Limits...
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.

Show MeSH

Related in: MedlinePlus

Mechanims of inhibition by EBNA3A of the MIZ-1-dependent activation of the CDKN2B transcription. (A) EBNA3A-dependent downregulation of CDKN2B expression is independent of CtBP. The CDKN2p-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A-CtBPmut 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 panels). (B) EBNA3A-dependent downregulation of CDKN2B expression is independent of MYC. The LCLwt and LCLΔ3A were treated or not with the 10058-F4 MYC inhibitor. Quantification of CDKN2B mRNA levels was performed by real time RT-PCR. Histogram bars represent values relative to housekeeping gene GAPDH. Error bars represent standard deviation from three replicate assays based on two different cDNAs. (C) EBNA3A inhibits MIZ-1 interaction with its coactivator Nucleophosmin. MIZ-1 was immunoprecipitated from eithe the LCLwt or the LCLΔ3A with an anti-MIZ-1 specific polyclonal antibody or with an unrelated antibody. Immunoprecipitated MIZ-1 and co-immunoprecipitated Nucleophosmin (NPM) proteins were analyzed by western blotting. Inputs correspond to 5% of the cell extract used for immunoprecipitation.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4150796&req=5

Figure 9: Mechanims of inhibition by EBNA3A of the MIZ-1-dependent activation of the CDKN2B transcription. (A) EBNA3A-dependent downregulation of CDKN2B expression is independent of CtBP. The CDKN2p-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A-CtBPmut 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 panels). (B) EBNA3A-dependent downregulation of CDKN2B expression is independent of MYC. The LCLwt and LCLΔ3A were treated or not with the 10058-F4 MYC inhibitor. Quantification of CDKN2B mRNA levels was performed by real time RT-PCR. Histogram bars represent values relative to housekeeping gene GAPDH. Error bars represent standard deviation from three replicate assays based on two different cDNAs. (C) EBNA3A inhibits MIZ-1 interaction with its coactivator Nucleophosmin. MIZ-1 was immunoprecipitated from eithe the LCLwt or the LCLΔ3A with an anti-MIZ-1 specific polyclonal antibody or with an unrelated antibody. Immunoprecipitated MIZ-1 and co-immunoprecipitated Nucleophosmin (NPM) proteins were analyzed by western blotting. Inputs correspond to 5% of the cell extract used for immunoprecipitation.

Mentions: Although we found a good correlation between the presence of EBNA3A and epigenetic modifications, these modifications do not likely account for the very rapid transcription shut-down of CDKN2B following Dox-induced expression of EBNA3A. Since EBNA3A has previously been shown to repress transcription by recruiting the co-repressor CTBP1 (13), we first asked whether the interaction of EBNA3A with CTBP1 was required for the repression of MIZ-1-dependent transcriptional activation. As EBNA3A has been shown to interact with CTBP1 via two motifs, ALDLS and VLDLS, located in the CT part of EBNA3A, we generated an EBNA3A mutant with these two motifs mutated (EBNA3A-CTBPmut) (13). We then compared the capacity of this mutant to inhibit MIZ-1-dependent transcriptional activation of our reporter construct with that of wild-type EBNA3A. As can be seen Figure 9A, EBNA3A-CTBPmut represses MIZ-1 transcriptional activation as efficiently as EBNA3A, indicating that CTBP binding to EBNA3A is not required for its downregulation of the CDKN2B promoter.


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)

Mechanims of inhibition by EBNA3A of the MIZ-1-dependent activation of the CDKN2B transcription. (A) EBNA3A-dependent downregulation of CDKN2B expression is independent of CtBP. The CDKN2p-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A-CtBPmut 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 panels). (B) EBNA3A-dependent downregulation of CDKN2B expression is independent of MYC. The LCLwt and LCLΔ3A were treated or not with the 10058-F4 MYC inhibitor. Quantification of CDKN2B mRNA levels was performed by real time RT-PCR. Histogram bars represent values relative to housekeeping gene GAPDH. Error bars represent standard deviation from three replicate assays based on two different cDNAs. (C) EBNA3A inhibits MIZ-1 interaction with its coactivator Nucleophosmin. MIZ-1 was immunoprecipitated from eithe the LCLwt or the LCLΔ3A with an anti-MIZ-1 specific polyclonal antibody or with an unrelated antibody. Immunoprecipitated MIZ-1 and co-immunoprecipitated Nucleophosmin (NPM) proteins were analyzed by western blotting. Inputs correspond to 5% 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 9: Mechanims of inhibition by EBNA3A of the MIZ-1-dependent activation of the CDKN2B transcription. (A) EBNA3A-dependent downregulation of CDKN2B expression is independent of CtBP. The CDKN2p-Luc construct was transfected into HeLa cells, together with a CMV-renilla LUC plasmid as internal control and expression plasmids for EBNA3A or EBNA3A-CtBPmut 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 panels). (B) EBNA3A-dependent downregulation of CDKN2B expression is independent of MYC. The LCLwt and LCLΔ3A were treated or not with the 10058-F4 MYC inhibitor. Quantification of CDKN2B mRNA levels was performed by real time RT-PCR. Histogram bars represent values relative to housekeeping gene GAPDH. Error bars represent standard deviation from three replicate assays based on two different cDNAs. (C) EBNA3A inhibits MIZ-1 interaction with its coactivator Nucleophosmin. MIZ-1 was immunoprecipitated from eithe the LCLwt or the LCLΔ3A with an anti-MIZ-1 specific polyclonal antibody or with an unrelated antibody. Immunoprecipitated MIZ-1 and co-immunoprecipitated Nucleophosmin (NPM) proteins were analyzed by western blotting. Inputs correspond to 5% of the cell extract used for immunoprecipitation.
Mentions: Although we found a good correlation between the presence of EBNA3A and epigenetic modifications, these modifications do not likely account for the very rapid transcription shut-down of CDKN2B following Dox-induced expression of EBNA3A. Since EBNA3A has previously been shown to repress transcription by recruiting the co-repressor CTBP1 (13), we first asked whether the interaction of EBNA3A with CTBP1 was required for the repression of MIZ-1-dependent transcriptional activation. As EBNA3A has been shown to interact with CTBP1 via two motifs, ALDLS and VLDLS, located in the CT part of EBNA3A, we generated an EBNA3A mutant with these two motifs mutated (EBNA3A-CTBPmut) (13). We then compared the capacity of this mutant to inhibit MIZ-1-dependent transcriptional activation of our reporter construct with that of wild-type EBNA3A. As can be seen Figure 9A, EBNA3A-CTBPmut represses MIZ-1 transcriptional activation as efficiently as EBNA3A, indicating that CTBP binding to EBNA3A is not required for its downregulation of the CDKN2B promoter.

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.

Show MeSH
Related in: MedlinePlus