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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 interacts with two distinct domains of Miz1. (A) Schematic representation of MIZ-1 and MIZ-1 deletion mutants. (B, C and D) Expression plasmids for Myc-EBNA3A and Flag-MIZ-1 or Flag-MIZ-1 deletion mutants 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 Flag antibody to detect MIZ-1 and the MIZ-1 deletion mutants or an anti-Myc 9E10 mAb to detect Myc-EBNA3A. Inputs correspond to 8% of the cell extract used for immunoprecipitation.
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Figure 2: EBNA3A interacts with two distinct domains of Miz1. (A) Schematic representation of MIZ-1 and MIZ-1 deletion mutants. (B, C and D) Expression plasmids for Myc-EBNA3A and Flag-MIZ-1 or Flag-MIZ-1 deletion mutants 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 Flag antibody to detect MIZ-1 and the MIZ-1 deletion mutants or an anti-Myc 9E10 mAb to detect Myc-EBNA3A. Inputs correspond to 8% of the cell extract used for immunoprecipitation.

Mentions: In order to precisely map the MIZ-1 domain(s) interacting with EBNA3A, we generated a series of MIZ-1 deletion mutants (Figure 2A) and performed co-immunoprecipitation assays. We first tested two complementary MIZ-1 mutants consisting of either the NT or the CT half of the protein. As can be seen in Figure 2B, both FL-MIZ-1 and MIZ-1CT interact with EBNA3A, whereas MIZ-1NT does not. This corroborates our finding that all MIZ-1 clones isolated in the Y2H screen contained the CT half of the protein albeit with a variable number of zinc finger motifs. We then tested a series of CT deletion mutants. As shown in Figure 2C, the mutants Δ1, Δ2 and Δ3 were all very inefficiently co-immunoprecipitated (lanes 3, 4, 5). By contrast, deletion of the zinc finger motif within the CT domain (mutant ΔZ13) had no effect (lane 6), neither did deletion of the domain between aa 638 and 716 previously shown to be required for the interaction with MYC (36) (Figure 2D, lane 9). Interestingly, we also found an interaction between the POZ domain of MIZ-1 and EBNA3A (Figure 2D, lane 12). This interaction appears to be as efficient as the interaction observed with FL-MIZ-1. This was unexpected since the mutants Δ1, Δ2, Δ3 and NT, that all contain the POZ domain do not interact efficiently with EBNA3A, suggesting that the capacity of the POZ domain to interact with EBNA3A is strongly perturbed in these deletion mutants. In conclusion, EBNA3A binds independently to both the NT POZ domain (aa 1–104) and the extreme CT (aa 739–803) of MIZ-1.


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 interacts with two distinct domains of Miz1. (A) Schematic representation of MIZ-1 and MIZ-1 deletion mutants. (B, C and D) Expression plasmids for Myc-EBNA3A and Flag-MIZ-1 or Flag-MIZ-1 deletion mutants 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 Flag antibody to detect MIZ-1 and the MIZ-1 deletion mutants or an anti-Myc 9E10 mAb to detect Myc-EBNA3A. Inputs correspond to 8% of the cell extract used for immunoprecipitation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150796&req=5

Figure 2: EBNA3A interacts with two distinct domains of Miz1. (A) Schematic representation of MIZ-1 and MIZ-1 deletion mutants. (B, C and D) Expression plasmids for Myc-EBNA3A and Flag-MIZ-1 or Flag-MIZ-1 deletion mutants 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 Flag antibody to detect MIZ-1 and the MIZ-1 deletion mutants or an anti-Myc 9E10 mAb to detect Myc-EBNA3A. Inputs correspond to 8% of the cell extract used for immunoprecipitation.
Mentions: In order to precisely map the MIZ-1 domain(s) interacting with EBNA3A, we generated a series of MIZ-1 deletion mutants (Figure 2A) and performed co-immunoprecipitation assays. We first tested two complementary MIZ-1 mutants consisting of either the NT or the CT half of the protein. As can be seen in Figure 2B, both FL-MIZ-1 and MIZ-1CT interact with EBNA3A, whereas MIZ-1NT does not. This corroborates our finding that all MIZ-1 clones isolated in the Y2H screen contained the CT half of the protein albeit with a variable number of zinc finger motifs. We then tested a series of CT deletion mutants. As shown in Figure 2C, the mutants Δ1, Δ2 and Δ3 were all very inefficiently co-immunoprecipitated (lanes 3, 4, 5). By contrast, deletion of the zinc finger motif within the CT domain (mutant ΔZ13) had no effect (lane 6), neither did deletion of the domain between aa 638 and 716 previously shown to be required for the interaction with MYC (36) (Figure 2D, lane 9). Interestingly, we also found an interaction between the POZ domain of MIZ-1 and EBNA3A (Figure 2D, lane 12). This interaction appears to be as efficient as the interaction observed with FL-MIZ-1. This was unexpected since the mutants Δ1, Δ2, Δ3 and NT, that all contain the POZ domain do not interact efficiently with EBNA3A, suggesting that the capacity of the POZ domain to interact with EBNA3A is strongly perturbed in these deletion mutants. In conclusion, EBNA3A binds independently to both the NT POZ domain (aa 1–104) and the extreme CT (aa 739–803) of MIZ-1.

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