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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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MIZ-1 interacts preferentially with the EBNA3 homology domain of EBNA3A. (A) Schematic representation of EBNA3A and EBNA3A deletion mutant. (B) Expression plasmids for Flag-MIZ-1 and Myc-EBNA3A or Myc-EBNA3A Δ141–238 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 MIZ-1 or an anti-Myc 9E10 mAb to detect Myc-EBNA3A and Myc-EBNA3A Δ141–238. Input corresponds to 8% of the cell extract used for immunoprecipitation.
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Figure 3: MIZ-1 interacts preferentially with the EBNA3 homology domain of EBNA3A. (A) Schematic representation of EBNA3A and EBNA3A deletion mutant. (B) Expression plasmids for Flag-MIZ-1 and Myc-EBNA3A or Myc-EBNA3A Δ141–238 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 MIZ-1 or an anti-Myc 9E10 mAb to detect Myc-EBNA3A and Myc-EBNA3A Δ141–238. Input corresponds to 8% of the cell extract used for immunoprecipitation.

Mentions: We then delimited the interaction domain of EBNA3A with MIZ-1. Because all three EBNA3s are able to interact with MIZ-1, we suspected the interaction domain to be localized within the homology domain of the EBNA3s (Figure 3A). We thus made use of an EBNA3A mutant with aa 141–238 deleted to test this hypothesis and indeed, co-immunoprecipitation of this deletion mutant with MIZ-1 was drastically reduced (Figure 3B). The importance of this domain for the interaction with both the MIZ-1 POZ domain and the MIZ-1 383–803 CT domain was then confirmed by GST-pulldown experiments (Supplementary Figure S2). Moreover, by using a series of NT and CT EBNA3A deletion mutants, we found that EBNA3A contacts the POZ domain of MIZ-1 via a minimum domain comprising aa 125–172 and that it also interacts with the CT of MIZ-1 via several regions—a major region is located in the first 224 NT amino acids of the protein and another in the CT half of the protein (Supplementary Figure S2).


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)

MIZ-1 interacts preferentially with the EBNA3 homology domain of EBNA3A. (A) Schematic representation of EBNA3A and EBNA3A deletion mutant. (B) Expression plasmids for Flag-MIZ-1 and Myc-EBNA3A or Myc-EBNA3A Δ141–238 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 MIZ-1 or an anti-Myc 9E10 mAb to detect Myc-EBNA3A and Myc-EBNA3A Δ141–238. Input corresponds to 8% of the cell extract used for immunoprecipitation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 3: MIZ-1 interacts preferentially with the EBNA3 homology domain of EBNA3A. (A) Schematic representation of EBNA3A and EBNA3A deletion mutant. (B) Expression plasmids for Flag-MIZ-1 and Myc-EBNA3A or Myc-EBNA3A Δ141–238 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 MIZ-1 or an anti-Myc 9E10 mAb to detect Myc-EBNA3A and Myc-EBNA3A Δ141–238. Input corresponds to 8% of the cell extract used for immunoprecipitation.
Mentions: We then delimited the interaction domain of EBNA3A with MIZ-1. Because all three EBNA3s are able to interact with MIZ-1, we suspected the interaction domain to be localized within the homology domain of the EBNA3s (Figure 3A). We thus made use of an EBNA3A mutant with aa 141–238 deleted to test this hypothesis and indeed, co-immunoprecipitation of this deletion mutant with MIZ-1 was drastically reduced (Figure 3B). The importance of this domain for the interaction with both the MIZ-1 POZ domain and the MIZ-1 383–803 CT domain was then confirmed by GST-pulldown experiments (Supplementary Figure S2). Moreover, by using a series of NT and CT EBNA3A deletion mutants, we found that EBNA3A contacts the POZ domain of MIZ-1 via a minimum domain comprising aa 125–172 and that it also interacts with the CT of MIZ-1 via several regions—a major region is located in the first 224 NT amino acids of the protein and another in the CT half of the protein (Supplementary Figure S2).

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