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2.
Mol Biol Cell ; 24(9): 1454-68, 2013 May.
Article in English | MEDLINE | ID: mdl-23485562

ABSTRACT

Promyelocytic leukemia nuclear bodies (PML-NBs)/nuclear domain 10s (ND10s) are nuclear structures that contain many transcriptional and chromatin regulatory factors. One of these, Sp100, is expressed from a single-copy gene and spliced into four isoforms (A, B, C, and HMG), which differentially regulate transcription. Here we evaluate Sp100 function in single cells using an inducible cytomegalovirus-promoter-regulated transgene, visualized as a chromatinized transcription site. Sp100A is the isoform most strongly recruited to the transgene array, and it significantly increases chromatin decondensation. However, Sp100A cannot overcome Daxx- and α-thalassemia mental retardation, X-linked (ATRX)-mediated transcriptional repression, which indicates that PML-NB/ND10 factors function within a regulatory hierarchy. Sp100A increases and Sp100B, which contains a SAND domain, decreases acetyl-lysine regulatory factor levels at activated sites, suggesting that Sp100 isoforms differentially regulate transcription by modulating lysine acetylation. In contrast to Daxx, ATRX, and PML, Sp100 is recruited to activated arrays in cells expressing the herpes simplex virus type 1 E3 ubiquitin ligase, ICP0, which degrades all Sp100 isoforms except unsumoylated Sp100A. The recruitment Sp100A(K297R), which cannot be sumoylated, further suggests that sumoylation plays an important role in regulating Sp100 isoform levels at transcription sites. This study provides insight into the ways in which viruses may modulate Sp100 to promote their replication cycles.


Subject(s)
Antigens, Nuclear/metabolism , Autoantigens/metabolism , Chromatin Assembly and Disassembly , Cytomegalovirus/physiology , Promoter Regions, Genetic , Acetylation , Adaptor Proteins, Signal Transducing/metabolism , Co-Repressor Proteins , DNA Helicases/metabolism , Epigenesis, Genetic , HeLa Cells , Humans , Molecular Chaperones , Nuclear Proteins/metabolism , Promyelocytic Leukemia Protein , Protein Isoforms/metabolism , Protein Transport , Proteolysis , Sumoylation , Transcription Factors/metabolism , Transcription Initiation Site , Transcription, Genetic , Tumor Suppressor Proteins/metabolism , Virus Latency , X-linked Nuclear Protein
3.
J Cell Sci ; 125(Pt 22): 5489-501, 2012 Nov 15.
Article in English | MEDLINE | ID: mdl-22976303

ABSTRACT

Histone H3.3 is a constitutively expressed H3 variant implicated in the epigenetic inheritance of chromatin structures. Recently, the PML-nuclear body (PML-NB)/Nuclear Domain 10 (ND10) proteins, Daxx and ATRX, were found to regulate replication-independent histone H3.3 chromatin assembly at telomeres and pericentric heterochromatin. As it is not completely understood how PML-NBs/ND10s regulate transcription and resistance to viral infection, we have used a CMV-promoter-regulated inducible transgene array, at which Daxx and ATRX are enriched, to delineate the mechanisms through which they regulate transcription. When integrated into HeLa cells, which express both Daxx and ATRX, the array is refractory to activation. However, transcription can be induced when ICP0, the HSV-1 E3 ubiquitin ligase required to reverse latency, is expressed. As ATRX and Daxx are depleted from the activated array in ICP0-expressing HeLa cells, this suggests that they are required to maintain a repressed chromatin environment. As histone H3.3 is strongly recruited to the ICP0-activated array but does not co-localize with the DNA, this also suggests that chromatin assembly is blocked during activation. The conclusion that the Daxx and ATRX pathway is required for transcriptional repression and chromatin assembly at this site is further supported by the finding that an array integrated into the ATRX-negative U2OS cell line can be robustly activated and that histone H3.3 is similarly recruited and unincorporated into the chromatin. Therefore, this study has important implications for understanding gene silencing, viral latency and PML-NB/ND10 function.


Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , DNA Helicases/metabolism , Nuclear Proteins/metabolism , Repressor Proteins/metabolism , Single-Cell Analysis/methods , Transcription, Genetic , Cell Line, Tumor , Chromatin/metabolism , Co-Repressor Proteins , Cytomegalovirus/genetics , DNA Helicases/chemistry , HeLa Cells , Histones/metabolism , Humans , Molecular Chaperones , Nuclear Proteins/chemistry , Promoter Regions, Genetic/genetics , Protein Structure, Tertiary , Transcriptional Activation/genetics , Transgenes , X-linked Nuclear Protein
4.
Cancer Res ; 70(23): 9991-10001, 2010 Dec 01.
Article in English | MEDLINE | ID: mdl-21118961

ABSTRACT

Identifying the functions of proteins, which associate with specific subnuclear structures, is critical to understanding eukaryotic nuclear dynamics. Sp100 is a prototypical protein of ND10/PML nuclear bodies, which colocalizes with Daxx and the proto-oncogenic PML. Sp100 isoforms contain SAND, PHD, Bromo, and HMG domains and are highly sumoylated, all characteristics suggestive of a role in chromatin-mediated gene regulation. A role for Sp100 in oncogenesis has not been defined previously. Using selective Sp100 isoform-knockdown approaches, we show that normal human diploid fibroblasts with reduced Sp100 levels rapidly senesce. Subsequently, small rapidly dividing Sp100 minus cells emerge from the senescing fibroblasts and are found to be highly tumorigenic in nude mice. The derivation of these tumorigenic cells from the parental fibroblasts is confirmed by microsatellite analysis. The small rapidly dividing Sp100 minus cells now also lack ND10/PML bodies, and exhibit genomic instability and p53 cytoplasmic sequestration. They have also activated MYC, RAS, and TERT pathways and express mesenchymal to epithelial transdifferentiation (MET) markers. Reintroduction of expression of only the Sp100A isoform is sufficient to maintain senescence and to inhibit emergence of the highly tumorigenic cells. Global transcriptome studies, quantitative PCR, and protein studies, as well as immunolocalization studies during the course of the transformation, reveal that a transient expression of stem cell markers precedes the malignant transformation. These results identify a role for Sp100 as a tumor suppressor in addition to its role in maintaining ND10/PML bodies and in the epigenetic regulation of gene expression.


Subject(s)
Antigens, Nuclear/genetics , Autoantigens/genetics , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Tumor Suppressor Proteins/genetics , Animals , Antigens, Nuclear/metabolism , Autoantigens/metabolism , Blotting, Western , Cell Transformation, Neoplastic/genetics , Cells, Cultured , Cellular Senescence/genetics , Epithelial-Mesenchymal Transition/genetics , Fibroblasts/cytology , Gene Expression Profiling , HEK293 Cells , Humans , Male , Mice , Mice, Nude , Neoplasms, Experimental/genetics , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Nuclear Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Promyelocytic Leukemia Protein , Proto-Oncogene Proteins c-myc/metabolism , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factors/metabolism , Transplantation, Heterologous , Tumor Suppressor Proteins/metabolism , ras Proteins/metabolism
5.
Proc Natl Acad Sci U S A ; 107(7): 2938-43, 2010 Feb 16.
Article in English | MEDLINE | ID: mdl-20133701

ABSTRACT

Corepressors play an essential role in nuclear receptor-mediated transcriptional repression. In general, corepressors directly bind to nuclear receptors via CoRNR boxes (L/I-X-X-I/V-I) in the absence of ligand and appear to act as scaffolds to further recruit chromatin remodeling complexes to specific target genes. Here, we describe the identification of the multiple LIM domain protein Ajuba as a unique corepressor for a subset of nuclear hormone receptors. Ajuba contains functional nuclear-receptor interacting motifs and selectively interacts with retinoic acid receptors (RARs) and rexinoid receptor (RXRs) subtypes in a ligand-dependent manner. Simultaneous mutation of these motifs abolishes RAR binding and concomitantly leads to loss of repression on RARE reporter genes. P19 cells depleted of Ajuba are highly sensitized to all-trans retinoic acid (atRA)-induced transcription and differentiation. In the absence of atRA, Ajuba can be readily found at the RARE control elements of RAR endogenous target genes. Stimulation of cells with atRA results in the dissociation of Ajuba from these regions. Moreover, we observed that coexpression of the known Ajuba binding partner Prmt5 (protein arginine methyltransferase-5) inhibited the Ajuba/RAR interaction. The high-affinity Ajuba-RAR/RXR interaction site overlaps the region responsible for Ajuba/Prmt5 binding, and thus binding appears to be mutually exclusive, providing a potential mechanism for these observations. Identification of Ajuba as a unique corepressor for nuclear receptors sheds new light on mechanisms for nuclear receptor-mediated repression and provides a unique target for developing more effective therapeutics to modulate this important pathway.


Subject(s)
Co-Repressor Proteins/metabolism , Gene Expression Regulation/physiology , Homeodomain Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction/physiology , Tretinoin/metabolism , Amino Acid Motifs/genetics , Amino Acid Motifs/physiology , Animals , Blotting, Western , Cell Line , Chromatin Immunoprecipitation , Humans , Immunoprecipitation , LIM Domain Proteins , Luciferases , Mice , Microscopy, Fluorescence , Protein Binding , Protein-Arginine N-Methyltransferases/metabolism , Receptors, Retinoic Acid/metabolism , Reverse Transcriptase Polymerase Chain Reaction
6.
J Virol ; 83(10): 5168-80, 2009 May.
Article in English | MEDLINE | ID: mdl-19279115

ABSTRACT

Cells have intrinsic defenses against virus infection, acting before the innate or the adaptive immune response. Preexisting antiviral proteins such as PML, Daxx, and Sp100 are stored in specific nuclear domains (ND10). In herpes simplex virus type 1 (HSV-1), the immediate-early protein ICP0 serves as a counterdefense through degradation of the detrimental protein PML. We asked whether interferon (IFN)-upregulated Sp100 is similarly antagonized by ICP0 in normal human fibroblasts by using a selective-knockdown approach. We find that of the four Sp100 isoforms, the three containing a SAND domain block the transcription of HSV-1 proteins ICP0 and ICP4 at the promoter level and that IFN changes the differential splicing of the Sp100 transcript in favor of the inhibitor Sp100C. At the protein level, ICP0 activity does not lead to the hydrolysis of any of the Sp100 isoforms. The SAND domain-containing isoforms are not general inhibitors of viral promoters, as the activity of the major immediate-early cytomegalovirus promoter is not diminished, whereas the long terminal repeat of a retrovirus, like the ICP0 promoter, is strongly inhibited. Since we could not find a specific promoter region in the ICP0 gene that responds to the SAND domain-containing isoforms, we questioned whether Sp100 could act through other antiviral proteins such as PML. We find that all four Sp100 isoforms stabilize ND10 and protect PML from ICP0-based hydrolysis. Loss of either all PML isoforms or all Sp100 isoforms reduces the opposite constituent ND10 protein, suggesting that various interdependent mechanisms of ND10-based proteins inhibit virus infection at the immediate-early level.


Subject(s)
Antigens, Nuclear/metabolism , Autoantigens/metabolism , Genes, Immediate-Early , Herpesvirus 1, Human/genetics , Immediate-Early Proteins/metabolism , Nuclear Proteins/metabolism , Transcription Factors/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Cell Line , Gene Expression Regulation, Viral , Gene Knockdown Techniques , Herpesvirus 1, Human/metabolism , Humans , Interferons/metabolism , Promoter Regions, Genetic , Promyelocytic Leukemia Protein , Protein Isoforms/metabolism , Protein Splicing , Transcription, Genetic , Up-Regulation
7.
Mol Cell ; 28(5): 823-37, 2007 Dec 14.
Article in English | MEDLINE | ID: mdl-18082607

ABSTRACT

Tandem PHD and bromodomains are often found in chromatin-associated proteins and have been shown to cooperate in gene silencing. Each domain can bind specifically modified histones: the mechanisms of cooperation between these domains are unknown. We show that the PHD domain of the KAP1 corepressor functions as an intramolecular E3 ligase for sumoylation of the adjacent bromodomain. The RING finger-like structure of the PHD domain is required for both Ubc9 binding and sumoylation and directs modification to specific lysine residues in the bromodomain. Sumoylation is required for KAP1-mediated gene silencing and functions by directly recruiting the SETDB1 histone methyltransferase and the CHD3/Mi2 component of the NuRD complex via SUMO-interacting motifs. Sumoylated KAP1 stimulates the histone methyltransferase activity of SETDB1. These data provide a mechanistic explanation for the cooperation of PHD and bromodomains in gene regulation and describe a function of the PHD domain as an intramolecular E3 SUMO ligase.


Subject(s)
DNA-Binding Proteins/genetics , Gene Expression Regulation , Gene Silencing , RING Finger Domains , Repressor Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Autoantigens/genetics , Autoantigens/metabolism , Bone Neoplasms/genetics , Bone Neoplasms/metabolism , Bone Neoplasms/pathology , Cells, Cultured , Chromatin/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/metabolism , Histone Deacetylases/genetics , Histone Deacetylases/metabolism , Histone-Lysine N-Methyltransferase , Humans , Kidney/metabolism , Lysine/chemistry , Mi-2 Nucleosome Remodeling and Deacetylase Complex , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Protein Methyltransferases/genetics , Protein Methyltransferases/metabolism , Protein Processing, Post-Translational , Repressor Proteins/antagonists & inhibitors , Repressor Proteins/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Transcription, Genetic , Tripartite Motif-Containing Protein 28 , Two-Hybrid System Techniques , Ubiquitin-Conjugating Enzymes/genetics , Ubiquitin-Conjugating Enzymes/metabolism , Ubiquitin-Protein Ligases/genetics
8.
J Virol ; 80(16): 8019-29, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16873258

ABSTRACT

Nuclear domains called ND10 or PML nuclear bodies contain interferon (IFN)-upregulated proteins like PML and Sp100. Paradoxically, herpes simplex virus 1 (HSV-1) begins its transcriptional cascade at aggregates of ND10-associated proteins, which in turn are destroyed by the HSV-1 immediate-early protein ICP0. While PML is essential in the formation of ND10, the function of Sp100 in the cells' defense against viral infection is unknown. In this study we investigated the potential antiviral effect of IFN-beta-induced Sp100. We found that IFN-beta treatment leads to a differential accumulation of four Sp100 isoforms in different cell lines. Using an HEK293 cell line derivative, 293-S, producing no detectable amounts of Sp100 even after IFN exposure, we analyzed individual Sp100 isoforms for their effect on HSV-1 infection. Sp100 isoforms B, C, and HMG, but not Sp100A, suppressed ICP0 and ICP4 early after infection. Isoforms B, C, and HMG suppressed expression from the ICP0 promoter in transient transfection, whereas Sp100A enhanced expression. Moreover, Sp100A localized in ND10, whereas the repressive isoforms were either dispersed within the nucleus or, at unphysiologically higher expression levels, formed new aggregates. The repressive activity was dependent on an intact SAND domain, since Sp100B bearing a W655Q mutation in the SAND domain lost this repressive activity and accumulated in ND10. Using RNA interference to knock down the repressive Sp100 isoforms B, C, and HMG, we find that they are an essential part of the IFN-beta-mediated suppression of ICP0 expression. These data suggest that repression by the Sp100 isoforms B, C, and HMG takes place outside of ND10 and raise the possibility that viral genomes at Sp100A accumulations are more likely to start their transcription program because of a more permissive local environment.


Subject(s)
Antigens, Nuclear/physiology , Autoantigens/physiology , Gene Expression Regulation, Viral , Herpesvirus 1, Human/drug effects , Immediate-Early Proteins/genetics , Interferon-beta/pharmacology , Nuclear Proteins/physiology , Repressor Proteins/physiology , Antigens, Nuclear/genetics , Autoantigens/genetics , Cell Line , Down-Regulation , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/metabolism , Humans , Immediate-Early Proteins/metabolism , Nuclear Proteins/agonists , Nuclear Proteins/genetics , Promoter Regions, Genetic , Protein Isoforms/agonists , Protein Isoforms/genetics , Protein Isoforms/physiology , Protein Structure, Tertiary , RNA Interference , Repressor Proteins/agonists , Repressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics
9.
J Cell Sci ; 116(Pt 3): 513-24, 2003 Feb 01.
Article in English | MEDLINE | ID: mdl-12508112

ABSTRACT

Nuclear domains called ND10 or PML bodies might function as nuclear depots by recruiting or releasing certain proteins. Although recruitment of proteins through interferon-induced upregulation and SUMO-1 modification level of PML had been defined, it is not known whether release of proteins is regulated and has physiological consequences. Exposure to sublethal environmental stress revealed a sequential release of ND10-associated proteins. Upon heat shock Daxx and Sp100 were released but PML remained, whereas exposure to subtoxic concentrations of CdCl(2) induced the release of ND10-associated proteins, including PML, with Sp100 remaining in a few sites. In both cases, recovery times were similar and were followed by a burst of mitotic activity. Cadmium-induced release of proteins from ND10 could be blocked by inhibiting activation of p38 MAPK or ERK1/2. By contrast, heat-shock-induced desumolation of PML and release of proteins from ND10 are unaffected by these inhibitors but can be recapitulated by overexpression of the SUMO isopeptidase SENP-1. Therefore, activation of SENP-1-like SUMO isopeptidase(s) during heat shock is not affected by these kinases. Thus, the release of ND10-associated proteins is not due to a general dispersal of nuclear domains but seems to be regulated by rapid desumolation during thermal stress and through the phosphorylation cascade of stress and mitogenic signaling pathways in the case of CdCl(2). Whether the release of certain proteins had consequences was tested for heat-shock-protein transcription and synthesis. Release of Daxx correlated with Hsp25 suppression, suggesting that Daxx normally inhibits immediate Hsp25 production. Release of PML correlated with lower production of Hsp70. These results suggest that segregation or release of PML or Daxx have differential physiological relevance during the stress response. The fact that enzymatic activation of protein release or segregation after stress modifies the heat-shock response strengthens the concept of ND10 as a regulated depot of effector proteins.


Subject(s)
Carrier Proteins/biosynthesis , Cell Nucleus Structures/metabolism , Eukaryotic Cells/metabolism , Heat-Shock Proteins/biosynthesis , Heat-Shock Response/physiology , Intracellular Signaling Peptides and Proteins , Neoplasm Proteins/biosynthesis , Nuclear Proteins/biosynthesis , Transcription Factors/biosynthesis , Animals , Cadmium/pharmacology , Carrier Proteins/drug effects , Carrier Proteins/genetics , Cell Nucleus Structures/drug effects , Cell Nucleus Structures/genetics , Cells, Cultured , Co-Repressor Proteins , Cysteine Endopeptidases , Endopeptidases/genetics , Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Eukaryotic Cells/drug effects , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , HSP70 Heat-Shock Proteins/biosynthesis , HSP70 Heat-Shock Proteins/genetics , Heat-Shock Proteins/genetics , Heat-Shock Response/drug effects , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/physiology , Macromolecular Substances , Mice , Molecular Chaperones , Neoplasm Proteins/drug effects , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Nuclear Proteins/drug effects , Nuclear Proteins/genetics , Promyelocytic Leukemia Protein , SUMO-1 Protein/genetics , SUMO-1 Protein/metabolism , Stress, Physiological/genetics , Stress, Physiological/metabolism , Transcription Factors/drug effects , Transcription Factors/genetics , Tumor Suppressor Proteins
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