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

Bazot Q, Deschamps T, Tafforeau L, Siouda M, Leblanc P, Harth-Hertle ML, Rabourdin-Combe C, Lotteau V, Kempkes B, Tommasino M, Gruffat H, Manet E - Nucleic Acids Res. (2014)

Bottom Line: Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus.Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A.Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

View Article: PubMed Central - PubMed

Affiliation: CIRI, International Center for Infectiology Research, Oncogenic Herpesviruses team, Université de Lyon, Lyon 69364, France Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon 69364, France CIRI, International Center for Infectiology Research, Cell Biology of Viral Infections team, Université de Lyon, Lyon 69364, France INSERM, U1111, Lyon 69364, France CNRS, UMR5308, Lyon 69364, France.

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EBNA3A relocalizes MIZ-1 into the nucleus. Expression plasmids for Flag-MIZ-1 alone (A) or Flag-MIZ-1 together with Myc-EBNA3A were transfected into HeLa cells (B and C). Proteins were visualized by indirect immunofluorescence using an anti-Flag polyclonal Ab (for detection of MIZ-1) (panels b) and an anti-tag Myc polyclonal Ab (for detection of EBNA3A) (panels c). A Fluorolink Cy3-labeled goat anti-mouse IgG (H + L) antibody and an Alexa Fluor 488 goat anti-rabbit antibody were used as secondary antibodies respectively. Cell nuclei were stained with Hoechst 33258 (Sigma) (panels a). (C) Quantification of the number of cells in which MIZ-1 is strictly nuclear versus cytoplasmic/nucleo-cytoplasmic in cells transfected with an expression vector for MIZ-1 or in cells co-transfected with expression vectors for MIZ-1 and EBNA3A. In the latter case, only cells co-expressing EBNA3A together with MIZ-1 are taken into consideration. The results are the combination of two different experiments in which 1333 cells altogether were observed.
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Figure 4: EBNA3A relocalizes MIZ-1 into the nucleus. Expression plasmids for Flag-MIZ-1 alone (A) or Flag-MIZ-1 together with Myc-EBNA3A were transfected into HeLa cells (B and C). Proteins were visualized by indirect immunofluorescence using an anti-Flag polyclonal Ab (for detection of MIZ-1) (panels b) and an anti-tag Myc polyclonal Ab (for detection of EBNA3A) (panels c). A Fluorolink Cy3-labeled goat anti-mouse IgG (H + L) antibody and an Alexa Fluor 488 goat anti-rabbit antibody were used as secondary antibodies respectively. Cell nuclei were stained with Hoechst 33258 (Sigma) (panels a). (C) Quantification of the number of cells in which MIZ-1 is strictly nuclear versus cytoplasmic/nucleo-cytoplasmic in cells transfected with an expression vector for MIZ-1 or in cells co-transfected with expression vectors for MIZ-1 and EBNA3A. In the latter case, only cells co-expressing EBNA3A together with MIZ-1 are taken into consideration. The results are the combination of two different experiments in which 1333 cells altogether were observed.

Mentions: MIZ-1 was previously described to be present mostly in the cytoplasm of HeLa cells with only a small proportion of the protein being localized in the nucleus. However, in the presence of overexpressed MYC, most cells show an exclusive nuclear localization pattern of MIZ-1 (36). Since EBNA3A, like MYC, is a nuclear protein, we asked whether the expression of EBNA3A would also induce nuclear localization of MIZ-1. We thus co-transfected HeLa cells with an expression vector for MIZ-1 tagged with a Flag-epitope, together or not with an expression vector for EBNA3A tagged with a Myc-epitope. Immunofluorescence staining of the cells demonstrates that, as expected, MIZ-1 expressed alone is either fully cytoplasmic or nucleo-cytoplasmic in a majority of the cells (Figure 4A and C). By contrast, in the presence of EBNA3A, MIZ-1 is exclusively nuclear in most of the cells (Figure 4B and C). From these experiments, we conclude that co-expression of the two proteins results in their functional interaction and consequent migration of MIZ-1 from the cytoplasm to the nucleus.


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

Bazot Q, Deschamps T, Tafforeau L, Siouda M, Leblanc P, Harth-Hertle ML, Rabourdin-Combe C, Lotteau V, Kempkes B, Tommasino M, Gruffat H, Manet E - Nucleic Acids Res. (2014)

EBNA3A relocalizes MIZ-1 into the nucleus. Expression plasmids for Flag-MIZ-1 alone (A) or Flag-MIZ-1 together with Myc-EBNA3A were transfected into HeLa cells (B and C). Proteins were visualized by indirect immunofluorescence using an anti-Flag polyclonal Ab (for detection of MIZ-1) (panels b) and an anti-tag Myc polyclonal Ab (for detection of EBNA3A) (panels c). A Fluorolink Cy3-labeled goat anti-mouse IgG (H + L) antibody and an Alexa Fluor 488 goat anti-rabbit antibody were used as secondary antibodies respectively. Cell nuclei were stained with Hoechst 33258 (Sigma) (panels a). (C) Quantification of the number of cells in which MIZ-1 is strictly nuclear versus cytoplasmic/nucleo-cytoplasmic in cells transfected with an expression vector for MIZ-1 or in cells co-transfected with expression vectors for MIZ-1 and EBNA3A. In the latter case, only cells co-expressing EBNA3A together with MIZ-1 are taken into consideration. The results are the combination of two different experiments in which 1333 cells altogether were observed.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4150796&req=5

Figure 4: EBNA3A relocalizes MIZ-1 into the nucleus. Expression plasmids for Flag-MIZ-1 alone (A) or Flag-MIZ-1 together with Myc-EBNA3A were transfected into HeLa cells (B and C). Proteins were visualized by indirect immunofluorescence using an anti-Flag polyclonal Ab (for detection of MIZ-1) (panels b) and an anti-tag Myc polyclonal Ab (for detection of EBNA3A) (panels c). A Fluorolink Cy3-labeled goat anti-mouse IgG (H + L) antibody and an Alexa Fluor 488 goat anti-rabbit antibody were used as secondary antibodies respectively. Cell nuclei were stained with Hoechst 33258 (Sigma) (panels a). (C) Quantification of the number of cells in which MIZ-1 is strictly nuclear versus cytoplasmic/nucleo-cytoplasmic in cells transfected with an expression vector for MIZ-1 or in cells co-transfected with expression vectors for MIZ-1 and EBNA3A. In the latter case, only cells co-expressing EBNA3A together with MIZ-1 are taken into consideration. The results are the combination of two different experiments in which 1333 cells altogether were observed.
Mentions: MIZ-1 was previously described to be present mostly in the cytoplasm of HeLa cells with only a small proportion of the protein being localized in the nucleus. However, in the presence of overexpressed MYC, most cells show an exclusive nuclear localization pattern of MIZ-1 (36). Since EBNA3A, like MYC, is a nuclear protein, we asked whether the expression of EBNA3A would also induce nuclear localization of MIZ-1. We thus co-transfected HeLa cells with an expression vector for MIZ-1 tagged with a Flag-epitope, together or not with an expression vector for EBNA3A tagged with a Myc-epitope. Immunofluorescence staining of the cells demonstrates that, as expected, MIZ-1 expressed alone is either fully cytoplasmic or nucleo-cytoplasmic in a majority of the cells (Figure 4A and C). By contrast, in the presence of EBNA3A, MIZ-1 is exclusively nuclear in most of the cells (Figure 4B and C). From these experiments, we conclude that co-expression of the two proteins results in their functional interaction and consequent migration of MIZ-1 from the cytoplasm to the nucleus.

Bottom Line: Focusing on the EBNA3A/MIZ-1 interaction we demonstrate that binding occurs in EBV-infected cells expressing both proteins at endogenous physiological levels and that in the presence of EBNA3A, a significant fraction of MIZ-1 translocates from the cytoplasm to the nucleus.Moreover, we show that a trimeric complex composed of a MIZ-1 recognition DNA element, MIZ-1 and EBNA3A can be formed, and that interaction of MIZ-1 with nucleophosmin (NPM), one of its coactivator, is prevented by EBNA3A.Finally, we show that, in the presence of EBNA3A, expression of the MIZ-1 target gene, CDKN2B, is downregulated and repressive H3K27 marks are established on its promoter region suggesting that EBNA3A directly counteracts the growth inhibitory action of MIZ-1.

View Article: PubMed Central - PubMed

Affiliation: CIRI, International Center for Infectiology Research, Oncogenic Herpesviruses team, Université de Lyon, Lyon 69364, France Université Lyon 1, Centre International de Recherche en Infectiologie, Lyon 69364, France CIRI, International Center for Infectiology Research, Cell Biology of Viral Infections team, Université de Lyon, Lyon 69364, France INSERM, U1111, Lyon 69364, France CNRS, UMR5308, Lyon 69364, France.

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Related in: MedlinePlus