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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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Epigenetic regulation of the CDKN2B locus by EBNA3A. (A) Schematic representation of the CDKN2B promoter region depicting the location of the fragments amplified in the ChIP-qPCR assays. The dark gray box represents the initiator element, the light gray boxes, the first two CDKN2B (p15INK4b) exons. (B and C) Comparative ChIP-qPCR analysis between LCLs transformed with either wt EBV or ΔEBNA3A EBV, quantifying the H3K27me3 (B) or the H3K4me2 (C) across the CDKN2B locus. The level of immunoprecipitated chromatin is expressed as a percentage of the input chromatin. Primers that amplify the promoter of inactive myoglobin (myo) were used as a control. Experiments were performed twice with two independent chromatin preparations.
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Figure 8: Epigenetic regulation of the CDKN2B locus by EBNA3A. (A) Schematic representation of the CDKN2B promoter region depicting the location of the fragments amplified in the ChIP-qPCR assays. The dark gray box represents the initiator element, the light gray boxes, the first two CDKN2B (p15INK4b) exons. (B and C) Comparative ChIP-qPCR analysis between LCLs transformed with either wt EBV or ΔEBNA3A EBV, quantifying the H3K27me3 (B) or the H3K4me2 (C) across the CDKN2B locus. The level of immunoprecipitated chromatin is expressed as a percentage of the input chromatin. Primers that amplify the promoter of inactive myoglobin (myo) were used as a control. Experiments were performed twice with two independent chromatin preparations.

Mentions: EBNA3 proteins have previously been found to trigger the recruitment of polycomb repressive complex 2 (PRC2) core subunits and the trimethylation of H3K27 around both the BCL2L11 promoter (27) and the CDKN2A locus encoding p16INK4a and p14ARF (17,16). We thus investigated the variation in H3K27 and H3K4 methylation levels at the CDKN2B promoter, depending on the presence or absence of EBNA3A. For this, ChIP, coupled with quantitative PCR (qPCR) assays directed across the CDKN2B locus (Figure 8A), were performed using EBVwt and EBVΔ3A LCLs. As can be seen in Figure 8B, the presence of EBNA3A correlates with a net increase in the repressive H3K27me3 mark, specifically centered on the site of the Inr motif. Concomitantly, as shown in Figure 8C, the H3K4 methylation was only marginally changed, which is reminiscent of what has also been observed in the case of the BCL2L11 and CDKN2A loci (27,17,16). From these experiments we conclude that the repression mechanism of CDKN2B expression by EBNA3A is due, at least partially, to epigenetic modifications, in particular the increase in H3K27 tri-methylation similar to what has been observed for the BCL2L11 and CDKN2A promoter regions.


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)

Epigenetic regulation of the CDKN2B locus by EBNA3A. (A) Schematic representation of the CDKN2B promoter region depicting the location of the fragments amplified in the ChIP-qPCR assays. The dark gray box represents the initiator element, the light gray boxes, the first two CDKN2B (p15INK4b) exons. (B and C) Comparative ChIP-qPCR analysis between LCLs transformed with either wt EBV or ΔEBNA3A EBV, quantifying the H3K27me3 (B) or the H3K4me2 (C) across the CDKN2B locus. The level of immunoprecipitated chromatin is expressed as a percentage of the input chromatin. Primers that amplify the promoter of inactive myoglobin (myo) were used as a control. Experiments were performed twice with two independent chromatin preparations.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 8: Epigenetic regulation of the CDKN2B locus by EBNA3A. (A) Schematic representation of the CDKN2B promoter region depicting the location of the fragments amplified in the ChIP-qPCR assays. The dark gray box represents the initiator element, the light gray boxes, the first two CDKN2B (p15INK4b) exons. (B and C) Comparative ChIP-qPCR analysis between LCLs transformed with either wt EBV or ΔEBNA3A EBV, quantifying the H3K27me3 (B) or the H3K4me2 (C) across the CDKN2B locus. The level of immunoprecipitated chromatin is expressed as a percentage of the input chromatin. Primers that amplify the promoter of inactive myoglobin (myo) were used as a control. Experiments were performed twice with two independent chromatin preparations.
Mentions: EBNA3 proteins have previously been found to trigger the recruitment of polycomb repressive complex 2 (PRC2) core subunits and the trimethylation of H3K27 around both the BCL2L11 promoter (27) and the CDKN2A locus encoding p16INK4a and p14ARF (17,16). We thus investigated the variation in H3K27 and H3K4 methylation levels at the CDKN2B promoter, depending on the presence or absence of EBNA3A. For this, ChIP, coupled with quantitative PCR (qPCR) assays directed across the CDKN2B locus (Figure 8A), were performed using EBVwt and EBVΔ3A LCLs. As can be seen in Figure 8B, the presence of EBNA3A correlates with a net increase in the repressive H3K27me3 mark, specifically centered on the site of the Inr motif. Concomitantly, as shown in Figure 8C, the H3K4 methylation was only marginally changed, which is reminiscent of what has also been observed in the case of the BCL2L11 and CDKN2A loci (27,17,16). From these experiments we conclude that the repression mechanism of CDKN2B expression by EBNA3A is due, at least partially, to epigenetic modifications, in particular the increase in H3K27 tri-methylation similar to what has been observed for the BCL2L11 and CDKN2A promoter regions.

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