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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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The EBNA3 proteins interact with the cellular protein MIZ-1. (A) Schematic representation of MIZ-1. The black box corresponds to the POZ domain of the protein and the dark gray boxes to zinc finger motifs. MIZ-1 380–803 corresponds to the protein expressed from the longest cDNA isolated in the Y2H screen. (B) Expression plasmids for Flag-EBNA3A (F.E3A), Flag-EBNA3B (F.E3B), Flag-EBNA3C (F.E3C) and MIZ-1 were transfected into HeLa cells as indicated. Cellular extracts were immunoprecipitated with an M2 anti-Flag mAb affinity gel and the immunoprecipitated complexes were analyzed by western blotting using an anti-Flag polyclonal antibody to detect the EBNA3 proteins (top panel) or an anti-Miz polyclonal antibody (bottom panel). Input corresponds to 8% of the cell extracts used for immunoprecipitation. (C) 35S-labeled EBNA3A, EBNA3B or EBNA3C were incubated with purified GST or GST-MIZ-1 383–803 bound to glutathione sepharose beads. The bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. In lane 1, the equivalent of one-twelfth of the EBNA3A, EBNA3B or EBNA3C-expressing rabbit reticulocyte lysates used in each assay was loaded onto the gel. (D) EBNA3A or MIZ-1 were immunoprecipitated (IP) from a lymphoblastoid cell line protein extract using anti-EBNA3A or anti-MIZ-1-specific polyclonal antibodies as indicated or with an anti-Flag antibody as control. Immuno and co-immunoprecipitated EBNA3A and MIZ-1 proteins were analyzed by western blotting. Input corresponds to 25% of the cell extract used for immunoprecipitation.
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Figure 1: The EBNA3 proteins interact with the cellular protein MIZ-1. (A) Schematic representation of MIZ-1. The black box corresponds to the POZ domain of the protein and the dark gray boxes to zinc finger motifs. MIZ-1 380–803 corresponds to the protein expressed from the longest cDNA isolated in the Y2H screen. (B) Expression plasmids for Flag-EBNA3A (F.E3A), Flag-EBNA3B (F.E3B), Flag-EBNA3C (F.E3C) and MIZ-1 were transfected into HeLa cells as indicated. Cellular extracts were immunoprecipitated with an M2 anti-Flag mAb affinity gel and the immunoprecipitated complexes were analyzed by western blotting using an anti-Flag polyclonal antibody to detect the EBNA3 proteins (top panel) or an anti-Miz polyclonal antibody (bottom panel). Input corresponds to 8% of the cell extracts used for immunoprecipitation. (C) 35S-labeled EBNA3A, EBNA3B or EBNA3C were incubated with purified GST or GST-MIZ-1 383–803 bound to glutathione sepharose beads. The bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. In lane 1, the equivalent of one-twelfth of the EBNA3A, EBNA3B or EBNA3C-expressing rabbit reticulocyte lysates used in each assay was loaded onto the gel. (D) EBNA3A or MIZ-1 were immunoprecipitated (IP) from a lymphoblastoid cell line protein extract using anti-EBNA3A or anti-MIZ-1-specific polyclonal antibodies as indicated or with an anti-Flag antibody as control. Immuno and co-immunoprecipitated EBNA3A and MIZ-1 proteins were analyzed by western blotting. Input corresponds to 25% of the cell extract used for immunoprecipitation.

Mentions: Since all MIZ-1 cDNAs isolated from the screen contain only sequences coding for the CT region of the protein, each possessing a variable number of internal zinc finger repeats (nine for the longest) (Figure 1A), we first checked if the EBNA3s interact with full-length MIZ-1 (FL-MIZ-1). For this, we performed a co-immunoprecipitation assay using HeLa cells transfected with expression vectors for FL-MIZ-1 tagged with a Myc epitope, and each one of the EBNA3s tagged with a Flag epitope. As can be seen in Figure 1B, FL-MIZ-1 was specifically co-immunoprecipitated with all three EBNA3s.


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)

The EBNA3 proteins interact with the cellular protein MIZ-1. (A) Schematic representation of MIZ-1. The black box corresponds to the POZ domain of the protein and the dark gray boxes to zinc finger motifs. MIZ-1 380–803 corresponds to the protein expressed from the longest cDNA isolated in the Y2H screen. (B) Expression plasmids for Flag-EBNA3A (F.E3A), Flag-EBNA3B (F.E3B), Flag-EBNA3C (F.E3C) and MIZ-1 were transfected into HeLa cells as indicated. Cellular extracts were immunoprecipitated with an M2 anti-Flag mAb affinity gel and the immunoprecipitated complexes were analyzed by western blotting using an anti-Flag polyclonal antibody to detect the EBNA3 proteins (top panel) or an anti-Miz polyclonal antibody (bottom panel). Input corresponds to 8% of the cell extracts used for immunoprecipitation. (C) 35S-labeled EBNA3A, EBNA3B or EBNA3C were incubated with purified GST or GST-MIZ-1 383–803 bound to glutathione sepharose beads. The bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. In lane 1, the equivalent of one-twelfth of the EBNA3A, EBNA3B or EBNA3C-expressing rabbit reticulocyte lysates used in each assay was loaded onto the gel. (D) EBNA3A or MIZ-1 were immunoprecipitated (IP) from a lymphoblastoid cell line protein extract using anti-EBNA3A or anti-MIZ-1-specific polyclonal antibodies as indicated or with an anti-Flag antibody as control. Immuno and co-immunoprecipitated EBNA3A and MIZ-1 proteins were analyzed by western blotting. Input corresponds to 25% of the cell extract used for immunoprecipitation.
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Related In: Results  -  Collection

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Figure 1: The EBNA3 proteins interact with the cellular protein MIZ-1. (A) Schematic representation of MIZ-1. The black box corresponds to the POZ domain of the protein and the dark gray boxes to zinc finger motifs. MIZ-1 380–803 corresponds to the protein expressed from the longest cDNA isolated in the Y2H screen. (B) Expression plasmids for Flag-EBNA3A (F.E3A), Flag-EBNA3B (F.E3B), Flag-EBNA3C (F.E3C) and MIZ-1 were transfected into HeLa cells as indicated. Cellular extracts were immunoprecipitated with an M2 anti-Flag mAb affinity gel and the immunoprecipitated complexes were analyzed by western blotting using an anti-Flag polyclonal antibody to detect the EBNA3 proteins (top panel) or an anti-Miz polyclonal antibody (bottom panel). Input corresponds to 8% of the cell extracts used for immunoprecipitation. (C) 35S-labeled EBNA3A, EBNA3B or EBNA3C were incubated with purified GST or GST-MIZ-1 383–803 bound to glutathione sepharose beads. The bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. In lane 1, the equivalent of one-twelfth of the EBNA3A, EBNA3B or EBNA3C-expressing rabbit reticulocyte lysates used in each assay was loaded onto the gel. (D) EBNA3A or MIZ-1 were immunoprecipitated (IP) from a lymphoblastoid cell line protein extract using anti-EBNA3A or anti-MIZ-1-specific polyclonal antibodies as indicated or with an anti-Flag antibody as control. Immuno and co-immunoprecipitated EBNA3A and MIZ-1 proteins were analyzed by western blotting. Input corresponds to 25% of the cell extract used for immunoprecipitation.
Mentions: Since all MIZ-1 cDNAs isolated from the screen contain only sequences coding for the CT region of the protein, each possessing a variable number of internal zinc finger repeats (nine for the longest) (Figure 1A), we first checked if the EBNA3s interact with full-length MIZ-1 (FL-MIZ-1). For this, we performed a co-immunoprecipitation assay using HeLa cells transfected with expression vectors for FL-MIZ-1 tagged with a Myc epitope, and each one of the EBNA3s tagged with a Flag epitope. As can be seen in Figure 1B, FL-MIZ-1 was specifically co-immunoprecipitated with all three EBNA3s.

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