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Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2.

Bazot Q, Paschos K, Skalska L, Kalchschmidt JS, Parker GA, Allday MJ - PLoS Pathog. (2015)

Bottom Line: We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters--widely reported to have cell transformation-associated activity--are regulated by EBNA3A and EBNA3C.ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28 kb upstream of the miR sequences.Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.

ABSTRACT
We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters--widely reported to have cell transformation-associated activity--are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours--including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28 kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.

No MeSH data available.


Related in: MedlinePlus

The increase of p57KIP2 level is associated with de-phosphorylation of Rb and reduced entry into S phase and proliferation.(A) Western blot analysis of extracts from LCL EBNA3A-ERT2 (line 5) cultured with (+) or without (-) 4HT for 30 days showing that after inactivation (and degradation) of the EBNA3A-ERT2 fusion protein, p57KIP2 expression increases, and the amount of hyperphosphorylated Rb (ppRb) is dramatically reduced and is no longer detected using a phospho-Rb-specific antibody. The blot was probed for γ-tubulin as a control for loading. (B) Cell cycle distribution of LCL EBNA3A-ERT2 (line 5) culture with or without 4HT for four weeks was determined by flow cytometry following exposure to EdU for 2 hours (2h pulse). (C) A comparison of the population growth rate between these cells cultured with or without 4HT was analysed by counting the number of viable cells every 2–3 days. Total cell numbers were plotted at each time point. Data are representative of two independent experiments.
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ppat.1005031.g012: The increase of p57KIP2 level is associated with de-phosphorylation of Rb and reduced entry into S phase and proliferation.(A) Western blot analysis of extracts from LCL EBNA3A-ERT2 (line 5) cultured with (+) or without (-) 4HT for 30 days showing that after inactivation (and degradation) of the EBNA3A-ERT2 fusion protein, p57KIP2 expression increases, and the amount of hyperphosphorylated Rb (ppRb) is dramatically reduced and is no longer detected using a phospho-Rb-specific antibody. The blot was probed for γ-tubulin as a control for loading. (B) Cell cycle distribution of LCL EBNA3A-ERT2 (line 5) culture with or without 4HT for four weeks was determined by flow cytometry following exposure to EdU for 2 hours (2h pulse). (C) A comparison of the population growth rate between these cells cultured with or without 4HT was analysed by counting the number of viable cells every 2–3 days. Total cell numbers were plotted at each time point. Data are representative of two independent experiments.

Mentions: In order to determine the consequences of up-regulating p57KIP2 in LCL cells that have grown out after infection of normal primary B cells with the minimum of selection, an early passage (<2 months post-infection) EBNA3A-ERT2 line produced from a mixed donor population of B cells (LCL 5) was used. This line was established in the presence of 4HT and the cells express little or no p57KIP2, but on removal of 4HT from the culture medium, they soon produce substantial amounts of p57KIP2 protein (Figs 11B and 12A). Consistent with the increase in p57KIP2 there was a pronounced reduction in the phosphorylation of the tumour suppressor Rb and this was associated with a gradual reduction in proliferation as revealed by reduced DNA synthesis (EdU incorporation) and reduced cell population growth (Fig 12). Similar analysis was repeated on several other independent EBNA3A-conditional LCLs and produced essentially identical results (S11 Fig). An important enzyme necessary for the phosphorylation of Rb is the cyclin-dependent kinase CDK6. Therefore the most likely explanation for the inhibition of Rb phosphorylation is the binding of p57KIP2 to CDK6 resulting in inactivation of the latter (see co-immunoprecipitations in S12 Fig). The effect of low levels of miR-221/miR-222 and high levels of p57KIP2 on the proliferation of these cells was surprisingly modest, rather less than has been reported previously in EBNA3A-conditional LCLs when EBNA3A was inactivated [10,78]; we do not know the reason for this, but as we have indicated above, different lines can have different properties because of clonal variation. The roles and interactions of the various CDKIs regulated by EBV are discussed in more detail below.


Epstein-Barr Virus Proteins EBNA3A and EBNA3C Together Induce Expression of the Oncogenic MicroRNA Cluster miR-221/miR-222 and Ablate Expression of Its Target p57KIP2.

Bazot Q, Paschos K, Skalska L, Kalchschmidt JS, Parker GA, Allday MJ - PLoS Pathog. (2015)

The increase of p57KIP2 level is associated with de-phosphorylation of Rb and reduced entry into S phase and proliferation.(A) Western blot analysis of extracts from LCL EBNA3A-ERT2 (line 5) cultured with (+) or without (-) 4HT for 30 days showing that after inactivation (and degradation) of the EBNA3A-ERT2 fusion protein, p57KIP2 expression increases, and the amount of hyperphosphorylated Rb (ppRb) is dramatically reduced and is no longer detected using a phospho-Rb-specific antibody. The blot was probed for γ-tubulin as a control for loading. (B) Cell cycle distribution of LCL EBNA3A-ERT2 (line 5) culture with or without 4HT for four weeks was determined by flow cytometry following exposure to EdU for 2 hours (2h pulse). (C) A comparison of the population growth rate between these cells cultured with or without 4HT was analysed by counting the number of viable cells every 2–3 days. Total cell numbers were plotted at each time point. Data are representative of two independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496050&req=5

ppat.1005031.g012: The increase of p57KIP2 level is associated with de-phosphorylation of Rb and reduced entry into S phase and proliferation.(A) Western blot analysis of extracts from LCL EBNA3A-ERT2 (line 5) cultured with (+) or without (-) 4HT for 30 days showing that after inactivation (and degradation) of the EBNA3A-ERT2 fusion protein, p57KIP2 expression increases, and the amount of hyperphosphorylated Rb (ppRb) is dramatically reduced and is no longer detected using a phospho-Rb-specific antibody. The blot was probed for γ-tubulin as a control for loading. (B) Cell cycle distribution of LCL EBNA3A-ERT2 (line 5) culture with or without 4HT for four weeks was determined by flow cytometry following exposure to EdU for 2 hours (2h pulse). (C) A comparison of the population growth rate between these cells cultured with or without 4HT was analysed by counting the number of viable cells every 2–3 days. Total cell numbers were plotted at each time point. Data are representative of two independent experiments.
Mentions: In order to determine the consequences of up-regulating p57KIP2 in LCL cells that have grown out after infection of normal primary B cells with the minimum of selection, an early passage (<2 months post-infection) EBNA3A-ERT2 line produced from a mixed donor population of B cells (LCL 5) was used. This line was established in the presence of 4HT and the cells express little or no p57KIP2, but on removal of 4HT from the culture medium, they soon produce substantial amounts of p57KIP2 protein (Figs 11B and 12A). Consistent with the increase in p57KIP2 there was a pronounced reduction in the phosphorylation of the tumour suppressor Rb and this was associated with a gradual reduction in proliferation as revealed by reduced DNA synthesis (EdU incorporation) and reduced cell population growth (Fig 12). Similar analysis was repeated on several other independent EBNA3A-conditional LCLs and produced essentially identical results (S11 Fig). An important enzyme necessary for the phosphorylation of Rb is the cyclin-dependent kinase CDK6. Therefore the most likely explanation for the inhibition of Rb phosphorylation is the binding of p57KIP2 to CDK6 resulting in inactivation of the latter (see co-immunoprecipitations in S12 Fig). The effect of low levels of miR-221/miR-222 and high levels of p57KIP2 on the proliferation of these cells was surprisingly modest, rather less than has been reported previously in EBNA3A-conditional LCLs when EBNA3A was inactivated [10,78]; we do not know the reason for this, but as we have indicated above, different lines can have different properties because of clonal variation. The roles and interactions of the various CDKIs regulated by EBV are discussed in more detail below.

Bottom Line: We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters--widely reported to have cell transformation-associated activity--are regulated by EBNA3A and EBNA3C.ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28 kb upstream of the miR sequences.Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.

View Article: PubMed Central - PubMed

Affiliation: Molecular Virology, Department of Medicine, Imperial College London, London, United Kingdom.

ABSTRACT
We show that two host-encoded primary RNAs (pri-miRs) and the corresponding microRNA (miR) clusters--widely reported to have cell transformation-associated activity--are regulated by EBNA3A and EBNA3C. Utilising a variety of EBV-transformed lymphoblastoid cell lines (LCLs) carrying knockout-, revertant- or conditional-EBV recombinants, it was possible to demonstrate unambiguously that EBNA3A and EBNA3C are both required for transactivation of the oncogenic miR-221/miR-222 cluster that is expressed at high levels in multiple human tumours--including lymphoma/leukemia. ChIP, ChIP-seq, and chromosome conformation capture analyses indicate that this activation results from direct targeting of both EBV proteins to chromatin at the miR-221/miR-222 genomic locus and activation via a long-range interaction between enhancer elements and the transcription start site of a long non-coding pri-miR located 28 kb upstream of the miR sequences. Reduced levels of miR-221/miR-222 produced by inactivation or deletion of EBNA3A or EBNA3C resulted in increased expression of the cyclin-dependent kinase inhibitor p57KIP2, a well-established target of miR-221/miR-222. MiR blocking experiments confirmed that miR-221/miR-222 target p57KIP2 expression in LCLs. In contrast, EBNA3A and EBNA3C are necessary to silence the tumour suppressor cluster miR-143/miR-145, but here ChIP-seq suggests that repression is probably indirect. This miR cluster is frequently down-regulated or deleted in human cancer, however, the targets in B cells are unknown. Together these data indicate that EBNA3A and EBNA3C contribute to B cell transformation by inhibiting multiple tumour suppressor proteins, not only by direct repression of protein-encoding genes, but also by the manipulation of host long non-coding pri-miRs and miRs.

No MeSH data available.


Related in: MedlinePlus