with the changes of the population doubling time induced by different temperatures. Conclusions The results support the hypothesis that the cell cycle time and the size of the ribogenesis machinery are coregulated and that measurements of AgNORs can thus be used as a static evaluation of the cell cycle duration in arbitrary units. Cytometry 43:110–116, 2001. © 2001 Wiley-Liss, Inc.

AgNORs are nucleolar proteins that interact specifically with silver salts. The size of silver precipitates measured by image analysis (ICM) in cycling cells proved to be inversely proportional to the cell cycle time and provided a significant correlation with prognosis for a large spectrum of cancers. Because ICM is time-consuming and poorly reproducible among laboratories using different imaging settings, this article presents a new approach to AgNOR quantitation based on flow cytometry (FCM). We report that silver precipitates caused a great decrease in the forward scattered light and that this effect was correlated with the AgNOR's relative area as measured by ICM. These results were confirmed by measuring cell lines having different cell cycle durations. Moreover, double staining using APase–Fast red fluorescence to reveal the Ki-67/MIB 1 antigen of cycling cells and silver nitrate to stain the AgNORs was successfully analyzed by FCM. The procedure makes it possible, for the first time, to validly and rapidly compare the growth fraction and cycling speed of partially proliferating cell populations, such as tumors.

International audience; The Saccharomyces cerevisiae CDC13 protein binds single-strand telomeric DNA. Here we report the isolation of new mutant alleles of CDC13 that confer either abnormal telomere lengthening or telomere shortening. This deregulation not only depended on telomerase (Est2/TLC1) and Est1, a direct regulator of telomerase, but also on the yeast Ku proteins, yKu70/Hdf1 and yKu80/Hdf2, that have been previously implicated in DNA repair and telomere maintenance. Expression of a Cdc13-yKu70 fusion protein resulted in telomere elongation, similar to that produced by a Cdc13-Est1 fusion, thus suggesting that yKu70 might promote Cdc13-mediated telomerase recruitment. We also demonstrate that Stn1 is an inhibitor of telomerase recruitment by Cdc13, based both on STN1 overexpression and Cdc13-Stn1 fusion experiments. We propose that accurate regulation of telomerase recruitment by Cdc13 results from a coordinated balance between positive control by yKu70 and negative control by Stn1. Our results represent the first evidence of a direct control of the telomerase-loading function of Cdc13 by a double-strand telomeric DNA-binding complex.

International audience; The functional regulation of chromatin is closely related to its spatial organization within the nucleus. In yeast, perinuclear chromatin domains constitute areas of transcriptional repression. These 'silent' domains are defined by the presence of perinuclear telomere clusters. The only protein found to be involved in the peripheral localization of telomeres is Yku70/Yku80. This conserved heterodimer can bind telomeres and functions in both repair of DNA double-strand breaks and telomere maintenance. These findings, however, do not address the underlying structural basis of perinuclear silent domains. Here we show that nuclear-pore-complex extensions formed by the conserved TPR homologues Mlp1 and Mlp2 are responsible for the structural and functional organization of perinuclear chromatin. Loss of MLP2 results in a severe deficiency in the repair of double-strand breaks. Furthermore, double deletion of MLP1 and MLP2 disrupts the clustering of perinuclear telomeres and releases telomeric gene repression. These effects are probably mediated through the interaction with Yku70. Mlp2 physically tethers Yku70 to the nuclear periphery, thus forming a link between chromatin and the nuclear envelope. We show, moreover, that this structural link is docked to nuclear-pore complexes through a cleavable nucleoporin, Nup145. We propose that, through these interactions, nuclear-pore complexes organize a nuclear subdomain that is intimately involved in the regulation of chromatin metabolism.

International audience; Ku70, a regulatory component of the DNA-dependent protein kinase, was identified by a yeast two-hybrid screen of a B lymphocyte cDNA library as a partner of p40phox, a regulatory component of the O2--producing NADPH oxidase. Truncated constructs of p40phox and Ku70 were used to map the interacting sites. The 186 C-terminal amino acids (aa) of Ku70 were found to interact with two distinct regions of p40phox, the central core region (aa 50-260) and the C-terminal extremity (aa 260-339). In complementary experiments, it was observed that Ku70 binds to immobilized recombinant p40phox fusion protein and that p40phox and Ku70 from a B lymphocyte cell extract comigrate in successive chromatographies on Q Separose, Superose 12 and hydroxylapatite columns. Moreover, we report that Ku70 and p40phox colocalize in B lymphocytes and in transfected Cos-7 cells. We also show that the two NADPH oxidase activating factors, p47phox and p67phox are substrates for DNA-PK in vitro and that they are present together with p40phox in the nucleus of B cells. These results may help solve the paradox that the phox protein triad, p40phox, p47phox and p67phox, is expressed equally in B lymphocytes and neutrophils, whereas the redox component of the NADPH oxidase, a flavocytochrome b, which is well expressed in neutrophils, is barely detectable in B lymphocytes.

International audience; The PML/SP100 nuclear bodies (NBs) were first described as discrete subnuclear structures containing the SP100 protein. Subsequently, they were shown to contain the PML protein which is part of the oncogenic PML-RARalpha hybrid produced by the t(15;17) chromosomal translocation characteristic of acute promyelocytic leukemia. Yet, the physiological role of these nuclear bodies remains unknown. Here, we show that SP100 binds to members of the heterochromatin protein 1 (HP1) families of non-histone chromosomal proteins. Further, we demonstrate that a naturally occurring splice variant of SP100, here called SP100-HMG, is a member of the high mobility group-1 (HMG-1) protein family and may thus possess DNA-binding potential. Both HP1 and SP100-HMG concentrate in the PML/SP100 NBs, and overexpression of SP100 leads to enhanced accumulation of endogenous HP1 in these structures. When bound to a promoter, SP100, SP100-HMG and HP1 behave as transcriptional repressors in transfected mammalian cells. These observations present molecular evidence for an association between the PML/SP100 NBs and the chromatin nuclear compartment. They support a model in which the NBs may play a role in certain aspects of chromatin dynamics.

The tumor-suppressive promyelocytic leukemia (PML) protein of acute promyelocytic leukemia (APL) has served as one of the defining components of a class of distinctive nuclear bodies (NBs). PML is delocalized from NBs in APL cells and is degraded in cells infected by several viruses. In these cells, NBs are disrupted, leading to the aberrant localization of NB proteins. These results have suggested a critical role for the NB in immune response and tumor suppression and raised the question of whether PML is crucial for the formation or stability of NB. In addition, PML is, among other proteins, covalently modified by SUMO-1. However, the functional relevance of this modification is unclear. Here, we show in primary PML(-/-) cells of various histologic origins, that in the absence of PML, several NB proteins such as Sp100, CBP, ISG20, Daxx, and SUMO-1 fail to accumulate in the NB and acquire aberrant localization patterns. Transfection of PML in PML(-/-) cells causes the relocalization of NB proteins. By contrast, a PML mutant that can no longer be modified by SUMO-1 fails to do so and displays an aberrant nuclear localization pattern. Therefore, PML is required for the proper formation of the NB. Conjugation to SUMO-1 is a prerequisite for PML to exert this function. These data shed new light on both the mechanisms underlying the formation of the NBs and the pathogenesis of APL. (Blood. 2000;95:2748-2752)

PML nuclear bodies (NBs) are subnuclear structures whose integrity is compromised in certain human diseases, including leukemia and neurodegenerative disorders. Infection by a number of DNA viruses similarly triggers the reorganization of these structures, suggesting an important role for the NBs in the viral infection process. While expression of the adenovirus E4 ORF3 protein leads to only a moderate redistribution of PML to filamentous structures, the herpes simplex virus (HSV) ICP0 protein and the cytomegalovirus (CMV) IE1 protein both induce a complete disruption of the NB structure. Recently, we and others have shown that the NB proteins PML and Sp100 are posttranslationally modified by covalent linkage with the ubiquitin-related SUMO-1 protein and that this modification may promote the assembly of these structures. Here we show that the HSV ICP0 and CMV IE1 proteins specifically abrogate the SUMO-1 modification of PML and Sp100, whereas the adenovirus E4 ORF3 protein does not affect this process. The potential of ICP0 and IE1 to alter SUMO-1 modification is directly linked to their capacity to disassemble NBs, thus strengthening the role for SUMO-1 conjugation in maintenance of the structural integrity of the NBs. This observation supports a model in which ICP0 and IE1 disrupt the NBs either by preventing the formation or by degrading of the SUMO-1-modified PML and Sp100 protein species. Finally, we show that the IE1 protein itself is a substrate for SUMO-1 modification, thus representing the first viral protein found to undergo this new type of posttranslational modification.