Epstein-Barr virus down-regulates tumor suppressor DOK1 expression

The DOK1 tumor suppressor gene encodes an adapter protein that acts as a negative regulator of several signaling pathways. We have previously reported that DOK1 expression is up-regulated upon cellular stress, via the transcription factor E2F1, and down-regulated in a variety of human malignancies due to aberrant hypermethylation of its promoter. Here we show that Epstein Barr virus (EBV) infection of primary human B-cells leads to the down-regulation of DOK1 gene expression via the viral oncoprotein LMP1. LMP1 alone induces recruitment to the DOK1 promoter of at least two independent inhibitory complexes, one containing E2F1/pRB/DNMT1 and another containing at least EZH2. These events result in tri-methylation of histone H3 at lysine 27 (H3K27me3) of the DOK1 promoter and gene expression silencing. We also present evidence that the presence of additional EBV proteins leads to further repression of DOK1 expression with an additional mechanism. Indeed, EBV infection of B-cells induces DNA methylation at the DOK1 promoter region including the E2F1 responsive elements that, in turn, lose the ability to interact with E2F complexes. Treatment of EBV-infected B-cell-lines with the methyl-transferase inhibitor 5-aza-2′-deoxycytidine rescues DOK1 expression. In summary, our data show the deregulation of DOK1 gene expression by EBV and provide novel insights into the regulation of the DOK1 tumor suppressor in viral-related carcinogenesis.Fil: Siouda, Maha. World Health Organization; FranciaFil: Frecha, Cecilia Ariana. World Health Organization; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Accardi, Rosita. World Health Organization; FranciaFil: Yue, Jiping. World Health Organization; FranciaFil: Cuenin, Cyrille. World Health Organization; FranciaFil: Grufat, Henri. Inserm; Francia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Ecole Normale Supérieure; FranciaFil: Manet, Evelyne. Inserm; Francia. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; Francia. Ecole Normale Supérieure; FranciaFil: Herceg, Zdenko. World Health Organization; FranciaFil: Sylla, Bakary S.. World Health Organization; FranciaFil: Tommasino, Massimo. World Health Organization; Franci

Epstein-Barr virus nuclear antigen 3A protein regulates CDKN2B transcription via interaction with MIZ-1.

peer reviewe

L'étude de la régulation transcriptionnelle et la répression épigénétique du gène suppresseur de tumeur DOK1 dans les carcinogenèses induites ou non par des oncovirus

Le suppresseur de tumeur DOK1 (downstream of tyrosine kinases1) est une protéine régulatrice de voies de signalisation impliquées dans des processus cellulaires tel que la prolifération, la migration et l'apoptose. Le rôle suppresseur de tumeur de DOK1 a été démontré dans des modèles animaux. Les souris knock-out pour DOK1 présentent une forte susceptibilité de développer des leucémies, des tumeurs malignes hématologiques, des adénocarcinomes pulmonaires, ainsi que des sarcomes histiocytaires agressifs. En outre, nous avons rapporté précédemment que le gène DOK1 peut être muté et son expression réprimée dans différentes tumeurs malignes humaines, telles que les lignées cellulaires de lymphome de Burkitt (BL) et la leucémie lymphoïde chronique (LLC). Cependant, les mécanismes de dérégulation de DOK1 restent inconnus, notamment dans les processus de carcinogenèse induite ou non par des oncovirus. Dans ce projet de thèse, nous avons d'abord caractérisé le promoteur de DOK1 et le rôle du facteur de transcription E2F1 comme le principal régulateur de l'expression de DOK1. Nous avons démontré pour la première fois la contribution de DOK1 dans la réponse cellulaire au stress par son rôle suppresseur de prolifération cellulaire et promoteur d'apoptose. Nous avons trouvé que l'expression du gène DOK1 est réprimée dans une variété de cancers humains, y compris le cancer de la tête et du cou, les lymphomes de Burkitt et les cancers du poumon. Cette répression est due à l'hyperméthylation aberrante de DOK1. Nous avons donc étudié les événements épigénétiques, qui sont souvent altérés dans les cancers, et leurs implications dans la répression de DOK1 dans les lignes cellulaire cancéreuses de la tête et du cou. Nous nous sommes par la suite intéressés aux mécanismes de dérégulation de DOK1 par le virus d'Epstein Barr dans le cadre de sa propriété oncogénique dans les lymphocytes B humains ainsi que dans les lignes cancéreuses du lymphome de Burkitt. Nos résultats apportent de nouvelles informations sur les mécanismes de régulation de l'expression de DOK1 dans la carcinogenèse induite ou non par des oncovirus, ce qui pourrait le définir comme un biomarqueur potentiel de cancer et comme une cible intéressante pour des thérapies épigénétiquesThe newly identified tumor suppressor DOK1 (downstream of tyrosine kinases1) inhibits cell proliferation, negatively regulates MAP kinase activity, opposes leukemogenesis, and promotes cell spreading, motility, and apoptosis. DOK1 also plays a role in the regulation of immune cell activation, including B cells. The tumor suppressor role of DOK1 was demonstrated in animal models. DOK1 knockout mice show a high susceptibility to develop leukemia, hematological malignancies as well as lung adenocarcinomas and aggressive histiocytic sarcoma. In addition, we previously reported that the DOK1 gene can be mutated and its expression is down-regulated in human malignancies such as Burkitt’s lymphoma cell lines (BL) and chronic lymphocytic leukemia (CLL). However, very little is known about the mechanisms underlying DOK1 gene regulation and silencing in viral- and non viral-related tumorigenesis. In the present project, we first characterized the DOK1 promoter. We have shown the role of E2F1 transcription factor as the major regulator of DOK1 expression and how DOK1 plays a role in DNA stress response though opposing cell proliferation and promoting apoptosis. We demonstrated that DOK1 gene expression is repressed in a variety of human cancers, including head and neck, Burkitt’s lymphoma and lung cancers, as a result of aberrant hypermethylation. We investigated the link between the epigenetic events and DOK1 silencing in non viral head and neck cancer cell lines, and by Epstein Barr virus in relation to its oncogenic activity in human B cells and neoplasia such as Burkitt’s lymphoma. These data provide novel insights into the regulation of DOK1 in viral and non viral-related carcinogenesis, and could define it as a potential cancer biomarker and an attractive target for epigeneticbased therap

Expression of the epidermodysplasia verruciformis-associated genes EVER1 and EVER2 is activated by exogenous DNA and inhibited by LMP1 oncoprotein from Epstein-Barr virus

EVER1 and EVER2 are mutated in epidermodysplasia verruciformis patients, who are susceptible to human betapapillomavirus (HPV) infection. It is unknown whether their products control the infection of other viruses. Here, we show that the expression of both genes in B cells is activated immediately after Epstein-Barr virus (EBV) infection, whereas at later stages, it is strongly repressed via activation of the NF-κB signaling pathway by latent membrane protein 1 (LMP1). Ectopic expression of EVER1 impairs the ability of EBV to infect B cells.Fil: Frecha, Cecilia Ariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Biología Celular y Neurociencia "Prof. Eduardo de Robertis". Universidad de Buenos Aires. Facultad de Medicina. Instituto de Biología Celular y Neurociencia; Argentina. International Agency For Research On Cancer; FranciaFil: Chevalier, Sébastien A.. International Agency For Research On Cancer; FranciaFil: van Uden, Patrick. International Agency For Research On Cancer; FranciaFil: Rubio, Ivonne. International Agency For Research On Cancer; FranciaFil: Siouda, Maha. International Agency For Research On Cancer; FranciaFil: Saidj, Djamel. International Agency For Research On Cancer; FranciaFil: Cohen, Camille. Université Claude Bernard Lyon 1; Francia. Centre National de la Recherche Scientifique; FranciaFil: Lomonte, Patrick. Centre National de la Recherche Scientifique; Francia. Université Claude Bernard Lyon 1; FranciaFil: Accardi, Rosita. International Agency For Research On Cancer; FranciaFil: Tommasino, Massimo. International Agency For Research On Cancer; Franci

Inactivation of the putative suppressor gene DOK1 by promoter hypermethylation in primary human cancers

The DOK1 gene is a putative tumour suppressor gene located on the human chromosome 2p13 which is frequently rearranged in leukaemia and other human tumours. We previously reported that the DOK1 gene can be mutated and its expression down-regulated in human malignancies. However, the mechanism underlying DOK1 silencing remains largely unknown. We show here that unscheduled silencing of DOK1 expression through aberrant hypermethylation is a frequent event in a variety of human malignancies. DOK1 was found to be silenced in nine head and neck cancer (HNC) cell lines studied and DOK1 CpG hypermethylation correlated with loss of gene expression in these cells. DOK1 expression could be restored via demethylating treatment using 5-aza-2′deoxycytidine. In addition, transduction of cancer cell lines with DOK1 impaired their proliferation, consistent with the critical role of epigenetic silencing of DOK1 in the development and maintenance of malignant cells. We further observed that DOK1 hyperm

(A) LCL cells were transfected with the indicated amounts of empty pcDNA3 (Vector) or expression vector pcDNA3-Flag-DOK1.

<p>After 24(<b>B</b>) LCL cells were monitored for cell cycle analysis 48 hours after being transfected with the indicated amounts of pcDNA3 empty (Vector) or expression vector pcDNA3-Flag-DOK1. Cells in different cycle phases (SubG0, G0/G1, S, or G2/M) are represented as percentage of total cells. (<b>C</b>) The same cells from (B) were monitored for apoptosis using Annexin V staining. Non transfected cells were used as control (NT). Error bars indicate the SD from two independent experiments. Data were analyzed using Student's t test (*, P<0.05).</p

LMP1-mediated NF-κB activation is required for EBV-related <i>DOK1</i> down-regulation.

<p>RPMI cells transduced with empty retroviral pLXSN (Vector), expression vector pLXSN-LMP1, or infected with GFP-EBV recombinant virus were treated with Bay11 or the equivalent volume of DMSO (Mock). (<b>A</b>) mRNA levels of <i>LMP1</i> and <i>DOK1</i> were measured by real time PCR, and normalized to <i>GAPDH</i> expression. (<b>B</b>) The indicated proteins were detected using western blotting. RPMI cells were transfected with pcDNA3 empty plasmid (Vector), expression vector pcDNA3-LMP1 and/or expressing the super-repressor IκBα (ΔIκBα), while RPMI cells infected with GFP-EBV recombinant virus were transfected only with pcDNA3 empty (Vector) or expression vector of the super-repressor IκBα (ΔIκBα). After 48 hours, cells were collected for analysis. (<b>C</b>) mRNA levels of <i>LMP1</i> and <i>DOK1</i> were measured by real time PCR, and normalized to <i>GAPDH</i> expression. (<b>D</b>) The indicated proteins were detected using western blotting. RPMI cells were transfected with empty pLXSN (Vector), or expression vector pLXSN-LMP1 wild type (WT), LMP1 mutant for the CTAR1 domain (AxAxA), and CTAR2 domain (378 stop), or both CRAT1 and 2 domains (AxAxA/378 stop). After 48 hours, cells were harvested for expression analysis. (<b>E</b>) mRNA levels of <i>LMP1</i>, <i>GAPDH</i> and <i>DOK1</i> were measured using real time PCR. (<b>F</b>) The indicated proteins were detected using western blotting. DOK1 protein levels were quantified from two independent immunoblots and normalized to the corresponding β-actin level (bottom of B, D and F). Stable RPMI cells with empty pLXSN (Vector), or expression vector pLXSN-LMP1, were treated with Bay11 or the equivalent volume of DMSO (Mock). (<b>G</b>) Cells were subjected to quantitative ChIP assay using the indicated antibody or IgG. The <i>DOK1</i> promoter was amplified by real-time PCR using specific primers flanking the E2F-response element located at −498/−486. Data were calculated as percentages of enrichment of total input. Error bars indicate the standard deviation from two independent experiments performed in triplicate. (<b>H</b>) <i>In vitro</i> DNA pull-down assay. The <i>DOK1</i> promoter region containing ERE1 was incubated with total lysate from RPMI cells expressing LMP1 treated with Mock or Bay11, and then pulled down using streptavidin-agarose beads. Immunoblotting was used to check the recruitment of E2F1, pRB, DNMT1, EZH2. β-Actin was used as a negative control of binding to DNA.</p

LMP1 plays a key role in EBV-mediated <i>DOK1</i> silencing.

<p>RPMI cells were infected with GFP recombinant EBV wild type (GFP-EBV) or lacking LMP1 (EBVΔLMP1). (<b>A</b>) The infection was monitored using flow cytometry for GFP expression. (<b>B and C</b>) mRNA levels of EBNA1, LMP1, <i>GAPDH</i> and <i>DOK1</i> in these cells were determined using real time PCR and the indicated proteins expression were analyzed using western blotting. Both RPMI and RPMI-EBVΔLMP1 cells were transduced using retroviral vector pLXSN empty (Vector) or expression vector pLXSN-LMP1. The cells were collected for mRNA and protein analysis. (<b>D and E</b>) The mRNA levels of EBNA1, LMP1, and <i>DOK1</i> in these cells were determined using real time PCR and normalized to <i>GAPDH</i> expression, while the indicated proteins expression were analyzed using western blot. RPMI cells were transiently transfected with increasing amounts of pcDNA3 empty plasmid (Vector) or expression vector pcDNA3-LMP1. (<b>F</b>) Cells were collected for mRNA and protein analysis. <i>LMP1</i>and <i>DOK1</i> gene expressions were measured using real time PCR for RNA levels and normalized to <i>GAPDH</i> expression. (<b>G</b>) The indicated protein levels were detected using western blotting. DOK1 protein levels were quantified from two independent immunoblots and normalized to the corresponding β-actin level (bottom of C, E and G).</p

Schematic model of <i>DOK1</i> gene regulation in EBV-infected cells.

<p>(<b>A</b>) In uninfected cells, <i>DOK1</i> expression is activated via the recruitment of the active form of the E2F1 transcription factor to its response element located at (−498/−486) on the <i>DOK1</i> promoter. (<b>B</b>) In cells expressing the oncoprotein LMP1, <i>DOK1</i> is down-regulated through the recruitment of the inhibitory complexes E2F1/pRB/DNMT1 and EZH2 to its promoter region. These complexes lead to the induction of partial DNA methylation and the increase of H3K27 trimethylation levels, respectively. (<b>C</b>) In EBV-infected cells, <i>DOK1</i> is repressed through heavy DNA methylation of its promoter region and the increase in H3K27 trimethylation level. These events likely induce conformational changes in the chromatin, which become less permissive to E2F1 transcription factor recruitment.</p