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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 activates the CDKN2B minimal promoter and specifically binds the INR in association with MIZ-1. (A) Schematic representation of the CDKN2Bp Luciferase reporter construct. The Firefly luciferase reporter gene was placed under the control of a −113/+160 bp fragment from the human CDKN2B promoter. INR: Initiator element. The arrow indicates the start of the transcription. (B) EBNA3A activates the CDKN2B minimal promoter. The CDKN2Bp-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A Δ141–238 and MIZ-1 as indicated in the figure. Results are plotted relative to the co-transfected CMV-renilla-LUC plasmid. Error bars represent standard deviations from three replicate assays. Protein expression levels were controled by western blotting using an anti-EBNA3A or an anti-MIZ-1 antibody for one representative experiment (bottom panel). (C) EBNA3A in association with MIZ-1 binds an oligonucleotide carrying the INR sequence. Whole LCLwt extracts were subjected to precipitation, using the CDKN2B promoter oligonucleotide—or a mutated control oligonucleotide that does not bind MIZ-1—and examined for the indicated proteins.
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Figure 7: EBNA3A activates the CDKN2B minimal promoter and specifically binds the INR in association with MIZ-1. (A) Schematic representation of the CDKN2Bp Luciferase reporter construct. The Firefly luciferase reporter gene was placed under the control of a −113/+160 bp fragment from the human CDKN2B promoter. INR: Initiator element. The arrow indicates the start of the transcription. (B) EBNA3A activates the CDKN2B minimal promoter. The CDKN2Bp-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A Δ141–238 and MIZ-1 as indicated in the figure. Results are plotted relative to the co-transfected CMV-renilla-LUC plasmid. Error bars represent standard deviations from three replicate assays. Protein expression levels were controled by western blotting using an anti-EBNA3A or an anti-MIZ-1 antibody for one representative experiment (bottom panel). (C) EBNA3A in association with MIZ-1 binds an oligonucleotide carrying the INR sequence. Whole LCLwt extracts were subjected to precipitation, using the CDKN2B promoter oligonucleotide—or a mutated control oligonucleotide that does not bind MIZ-1—and examined for the indicated proteins.

Mentions: We next wanted to determine whether the repression effect of EBNA3A on CDKN2B expression was directly linked to MIZ-1 regulation of the CDKN2B promoter. To this end, we used a reporter construct in which the Firefly luciferase coding sequence was placed under the control of a minimal CDKN2B promoter (−113/+160). This contains an Initiator element (Inr) previously shown to recruit MIZ-1 to the promoter by direct binding of the protein to this sequence (37) (Figure 7A). As shown in Figure 7B, the reporter construct was activated by MIZ-1 in HeLa cells and this activation was efficiently repressed by EBNA3A but not by EBNA3A Δ141–238, a deletion mutant whose interaction with MIZ-1 is drastically reduced. These results indicate that EBNA3A specifically represses CDKN2B transcriptional activation by 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 activates the CDKN2B minimal promoter and specifically binds the INR in association with MIZ-1. (A) Schematic representation of the CDKN2Bp Luciferase reporter construct. The Firefly luciferase reporter gene was placed under the control of a −113/+160 bp fragment from the human CDKN2B promoter. INR: Initiator element. The arrow indicates the start of the transcription. (B) EBNA3A activates the CDKN2B minimal promoter. The CDKN2Bp-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A Δ141–238 and MIZ-1 as indicated in the figure. Results are plotted relative to the co-transfected CMV-renilla-LUC plasmid. Error bars represent standard deviations from three replicate assays. Protein expression levels were controled by western blotting using an anti-EBNA3A or an anti-MIZ-1 antibody for one representative experiment (bottom panel). (C) EBNA3A in association with MIZ-1 binds an oligonucleotide carrying the INR sequence. Whole LCLwt extracts were subjected to precipitation, using the CDKN2B promoter oligonucleotide—or a mutated control oligonucleotide that does not bind MIZ-1—and examined for the indicated proteins.
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Figure 7: EBNA3A activates the CDKN2B minimal promoter and specifically binds the INR in association with MIZ-1. (A) Schematic representation of the CDKN2Bp Luciferase reporter construct. The Firefly luciferase reporter gene was placed under the control of a −113/+160 bp fragment from the human CDKN2B promoter. INR: Initiator element. The arrow indicates the start of the transcription. (B) EBNA3A activates the CDKN2B minimal promoter. The CDKN2Bp-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A Δ141–238 and MIZ-1 as indicated in the figure. Results are plotted relative to the co-transfected CMV-renilla-LUC plasmid. Error bars represent standard deviations from three replicate assays. Protein expression levels were controled by western blotting using an anti-EBNA3A or an anti-MIZ-1 antibody for one representative experiment (bottom panel). (C) EBNA3A in association with MIZ-1 binds an oligonucleotide carrying the INR sequence. Whole LCLwt extracts were subjected to precipitation, using the CDKN2B promoter oligonucleotide—or a mutated control oligonucleotide that does not bind MIZ-1—and examined for the indicated proteins.
Mentions: We next wanted to determine whether the repression effect of EBNA3A on CDKN2B expression was directly linked to MIZ-1 regulation of the CDKN2B promoter. To this end, we used a reporter construct in which the Firefly luciferase coding sequence was placed under the control of a minimal CDKN2B promoter (−113/+160). This contains an Initiator element (Inr) previously shown to recruit MIZ-1 to the promoter by direct binding of the protein to this sequence (37) (Figure 7A). As shown in Figure 7B, the reporter construct was activated by MIZ-1 in HeLa cells and this activation was efficiently repressed by EBNA3A but not by EBNA3A Δ141–238, a deletion mutant whose interaction with MIZ-1 is drastically reduced. These results indicate that EBNA3A specifically represses CDKN2B transcriptional activation by 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