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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 down-regulates CDKN2B's transcript expression levels in cells treated with cisplatin. LCLwt and LCLΔ3A were treated with 33 μM cisplatin or an equivalent amount of carrier (DMF). Cell aliquots were collected at 0, 2, 4, 6, 8 and 12 h for RNA analysis and at 0, 8, 12 and 24 h for cell cycle analysis. (A) CDKN2B transcripts in total RNA were quantified by RTqPCR normalized to GAPDH RNA. (B) Cells were stained with propidium iodide and analysed by FACS. The data shown are derived from a single experiment but are representative of three experiments. Error bars represent standard deviation from three qPCR replicates.
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Figure 6: EBNA3A down-regulates CDKN2B's transcript expression levels in cells treated with cisplatin. LCLwt and LCLΔ3A were treated with 33 μM cisplatin or an equivalent amount of carrier (DMF). Cell aliquots were collected at 0, 2, 4, 6, 8 and 12 h for RNA analysis and at 0, 8, 12 and 24 h for cell cycle analysis. (A) CDKN2B transcripts in total RNA were quantified by RTqPCR normalized to GAPDH RNA. (B) Cells were stained with propidium iodide and analysed by FACS. The data shown are derived from a single experiment but are representative of three experiments. Error bars represent standard deviation from three qPCR replicates.

Mentions: However, the above experiments were made using normal proliferating cells in which the level of CDKN2B transcript is expected to be low. By contrast, CDKN2B expression is known to be activated at the transcriptional level in a MIZ-1-dependent manner in response to UV irradiation, DNA damage or treatment with TGFβ (39,70,71). In order to test the impact of EBNA3A in conditions where CDKN2B expression is activated, we used cisplatin as a DNA-damage inducing agent. Cell cycle analysis revealed that treatment with cisplatin of either LCLwt or LCLΔ3A results in cell growth arrest and apoptosis as previously reported (72,73), but the response to the cisplatin treatment is clearly delayed in the case of LCLwt compared to LCLΔ3A (Figure 6B). In parallel, quantification by RTqPCR (Figure 6A) of CDKN2B transcript levels in the case of LCLΔ3A over a 12 h time course period, shows a very rapid increase—within 4 h—in CDKN2B transcript levels. The maximum—a 10-fold increase—is reached after 12 h, beyond which the state of the cells did not allow further RNA quantification. In the case of LCLwt, the levels of the CDKN2B transcript are also induced following treatment with cisplatin—with a pattern similar to that observed in LCLΔ3A—but the levels remain very low, staying in the range observed in LCLΔ3A before treatment with cisplatin.


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 down-regulates CDKN2B's transcript expression levels in cells treated with cisplatin. LCLwt and LCLΔ3A were treated with 33 μM cisplatin or an equivalent amount of carrier (DMF). Cell aliquots were collected at 0, 2, 4, 6, 8 and 12 h for RNA analysis and at 0, 8, 12 and 24 h for cell cycle analysis. (A) CDKN2B transcripts in total RNA were quantified by RTqPCR normalized to GAPDH RNA. (B) Cells were stained with propidium iodide and analysed by FACS. The data shown are derived from a single experiment but are representative of three experiments. Error bars represent standard deviation from three qPCR replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 6: EBNA3A down-regulates CDKN2B's transcript expression levels in cells treated with cisplatin. LCLwt and LCLΔ3A were treated with 33 μM cisplatin or an equivalent amount of carrier (DMF). Cell aliquots were collected at 0, 2, 4, 6, 8 and 12 h for RNA analysis and at 0, 8, 12 and 24 h for cell cycle analysis. (A) CDKN2B transcripts in total RNA were quantified by RTqPCR normalized to GAPDH RNA. (B) Cells were stained with propidium iodide and analysed by FACS. The data shown are derived from a single experiment but are representative of three experiments. Error bars represent standard deviation from three qPCR replicates.
Mentions: However, the above experiments were made using normal proliferating cells in which the level of CDKN2B transcript is expected to be low. By contrast, CDKN2B expression is known to be activated at the transcriptional level in a MIZ-1-dependent manner in response to UV irradiation, DNA damage or treatment with TGFβ (39,70,71). In order to test the impact of EBNA3A in conditions where CDKN2B expression is activated, we used cisplatin as a DNA-damage inducing agent. Cell cycle analysis revealed that treatment with cisplatin of either LCLwt or LCLΔ3A results in cell growth arrest and apoptosis as previously reported (72,73), but the response to the cisplatin treatment is clearly delayed in the case of LCLwt compared to LCLΔ3A (Figure 6B). In parallel, quantification by RTqPCR (Figure 6A) of CDKN2B transcript levels in the case of LCLΔ3A over a 12 h time course period, shows a very rapid increase—within 4 h—in CDKN2B transcript levels. The maximum—a 10-fold increase—is reached after 12 h, beyond which the state of the cells did not allow further RNA quantification. In the case of LCLwt, the levels of the CDKN2B transcript are also induced following treatment with cisplatin—with a pattern similar to that observed in LCLΔ3A—but the levels remain very low, staying in the range observed in LCLΔ3A before treatment with cisplatin.

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