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1.
J Cell Sci ; 133(10)2020 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-32317397

RESUMO

A large fraction of epigenetically silent heterochromatin is anchored to the nuclear periphery via 'tethering proteins' that function to bridge heterochromatin and the nuclear membrane or nuclear lamina. We previously identified a human tethering protein, PRR14, that binds heterochromatin through an N-terminal domain, but the mechanism and regulation of nuclear lamina association remained to be investigated. Here we identify an evolutionarily conserved PRR14 nuclear lamina binding domain (LBD) that is both necessary and sufficient for positioning of PRR14 at the nuclear lamina. We show that PRR14 associates dynamically with the nuclear lamina, and provide evidence that such dynamics are regulated through phosphorylation and dephosphorylation of the LBD. Furthermore, we identify a PP2A phosphatase recognition motif within the evolutionarily conserved C-terminal Tantalus domain of PRR14. Disruption of this motif affects PRR14 localization to the nuclear lamina. The overall findings demonstrate a heterochromatin anchoring mechanism whereby the PRR14 tether simultaneously binds heterochromatin and the nuclear lamina through two separable modular domains. Our findings also describe an optimal PRR14 LBD fragment that could be used for efficient targeting of fusion proteins to the nuclear lamina.


Assuntos
Heterocromatina , Lâmina Nuclear , Núcleo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Lâmina Nuclear/genética , Lâmina Nuclear/metabolismo , Fosforilação
2.
Postepy Biochem ; 62(3): 280-285, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-28132482

RESUMO

Collaborations between the Wlodawer and Skalka laboratories have covered a period of almost 30 years. During that time our groups have co-authored 18 publications, including several much cited journal articles, book chapters, and scholarly reviews. It has therefore been most rewarding for us to share enthusiasm, insights, and expertise with our Frederick colleagues over the years, and also to enjoy lasting friendships.


Assuntos
Bioquímica/história , Cristalografia/história , Proteínas dos Retroviridae/química , Retroviridae/enzimologia , Cristalografia/métodos , História do Século XX , História do Século XXI , Conformação Proteica , Proteínas dos Retroviridae/metabolismo , Estados Unidos
3.
J Virol ; 87(4): 2137-50, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23221555

RESUMO

Integrated retroviral DNA is subject to epigenetic transcriptional silencing at different frequencies. This process is mediated by repressive DNA methylation and histone modifications on viral chromatin. However, the detailed mechanisms by which retroviral silencing is initiated and maintained are not well understood. Using a model system in which avian sarcoma virus (ASV) DNA is epigenetically repressed in mammalian cells, we previously found that a cellular scaffolding protein, Daxx, acts as an antiretroviral factor that promotes epigenetic repression through recruitment of histone deacetylases (HDACs). Here we show that human Daxx protein levels are increased in response to retroviral infection and that Daxx acts at the time of infection to initiate epigenetic repression. Consistent with a rapid and active antiviral epigenetic response, we found that repressive histone marks and long terminal repeat (LTR) DNA methylation could be detected within 12 h to 3 days postinfection, respectively. Daxx was also found to be required for long-term ASV silencing maintenance and full viral DNA methylation, and it was physically associated with both viral DNA and DNA methyltransferases (DNMTs). These findings support a model in which incoming retroviral protein-DNA complexes are detected by Daxx, and the integrated provirus is rapidly chromatinized and repressed by DNA methylation and histone modification as part of an antiviral response. These results uncover a possible direct and active antiviral mechanism by which DNMTs can be recruited to retroviral DNA.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Vírus do Sarcoma Aviário/genética , Metilação de DNA , Repressão Epigenética , Regulação Viral da Expressão Gênica , Interações Hospedeiro-Patógeno , Proteínas Nucleares/metabolismo , Animais , Vírus do Sarcoma Aviário/fisiologia , Linhagem Celular , Proteínas Correpressoras , Inativação Gênica , Humanos , Chaperonas Moleculares
4.
Virol J ; 11: 100, 2014 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-24884573

RESUMO

BACKGROUND: The antiviral protein Daxx acts as a restriction factor of avian sarcoma virus (ASV; Retroviridae) in mammalian cells by promoting epigenetic silencing of integrated proviral DNA. Although Daxx is encoded by a type I (α/ß) interferon-stimulated gene, the requirement for Daxx in the interferon anti-retroviral response has not been elucidated. In this report, we describe the results of experiments designed to investigate the role of Daxx in the type I interferon-induced anti-ASV response. FINDINGS: Using an ASV reporter system, we show that type I interferons are potent inhibitors of ASV replication. We demonstrate that, while Daxx is necessary to silence ASV gene expression in the absence of interferons, type I interferons are fully-capable of inducing an antiviral state in the absence of Daxx. CONCLUSIONS: These results provide evidence that Daxx is not essential for the anti-ASV interferon response in mammalian cells, and that interferons deploy multiple, redundant antiviral mechanisms to protect cells from ASV.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Vírus do Sarcoma Aviário/imunologia , Vírus do Sarcoma Aviário/fisiologia , Interferon Tipo I/imunologia , Proteínas Nucleares/imunologia , Replicação Viral , Animais , Aves , Linhagem Celular , Proteínas Correpressoras , Humanos , Chaperonas Moleculares
5.
Nucleus ; 14(1): 2165602, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-36633363

RESUMO

The eukaryotic genome is organized in three dimensions within the nucleus. Transcriptionally active chromatin is spatially separated from silent heterochromatin, a large fraction of which is located at the nuclear periphery. However, the mechanisms by which chromatin is localized at the nuclear periphery remain poorly understood. Here we demonstrate that Proline Rich 14 (PRR14) protein organizes H3K9me3-modified heterochromatin at the nuclear lamina. We show that PRR14 dynamically associates with both the nuclear lamina and heterochromatin, and is able to reorganize heterochromatin in the nucleus of interphase cells independent of mitosis. We characterize two functional HP1-binding sites within PRR14 that contribute to its association with heterochromatin. We also demonstrate that PPR14 forms an anchoring surface for heterochromatin at the nuclear lamina where it interacts dynamically with HP1-associated chromatin. Our study proposes a model of dynamic heterochromatin organization at the nuclear lamina via the PRR14 tethering protein.


Assuntos
Heterocromatina , Lâmina Nuclear , Heterocromatina/metabolismo , Lâmina Nuclear/metabolismo , Núcleo Celular/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/metabolismo
6.
J Biol Chem ; 286(29): 25710-8, 2011 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-21622554

RESUMO

In the initial step of integration, retroviral integrase (IN) introduces precise nicks in the degenerate, short inverted repeats at the ends of linear viral DNA. The scissile phosphodiester bond is located immediately 3' of a highly conserved CA/GT dinucleotide, usually 2 bp from the ends. These nicks create new recessed 3'-OH viral DNA ends that are required for joining to host cell DNA. Previous studies have indicated that unpairing, "fraying," of the viral DNA ends by IN contributes to end recognition or catalysis. Here, we report that end fraying can be detected independently of catalysis with both avian sarcoma virus (ASV) and human immunodeficiency virus type 1 (HIV-1) IN proteins by use of fluorescence resonance energy transfer (FRET). The results were indicative of an IN-induced intramolecular conformational change in the viral DNA ends (cis FRET). Fraying activity is tightly coupled to the DNA binding capabilities of these enzymes, as follows: an inhibitor effective against both IN proteins was shown to block ASV IN DNA binding and end fraying, with similar dose responses; ASV IN substitutions that reduced DNA binding also reduced end fraying activity; and HIV-1 IN DNA binding and end fraying were both undetectable in the absence of a metal cofactor. Consistent with our previous results, end fraying is sequence-independent, suggesting that the DNA terminus per se is a major structural determinant for recognition. We conclude that frayed ends represent a functional intermediate in which DNA termini can be sampled for suitability for endonucleolytic processing.


Assuntos
Vírus do Sarcoma Aviário/enzimologia , Pareamento de Bases , DNA Viral/química , Integrase de HIV/metabolismo , HIV-1/enzimologia , Vírus do Sarcoma Aviário/genética , Vírus do Sarcoma Aviário/metabolismo , Sequência de Bases , Domínio Catalítico , Coenzimas/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Transferência Ressonante de Energia de Fluorescência , Integrase de HIV/química , HIV-1/genética , HIV-1/metabolismo , Metais/metabolismo , Reprodutibilidade dos Testes
7.
J Biol Chem ; 285(1): 422-33, 2010 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-19880521

RESUMO

Epigenetic silencing is mediated by families of factors that place, remove, read, and transmit repressive histone and DNA methylation marks on chromatin. How the roles for these functionally diverse factors are specified and integrated is the subject of intense study. To address these questions, HeLa cells harboring epigenetically silent green fluorescent protein reporter genes were interrogated with a small interference RNA library targeting 200 predicted epigenetic regulators, including potential activators, silencers, chromatin remodelers, and ancillary factors. Using this approach, individual, or combinatorial requirements for specific epigenetic silencing factors could be detected by measuring green fluorescent protein reactivation after small interference RNA-based factor knockdown. In our analyses, we identified a specific subset of 15 epigenetic factors that are candidates for participation in a functional epigenetic silencing network in human cells. These factors include histone deacetylase 1, de novo DNA methyltransferase 3A, components of the polycomb PRC1 complex (RING1 and HPH2), and the histone lysine methyltransferases KMT1E and KMT5C. Roles were also detected for two TRIM protein family members, the cohesin component Rad21, and the histone chaperone CHAF1A (CAF-1 p150). Remarkably, combinatorial knockdown of factors was not required for reactivation, indicating little functional redundancy. Consistent with this interpretation, knockdown of either KMT1E or CHAF1A resulted in a loss of multiple histone-repressive marks and concomitant gain of activation marks on the promoter during reactivation. These results reveal how functionally diverse factors may cooperate to maintain gene silencing during normal development or in disease. Furthermore, the findings suggest an avenue for discovery of new targets for epigenetic therapies.


Assuntos
Inativação Gênica , Proteínas Nucleares/metabolismo , Azacitidina/farmacologia , Separação Celular , Fator 1 de Modelagem da Cromatina/metabolismo , Células Clonais , Citomegalovirus/genética , DNA Metiltransferase 3A , Técnicas de Silenciamento de Genes , Inativação Gênica/efeitos dos fármacos , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Ensaios de Triagem em Larga Escala , Histonas/metabolismo , Humanos , Modelos Genéticos , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , Fase S/efeitos dos fármacos , Fatores de Transcrição
8.
J Virol ; 82(5): 2313-23, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18094192

RESUMO

Integrated retroviral DNA is subject to epigenetic gene silencing, resulting in loss of expression of viral genes as well as reporter or therapeutic genes transduced by retroviral vectors. Possible mediators of such silencing include the histone deacetylase (HDAC) family of cellular proteins. We previously isolated HeLa cell populations that harbored silent avian sarcoma virus-based green fluorescent protein (GFP) vectors that could be reactivated by treatment with HDAC inhibitors. Here, we developed a small interfering RNA (siRNA)-based approach to identify specific host factors that participate in the maintenance of silencing. Knockdown of HDAC1, the transcriptional repressor Daxx (a binding partner of HDAC1), or heterochromatin protein 1 gamma resulted in robust and specific GFP reporter gene reactivation. Analyses of cell clones and diverse GFP vector constructs revealed that the roles of HDAC1 and Daxx in retroviral silencing are largely independent of the integration site or the promoter controlling the silent GFP reporter gene. Previous findings from our laboratory and those of others have suggested that Daxx and HDAC proteins may act broadly as part of an antiviral response to repress viral gene transcription. Expression of presumptive viral "countermeasure" proteins that are known to inhibit Daxx or HDACs (pp71, IE2, and Gam1) resulted in the reactivation of GFP reporter gene expression. This study has identified individual host factors that maintain retroviral silencing and supports the proposal that these factors participate in an antiviral response. Furthermore, our results indicate that siRNAs can be used as specific reagents to interrupt the maintenance of epigenetic silencing.


Assuntos
Epigênese Genética/fisiologia , Inativação Gênica/fisiologia , Proteínas/fisiologia , Retroviridae/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Sequência de Bases , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/fisiologia , Proteínas Correpressoras , Primers do DNA , Células HeLa , Inibidores de Histona Desacetilases , Histona Desacetilases/genética , Histona Desacetilases/fisiologia , Humanos , Chaperonas Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , Regiões Promotoras Genéticas , RNA Interferente Pequeno , Reação em Cadeia da Polimerase Via Transcriptase Reversa
9.
J Acoust Soc Am ; 125(2): 958-67, 2009 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19206872

RESUMO

An analysis of cicada mating calls, measured in field experiments, indicates that the very high levels of acoustic energy radiated by this relatively small insect are mainly attributed to the nonlinear characteristics of the signal. The cicada emits one of the loudest sounds in all of the insect population with a sound production system occupying a physical space typically less than 3 cc. The sounds made by tymbals are amplified by the hollow abdomen, functioning as a tuned resonator, but models of the signal based solely on linear techniques do not fully account for a sound radiation capability that is so disproportionate to the insect's size. The nonlinear behavior of the cicada signal is demonstrated by combining the mutual information and surrogate data techniques; the results obtained indicate decorrelation when the phase-randomized and non-phase-randomized data separate. The Volterra expansion technique is used to fit the nonlinearity in the insect's call. The second-order Volterra estimate provides further evidence that the cicada mating calls are dominated by nonlinear characteristics and also suggests that the medium contributes to the cicada's efficient sound propagation. Application of the same principles has the potential to improve radiated sound levels for sonar applications.


Assuntos
Acústica , Hemípteros/fisiologia , Modelos Biológicos , Dinâmica não Linear , Comportamento Sexual Animal , Vocalização Animal , Animais , Feminino , Masculino , Movimento (Física) , Processamento de Sinais Assistido por Computador , Som , Espectrografia do Som
10.
Oncogene ; 38(19): 3710-3728, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30674989

RESUMO

Melanoma is an aggressive neoplasm with increasing incidence that is classified by the NCI as a recalcitrant cancer, i.e., a cancer with poor prognosis, lacking progress in diagnosis and treatment. In addition to conventional therapy, melanoma treatment is currently based on targeting the BRAF/MEK/ERK signaling pathway and immune checkpoints. As drug resistance remains a major obstacle to treatment success, advanced therapeutic approaches based on novel targets are still urgently needed. We reasoned that the base excision repair enzyme thymine DNA glycosylase (TDG) could be such a target for its dual role in safeguarding the genome and the epigenome, by performing the last of the multiple steps in DNA demethylation. Here we show that TDG knockdown in melanoma cell lines causes cell cycle arrest, senescence, and death by mitotic alterations; alters the transcriptome and methylome; and impairs xenograft tumor formation. Importantly, untransformed melanocytes are minimally affected by TDG knockdown, and adult mice with conditional knockout of Tdg are viable. Candidate TDG inhibitors, identified through a high-throughput fluorescence-based screen, reduced viability and clonogenic capacity of melanoma cell lines and increased cellular levels of 5-carboxylcytosine, the last intermediate in DNA demethylation, indicating successful on-target activity. These findings suggest that TDG may provide critical functions specific to cancer cells that make it a highly suitable anti-melanoma drug target. By potentially disrupting both DNA repair and the epigenetic state, targeting TDG may represent a completely new approach to melanoma therapy.


Assuntos
Inibidores Enzimáticos/farmacologia , Melanoma/patologia , Timina DNA Glicosilase/genética , Animais , Ciclo Celular/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos Knockout , Camundongos SCID , Camundongos Transgênicos , Terapia de Alvo Molecular/métodos , Timina DNA Glicosilase/antagonistas & inibidores , Timina DNA Glicosilase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
11.
EBioMedicine ; 22: 28-43, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28687497

RESUMO

Genome-Wide Association Studies (GWAS) and subsequent fine-mapping studies (>50) have implicated single nucleotide polymorphisms (SNPs) located at the CCDC170/C6ORF97-ESR1 locus (6q25.1) as being associated with the risk of breast cancer. Surprisingly, our analysis using genome-wide differential allele-specific expression (DASE), an indicator for breast cancer susceptibility, suggested that the genetic alterations of CCDC170, but not ESR1, account for GWAS-associated breast cancer risk at this locus. Breast cancer-associated CCDC170 nonsense mutations and rearrangements have also been detected, with the latter being specifically implicated in driving breast cancer. Here we report that the wild type CCDC170 protein localizes to the region of the Golgi apparatus and binds Golgi-associated microtubules (MTs), and that breast cancer-linked truncations of CCDC170 result in loss of Golgi localization. Overexpression of wild type CCDC170 triggers Golgi reorganization, and enhances Golgi-associated MT stabilization and acetyltransferase ATAT1-dependent α-tubulin acetylation. Golgi-derived MTs regulate cellular polarity and motility, and we provide evidence that dysregulation of CCDC170 affects polarized cell migration. Taken together, our findings demonstrate that CCDC170 plays an essential role in Golgi-associated MT organization and stabilization, and implicate a mechanism for how perturbations in the CCDC170 gene may contribute to the hallmark changes in cell polarity and motility seen in breast cancer.


Assuntos
Neoplasias da Mama/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Microtúbulos/metabolismo , Acetilação , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Polaridade Celular , Feminino , Predisposição Genética para Doença , Humanos , Células MCF-7 , Polimorfismo de Nucleotídeo Único , Ligação Proteica
13.
PLoS One ; 10(2): e0116839, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25642713

RESUMO

Melanoma tissues and cell lines are heterogeneous, and include cells with invasive, proliferative, stem cell-like, and differentiated properties. Such heterogeneity likely contributes to the aggressiveness of the disease and resistance to therapy. One model suggests that heterogeneity arises from rare cancer stem cells (CSCs) that produce distinct cancer cell lineages. Another model suggests that heterogeneity arises through reversible cellular plasticity, or phenotype-switching. Recent work indicates that phenotype-switching may include the ability of cancer cells to dedifferentiate to a stem cell-like state. We set out to investigate the phenotype-switching capabilities of melanoma cells, and used unbiased methods to identify genes that may control such switching. We developed a system to reversibly synchronize melanoma cells between 2D-monolayer and 3D-stem cell-like growth states. Melanoma cells maintained in the stem cell-like state showed a striking upregulation of a gene set related to development and neural stem cell biology, which included SRY-box 2 (SOX2) and Inhibitor of DNA Binding 4 (ID4). A gene set related to cancer cell motility and invasiveness was concomitantly downregulated. Intense and pervasive ID4 protein expression was detected in human melanoma tissue samples, suggesting disease relevance for this protein. SiRNA knockdown of ID4 inhibited switching from monolayer to 3D-stem cell-like growth, and instead promoted switching to a highly differentiated, neuronal-like morphology. We suggest that ID4 is upregulated in melanoma as part of a stem cell-like program that facilitates further adaptive plasticity. ID4 may contribute to disease by preventing stem cell-like melanoma cells from progressing to a normal differentiated state. This interpretation is guided by the known role of ID4 as a differentiation inhibitor during normal development. The melanoma stem cell-like state may be protected by factors such as ID4, thereby potentially identifying a new therapeutic vulnerability to drive differentiation to the normal cell phenotype.


Assuntos
Diferenciação Celular , Regulação Neoplásica da Expressão Gênica , Proteínas Inibidoras de Diferenciação/metabolismo , Melanoma/metabolismo , Melanoma/patologia , Adesão Celular , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Proteínas Inibidoras de Diferenciação/deficiência , Proteínas Inibidoras de Diferenciação/genética , Melanoma/genética , Invasividade Neoplásica , Células-Tronco Neoplásicas/patologia , Fenótipo , RNA Interferente Pequeno/genética , Fatores de Transcrição SOXB1/deficiência , Fatores de Transcrição SOXB1/genética , Transcrição Gênica
14.
Nucleus ; 5(1): 32-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24637393

RESUMO

A conserved organizational feature of eukaryotic nuclei is the peripheral heterochromatin compartment, which provides a protected area for epigenetically silent genes and gene-poor DNA. In metazoan cells this compartment is associated with the nuclear lamina, the protein meshwork at the inner edge of the nucleus. Heterochromatin-nuclear lamina interactions promote epigenetic gene silencing, which may drive many normal and diseased biological processes. We recently obtained evidence that a previously unstudied human protein, PRR14, participates in the tethering of heterochromatin to the inner nuclear periphery. PRR14 associates with the nuclear lamina and attaches to heterochromatin through its binding partner, heterochromatin protein 1 (HP1). After disassembly early in mitosis, PRR14 reassembles in two steps, first binding to anaphase chromosomes through HP1, followed by association with the nuclear lamina in telophase. PRR14 may thereby play a role in specifying HP1-bound heterochromatin for reattachment to the nuclear lamina at mitotic exit. Here we review the relevant literature, summarize our initial work, and provide additional comments and findings.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Heterocromatina/metabolismo , Lâmina Nuclear/metabolismo , Núcleo Celular/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas de Ligação a DNA/genética , Epigênese Genética , Inativação Gênica , Heterocromatina/genética , Humanos , Microscopia Confocal , Mitose/genética , Mitose/fisiologia , Lâmina Nuclear/genética
15.
Epigenetics ; 9(9): 1280-9, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25147916

RESUMO

Cellular identity in both normal and disease processes is determined by programmed epigenetic activation or silencing of specific gene subsets. Here, we have used human cells harboring epigenetically silent GFP-reporter genes to perform a genome-wide siRNA knockdown screen for the identification of cellular factors that are required to maintain epigenetic gene silencing. This unbiased screen interrogated 21,121 genes, and we identified and validated a set of 128 protein factors. This set showed enrichment for functional categories, and protein-protein interactions. Among this set were known epigenetic silencing factors, factors with no previously identified role in epigenetic gene silencing, as well as unstudied factors. The set included non-nuclear factors, for example, components of the integrin-adhesome. A key finding was that the E1 and E2 enzymes of the small ubiquitin-like modifier (SUMO) pathway (SAE1, SAE2/UBA2, UBC9/UBE2I) are essential for maintenance of epigenetic silencing. This work provides the first genome-wide functional view of human factors that mediate epigenetic gene silencing. The screen output identifies novel epigenetic factors, networks, and mechanisms, and provides a set of candidate targets for epigenetic therapy and cellular reprogramming.


Assuntos
Epigênese Genética , Inativação Gênica , Proteínas/metabolismo , Transdução de Sinais , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas/genética , RNA Interferente Pequeno/genética , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo
16.
Cell Rep ; 5(2): 292-301, 2013 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-24209742

RESUMO

The nuclear lamina is a protein meshwork that lies under the inner nuclear membrane of metazoan cells. One function of the nuclear lamina is to organize heterochromatin at the inner nuclear periphery. However, very little is known about how heterochromatin attaches to the nuclear lamina and how such attachments are restored at mitotic exit. Here, we show that a previously unstudied human protein, PRR14, functions to tether heterochromatin to the nuclear periphery during interphase, through associations with heterochromatin protein 1 (HP1) and the nuclear lamina. During early mitosis, PRR14 is released from the nuclear lamina and chromatin and remains soluble. Strikingly, at the onset of anaphase, PRR14 is incorporated rapidly into chromatin through HP1 binding. Finally, in telophase, PRR14 relocalizes to the reforming nuclear lamina. This stepwise reassembly of PRR14 suggests a function in the selection of HP1-bound heterochromatin for reattachment to the nuclear lamina as cells exit mitosis.


Assuntos
Núcleo Celular/metabolismo , Heterocromatina/metabolismo , Lâmina Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Células HeLa , Heterocromatina/química , Humanos , Interfase , Microscopia Confocal , Mitose , Lâmina Nuclear/química , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
17.
Cell Cycle ; 7(22): 3539-47, 2008 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19001877

RESUMO

Heterochromatin plays an essential role in the preservation of epigenetic information, the transcriptional repression of repetitive DNA elements and inactive genes, and the proper segregation of chromosomes during mitosis. Here we identify KDM2A, a JmjC-domain containing histone demethylase, as a heterochromatin-associated and HP1-interacting protein that promotes HP1 localization to chromatin. We show that KDM2A is required to maintain the heterochromatic state, as determined using a candidate-based approach coupled to an in vivo epigenetic reporter system. Remarkably, a parallel and independent siRNA screen also detected a role for KDM2A in epigenetic silencing. Moreover, we demonstrate that KDM2A associates with centromeres and represses transcription of small non-coding RNAs that are encoded by the clusters of satellite repeats at the centromere. Dissecting the relationship between heterochromatin and centromeric RNA transcription is the basis of ongoing studies. We demonstrate that forced expression of these satellite RNA transcripts compromise the heterochromatic state and HP1 localization to chromatin. Finally, we show that KDM2A is required to sustain centromeric integrity and genomic stability, particularly during mitosis. Since the disruption of epigenetic control mechanisms contributes to cellular transformation, these results, together with the low levels of KDM2A found in prostate carcinomas, suggest a role for KDM2A in cancer development.


Assuntos
DNA Satélite/genética , Heterocromatina/genética , Oxirredutases N-Desmetilantes/fisiologia , Transcrição Gênica , Células 3T3 , Animais , Transformação Celular Neoplásica , Centrômero/genética , Montagem e Desmontagem da Cromatina/genética , Proteínas F-Box , Instabilidade Genômica , Células HeLa , Histonas/metabolismo , Humanos , Histona Desmetilases com o Domínio Jumonji , Camundongos , Transfecção
18.
J Virol ; 81(6): 2592-604, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17202206

RESUMO

Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(-)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(-) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(-) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(-) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(-) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.


Assuntos
Epigênese Genética , Inativação Gênica , Inibidores de Histona Desacetilases , Retroviridae/metabolismo , Transativadores/metabolismo , Células Clonais/metabolismo , Inibidores Enzimáticos/farmacologia , Genes Reporter , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Ésteres de Forbol/farmacologia , Retroviridae/genética
19.
J Cell Biochem ; 94(5): 880-9, 2005 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-15669021

RESUMO

The study of retroviruses over the last century has revealed a wide variety of disease-producing mechanisms, as well as apparently harmless interactions with animal hosts. Despite their potential pathogenic properties, the intrinsic features of retroviruses have been harnessed to create gene transfer vectors that may be useful for the treatment of disease. Retroviruses, as all viruses, have evolved to infect specific cells within the host, and such specificities are relevant to both pathogenesis and retrovirus-based vector design. The majority of cells of an animal host are not progressing rapidly through the cell cycle, and such a cellular environment appears to be suboptimal for replication of all retroviruses. Retrovirus-based vectors can therefore be restricted in many important target cells, such as post-mitotic differentiated cells or stem cells that may divide only infrequently. Despite intense interest, our understanding of how cell cycle status influences retroviral infection is still quite limited. In this review, we focus on the importance of the cell cycle as it relates to the early steps in retroviral replication. Retroviruses have been categorized based on their abilities to complete these early steps in non-cycling cells. However, all retroviruses are subject to a variety of cell cycle restrictions. Here, we discuss such restrictions, and how they may block retroviral replication, be tolerated, or overcome.


Assuntos
Ciclo Celular , Retroviridae/fisiologia , Replicação Viral , Animais , Genoma Viral , Retroviridae/genética
20.
J Virol ; 79(8): 4610-8, 2005 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15795247

RESUMO

The cellular protein Daxx was identified as an interactor with avian sarcoma virus (ASV) integrase (IN) in a yeast two-hybrid screen. After infection, Daxx-IN interactions were detected by coimmunoprecipitation. An association between Daxx and viral DNA, likely mediated by IN, was also detected by chromatin immunoprecipitation. Daxx was not required for early events in ASV replication, including integration, as Daxx-null cells were transduced as efficiently as Daxx-expressing cells. However, viral reporter gene expression from ASV-based vectors was substantially higher in the Daxx-null cells than in Daxx-complemented cells. Consistent with this observation, histone deacetylases (HDACs) were found to associate with viral DNA in Daxx-complemented cells but not in Daxx-null cells. Furthermore, Daxx protein was induced in an interferon-like manner upon ASV infection. We conclude that Daxx interacts with an IN-viral DNA complex early after infection and may mediate the repression of viral gene expression via the recruitment of HDACs. Our findings provide a novel example of cellular immunity against viral replication in which viral transcription is repressed via the recruitment of antiviral proteins to the viral DNA.


Assuntos
Vírus do Sarcoma Aviário/enzimologia , Proteínas de Transporte/metabolismo , DNA Viral/metabolismo , Integrases/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Nucleares/metabolismo , Transcrição Gênica , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Embrião de Galinha , Proteínas Correpressoras , Primers do DNA , Fibroblastos/virologia , Células HeLa , Inibidores de Histona Desacetilases , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Chaperonas Moleculares , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase
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