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1.
J Virol ; 92(17)2018 09 01.
Article in English | MEDLINE | ID: mdl-29950415

ABSTRACT

EBV latent membrane protein 1 (LMP1) is released from latently infected tumor cells in small membrane-enclosed extracellular vesicles (EVs). Accumulating evidence suggests that LMP1 is a major driver of EV content and functions. LMP1-modified EVs have been shown to influence recipient cell growth, migration, differentiation, and regulation of immune cell function. Despite the significance of LMP1-modified exosomes, very little is known about how this viral protein enters or manipulates the host EV pathway. In this study, LMP1 deletion mutants were generated to assess protein regions required for EV trafficking. Following transfection of LMP1 or mutant plasmids, EVs were collected by differential centrifugation, and the levels of specific cargo were evaluated by immunoblot analysis. The results demonstrate that, together, the N terminus and transmembrane region 1 of LMP1 are sufficient for efficient sorting into EVs. Consistent with these findings, a mutant lacking the N terminus and transmembrane domains 1 through 4 (TM5-6) failed to be packaged into EVs, and exhibited higher colocalization with endoplasmic reticulum and early endosome markers than the wild-type protein. Surprisingly, TM5-6 maintained the ability to colocalize and form a complex with CD63, an abundant exosome protein that is important for the incorporation of LMP1 into EVs. Other mutations within LMP1 resulted in enhanced levels of secretion, pointing to potential positive and negative regulatory mechanisms for extracellular vesicle sorting of LMP1. These data suggest new functions of the N terminus and transmembrane domains in LMP1 intra- and extracellular trafficking that are likely downstream of an interaction with CD63.IMPORTANCE EBV infection contributes to the development of cancers, such as nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin's disease, and posttransplant lymphomas, in immunocompromised or genetically susceptible individuals. LMP1 is an important viral protein expressed by EBV in these cancers. LMP1 is secreted in extracellular vesicles (EVs), and the transfer of LMP1-modified EVs to uninfected cells can alter their physiology. Understanding the cellular machinery responsible for sorting LMP1 into EVs is limited, despite the importance of LMP1-modified EVs. Here, we illustrate the roles of different regions of LMP1 in EV packaging. Our results show that the N terminus and TM1 are sufficient to drive LMP1 EV trafficking. We further show the existence of potential positive and negative regulatory mechanisms for LMP1 vesicle sorting. These findings provide a better basis for future investigations to identify the mechanisms of LMP1 targeting to EVs, which could have broad implications in understanding EV cargo sorting.


Subject(s)
Exosomes/metabolism , Herpesvirus 4, Human/physiology , Protein Transport , Viral Matrix Proteins/metabolism , DNA Mutational Analysis , HEK293 Cells , Herpesvirus 4, Human/genetics , Humans , Viral Matrix Proteins/genetics
2.
Virology ; 516: 55-70, 2018 03.
Article in English | MEDLINE | ID: mdl-29329079

ABSTRACT

Epstein-Barr virus LMP1 is an oncoprotein required for immortalizing B lymphocytes and also plays important roles in transforming non-lymphoid tissue. The discovery of LMP1 protein interactions will likely generate targets to treat EBV-associated cancers. Here, we define the broader LMP1 interactome using the recently developed BioID method. Combined with mass spectrometry, we identified over 1000 proteins across seven independent experiments with direct or indirect relationships to LMP1. Pathway analysis suggests that a significant number of the proteins identified are involved in signal transduction and protein or vesicle trafficking. Interestingly, a large number of proteins thought to be important in the formation of exosomes and protein targeting were recognized as probable LMP1 interacting partners, including CD63, syntenin-1, ALIX, TSG101, HRS, CHMPs, and sorting nexins. Therefore, it is likely that LMP1 modifies protein trafficking and exosome biogenesis pathways. In support of this, knock-down of syntenin-1 and ALIX resulted in reduced exosomal LMP1.


Subject(s)
Epstein-Barr Virus Infections/metabolism , Herpesvirus 4, Human/metabolism , Viral Matrix Proteins/metabolism , Biotin/metabolism , Calcium-Binding Proteins/genetics , Calcium-Binding Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Endosomal Sorting Complexes Required for Transport/genetics , Endosomal Sorting Complexes Required for Transport/metabolism , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/virology , Exosomes/metabolism , Exosomes/virology , Herpesvirus 4, Human/genetics , Host-Pathogen Interactions , Humans , Mass Spectrometry , Protein Binding , Protein Interaction Mapping , Signal Transduction , Syntenins/genetics , Syntenins/metabolism , Tetraspanin 30/genetics , Tetraspanin 30/metabolism , Viral Matrix Proteins/genetics
3.
J Virol ; 91(5)2017 03 01.
Article in English | MEDLINE | ID: mdl-27974566

ABSTRACT

Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma, Burkitt's lymphoma, and Hodgkin's lymphoma. In the context of cancer, EBV hijacks the exosomal pathway to modulate cell-to-cell signaling by secreting viral components such as an oncoprotein, LMP1, into host cell membrane-bound EVs. Trafficking of LMP1 into exosomes is associated with increased oncogenicity of these secreted vesicles. However, we have only a limited understanding of the mechanisms surrounding exosomal cargo packaging, including viral proteins. Here, we describe a role of LMP1 in EV production that requires CD63 and provide an extensive demonstration of CD63-mediated exosomal LMP1 release that is distinct from lipid raft trafficking. Finally, we present further evidence of the role of CD63 in limiting LMP1-induced noncanonical NF-κB and ERK activation. Our findings have implications for future investigations of physiological and pathological mechanisms of exosome biogenesis, protein trafficking, and signal transduction, especially in viral-associated tumorigenesis.


Subject(s)
Exosomes/metabolism , Herpesvirus 4, Human/physiology , Signal Transduction , Tetraspanin 30/physiology , Viral Matrix Proteins/metabolism , Animals , HEK293 Cells , Humans , Membrane Microdomains/metabolism , NF-kappa B/metabolism , Protein Transport , Rats , Secretory Vesicles/metabolism
4.
Genome Biol ; 13(8): R70, 2012 Aug 20.
Article in English | MEDLINE | ID: mdl-22906166

ABSTRACT

BACKGROUND: The X-linked macrosatellite DXZ4 is a large homogenous tandem repeat that in females adopts an alternative chromatin organization on the primate X chromosome in response to X-chromosome inactivation. It is packaged into heterochromatin on the active X chromosome but into euchromatin and bound by the epigenetic organizer protein CTCF on the inactive X chromosome. Because its DNA sequence diverges rapidly beyond the New World monkeys, the existence of DXZ4 outside the primate lineage is unknown. RESULTS: Here we extend our comparative genome analysis and report the identification and characterization of the mouse homolog of the macrosatellite. Furthermore, we provide evidence of DXZ4 in a conserved location downstream of the PLS3 gene in a diverse group of mammals, and reveal that DNA sequence conservation is restricted to the CTCF binding motif, supporting a central role for this protein at this locus. However, many features that characterize primate DXZ4 differ in mouse, including the overall size of the array, the mode of transcription, the chromatin organization and conservation between adjacent repeat units of DNA sequence and length. Ctcf binds Dxz4 but is not exclusive to the inactive X chromosome, as evidenced by association in some males and equal binding to both X chromosomes in trophoblast stem cells. CONCLUSIONS: Characterization of Dxz4 reveals substantial differences in the organization of DNA sequence, chromatin packaging, and the mode of transcription, so the potential roles performed by this sequence in mouse have probably diverged from those on the primate X chromosome.


Subject(s)
Conserved Sequence , DNA, Satellite/genetics , Repressor Proteins/metabolism , Animals , BALB 3T3 Cells , Binding Sites , CCCTC-Binding Factor , Chromatin/genetics , Cloning, Molecular , Comparative Genomic Hybridization , DNA Methylation , Female , Gene Expression Regulation , Genetic Loci , Humans , Male , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , Microfilament Proteins/genetics , NIH 3T3 Cells , Promoter Regions, Genetic , Sequence Analysis, DNA , Tandem Repeat Sequences , X Chromosome/genetics
5.
Hum Mol Genet ; 21(20): 4367-77, 2012 Oct 15.
Article in English | MEDLINE | ID: mdl-22791747

ABSTRACT

The human X-linked macrosatellite DXZ4 is a large tandem repeat located at Xq23 that is packaged into heterochromatin on the male X chromosome and female active X chromosome and, in response to X chromosome, inactivation is organized into euchromatin bound by the insulator protein CCCTC-binding factor (CTCF) on the inactive X chromosome (Xi). The purpose served by this unusual epigenetic regulation is unclear, but suggests a Xi-specific gain of function for DXZ4. Other less extensive bands of euchromatin can be observed on the Xi, but the identity of the underlying DNA sequences is unknown. Here, we report the identification of two novel human X-linked tandem repeats, located 58 Mb proximal and 16 Mb distal to the macrosatellite DXZ4. Both tandem repeats are entirely contained within the transcriptional unit of novel spliced transcripts. Like DXZ4, the tandem repeats are packaged into Xi-specific CTCF-bound euchromatin. These sequences undergo frequent CTCF-dependent interactions with DXZ4 on the Xi, implicating DXZ4 as an epigenetically regulated Xi-specific structural element and providing the first putative functional attribute of a macrosatellite in the human genome.


Subject(s)
Chromosomes, Human, X/genetics , Repressor Proteins/genetics , Tandem Repeat Sequences/genetics , Binding Sites , CCCTC-Binding Factor , Cell Line, Tumor , Epigenesis, Genetic , Female , Heterochromatin/genetics , Heterochromatin/metabolism , Humans , Male
6.
Nucleic Acids Res ; 40(4): 1596-608, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22064860

ABSTRACT

DXZ4 is an X-linked macrosatellite composed of 12-100 tandemly arranged 3-kb repeat units. In females, it adopts opposite chromatin arrangements at the two alleles in response to X-chromosome inactivation. In males and on the active X chromosome, it is packaged into heterochromatin, but on the inactive X chromosome (Xi), it adopts a euchromatic conformation bound by CTCF. Here we report that the ubiquitous transcription factor YY1 associates with the euchromatic form of DXZ4 on the Xi. The binding of YY1 close to CTCF is reminiscent of that at other epigenetically regulated sequences, including sites of genomic imprinting, and at the X-inactivation centre, suggesting a common mode of action in this arrangement. As with CTCF, binding of YY1 to DXZ4 in vitro is not blocked by CpG methylation, yet in vivo both proteins are restricted to the hypomethylated form. In several male carcinoma cell lines, DXZ4 can adopt a Xi-like conformation in response to cellular transformation, characterized by CpG hypomethylation and binding of YY1 and CTCF. Analysis of a male melanoma cell line and normal skin cells from the same individual confirmed that a transition in chromatin state occurred in response to transformation.


Subject(s)
Carcinoma/genetics , Chromosomes, Human, X/metabolism , Repressor Proteins/metabolism , Tandem Repeat Sequences , YY1 Transcription Factor/metabolism , Base Sequence , CCCTC-Binding Factor , Cell Line, Tumor , Cell Transformation, Neoplastic , Cells, Cultured , Chromatin/metabolism , Chromosomes, Human, X/chemistry , Consensus Sequence , CpG Islands , DNA Methylation , Female , Histones/metabolism , Humans , Male , YY1 Transcription Factor/analysis
7.
PLoS One ; 6(4): e18969, 2011 Apr 22.
Article in English | MEDLINE | ID: mdl-21544201

ABSTRACT

Macrosatellites are some of the most polymorphic regions of the human genome, yet many remain uncharacterized despite the association of some arrays with disease susceptibility. This study sought to explore the polymorphic nature of the X-linked macrosatellite DXZ4. Four aspects of DXZ4 were explored in detail, including tandem repeat copy number variation, array instability, monomer sequence polymorphism and array expression. DXZ4 arrays contained between 12 and 100 3.0 kb repeat units with an average array containing 57. Monomers were confirmed to be arranged in uninterrupted tandem arrays by restriction digest analysis and extended fiber FISH, and therefore DXZ4 encompasses 36-288 kb of Xq23. Transmission of DXZ4 through three generations in three families displayed a high degree of meiotic instability (8.3%), consistent with other macrosatellite arrays, further highlighting the unstable nature of these sequences in the human genome. Subcloning and sequencing of complete DXZ4 monomers identified numerous single nucleotide polymorphisms and alleles for the three microsatellite repeats located within each monomer. Pairwise comparisons of DXZ4 monomer sequences revealed that repeat units from an array are more similar to one another than those originating from different arrays. RNA fluorescence in situ hybridization revealed significant variation in DXZ4 expression both within and between cell lines. DXZ4 transcripts could be detected originiating from both the active and inactive X chromosome. Expression levels of DXZ4 varied significantly between males, but did not relate to the size of the array, nor did inheritance of the same array result in similar expression levels. Collectively, these studies provide considerable insight into the polymorphic nature of DXZ4, further highlighting the instability and variation potential of macrosatellites in the human genome.


Subject(s)
Repetitive Sequences, Nucleic Acid/genetics , DNA Copy Number Variations/genetics , Female , Genome, Human/genetics , Humans , Male , Phylogeny
8.
BMC Genomics ; 11: 632, 2010 Nov 15.
Article in English | MEDLINE | ID: mdl-21078170

ABSTRACT

BACKGROUND: Macrosatellites are some of the largest variable number tandem repeats in the human genome, but what role these unusual sequences perform is unknown. Their importance to human health is clearly demonstrated by the 4q35 macrosatellite D4Z4 that is associated with the onset of the muscle degenerative disease facioscapulohumeral muscular dystrophy. Nevertheless, many other macrosatellite arrays in the human genome remain poorly characterized. RESULTS: Here we describe the organization, tandem repeat copy number variation, transmission stability and expression of four macrosatellite arrays in the human genome: the TAF11-Like array located on chromosomes 5p15.1, the SST1 arrays on 4q28.3 and 19q13.12, the PRR20 array located on chromosome 13q21.1, and the ZAV array at 9q32. All are polymorphic macrosatellite arrays that at least for TAF11-Like and SST1 show evidence of meiotic instability. With the exception of the SST1 array that is ubiquitously expressed, all are expressed at high levels in the testis and to a lesser extent in the brain. CONCLUSIONS: Our results extend the number of characterized macrosatellite arrays in the human genome and provide the foundation for formulation of hypotheses to begin assessing their functional role in the human genome.


Subject(s)
Chromosomal Instability/genetics , DNA Copy Number Variations/genetics , Gene Expression Regulation , Genome, Human/genetics , Minisatellite Repeats/genetics , Oligonucleotide Array Sequence Analysis/methods , Amino Acid Sequence , Animals , Cell Line , Chromosomes, Human/genetics , Conserved Sequence/genetics , Female , Gene Expression Profiling , Humans , Male , Molecular Sequence Data , Pedigree , Primates/genetics , TATA-Binding Protein Associated Factors/chemistry , TATA-Binding Protein Associated Factors/genetics , Transcription Factor TFIID/chemistry , Transcription Factor TFIID/genetics
9.
Dev Cell ; 18(2): 191-202, 2010 Feb 16.
Article in English | MEDLINE | ID: mdl-20159591

ABSTRACT

Human developmental disorders caused by chromatin dysfunction often display overlapping clinical manifestations, such as cognitive deficits, but the underlying molecular links are poorly defined. Here, we show that ATRX, MeCP2, and cohesin, chromatin regulators implicated in ATR-X, RTT, and CdLS syndromes, respectively, interact in the brain and colocalize at the H19 imprinting control region (ICR) with preferential binding on the maternal allele. Importantly, we show that ATRX loss of function alters enrichment of cohesin, CTCF, and histone modifications at the H19 ICR, without affecting DNA methylation on the paternal allele. ATRX also affects cohesin, CTCF, and MeCP2 occupancy within the Gtl2/Dlk1 imprinted domain. Finally, we show that loss of ATRX interferes with the postnatal silencing of the maternal H19 gene along with a larger network of imprinted genes. We propose that ATRX, cohesin, and MeCP2 cooperate to silence a subset of imprinted genes in the postnatal mouse brain.


Subject(s)
Brain/growth & development , Brain/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromosomal Proteins, Non-Histone/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , Gene Silencing , Genomic Imprinting , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Animals , CCCTC-Binding Factor , Calcium-Binding Proteins , Chromatin/genetics , Chromatin/metabolism , Cognition Disorders/genetics , Cognition Disorders/metabolism , DNA Helicases/deficiency , DNA Methylation , Female , Gene Regulatory Networks , Histones/metabolism , Humans , Insulin-Like Growth Factor II/genetics , Insulin-Like Growth Factor II/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Nuclear Proteins/deficiency , Proteins/genetics , Proteins/metabolism , RNA, Long Noncoding , RNA, Untranslated/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , X-linked Nuclear Protein , Cohesins
10.
BMC Genomics ; 9: 468, 2008 Oct 08.
Article in English | MEDLINE | ID: mdl-18842153

ABSTRACT

BACKGROUND: Pseudoautosomal regions (PAR1 and PAR2) in eutherians retain homologous regions between the X and Y chromosomes that play a critical role in the obligatory X-Y crossover during male meiosis. Genes that reside in the PAR1 are exceptional in that they are rich in repetitive sequences and undergo a very high rate of recombination. Remarkably, murine PAR1 homologs have translocated to various autosomes, reflecting the complex recombination history during the evolution of the mammalian X chromosome. RESULTS: We now report that the SNF2-type chromatin remodeling protein ATRX controls the expression of eutherian ancestral PAR1 genes that have translocated to autosomes in the mouse. In addition, we have identified two potentially novel mouse PAR1 orthologs. CONCLUSION: We propose that the ancestral PAR1 genes share a common epigenetic environment that allows ATRX to control their expression.


Subject(s)
Chromatin Assembly and Disassembly , DNA Helicases/genetics , Genome , Nuclear Proteins/genetics , Translocation, Genetic , Amino Acid Sequence , Animals , Cells, Cultured , Evolution, Molecular , Gene Deletion , Gene Expression Profiling , Humans , Mice , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Phylogeny , Prosencephalon/growth & development , RNA/genetics , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , Sequence Alignment , X-linked Nuclear Protein
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