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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

Activation of EBNA3A represses miR-143/miR-145 expression.(A) MiR-143/miR-145 and RNU48 expression were determined by qPCR in EBNA3A-ERT2 LCL established without the presence of 4HT (never HT) and 28 days after addition of 4HT to culture medium (+HT). (B) As in (A) but analysing the expression of ALAS1, CXCL9 and CXCL10 as a controls for activation of EBNA3A following the addition of 4HT.
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ppat.1005031.g005: Activation of EBNA3A represses miR-143/miR-145 expression.(A) MiR-143/miR-145 and RNU48 expression were determined by qPCR in EBNA3A-ERT2 LCL established without the presence of 4HT (never HT) and 28 days after addition of 4HT to culture medium (+HT). (B) As in (A) but analysing the expression of ALAS1, CXCL9 and CXCL10 as a controls for activation of EBNA3A following the addition of 4HT.

Mentions: Several EBNA3A-ERT2 LCLs produced from two different individual B cell donors (D11 for LCLs number 1–2 and D13 for cell lines number 3–4) and a mixed donor population of B cells (LCL number 5) were established in the presence of 4HT and then analyzed ~30 days after removal (-4HT) or leaving in 4HT (+4HT) (Fig 4A). Consistently on removal of 4HT (washed), miR-221 and miR-222 were expressed at a lower level (Fig 4B), whereas miR-143 and miR-145 were modestly induced (Fig 4C). As with the experiments using conditional EBNA3C, when EBNA3A-conditional cells that had been grown into LCLs in the absence of 4HT (never HT), there was a substantial repression of miR-143 and miR-145 when 4HT was added to the culture medium (Fig 5A and S1 Fig). All these experiments showed that—like EBNA3C –active EBNA3A is necessary for the regulation of both miR clusters, ruling out the possibility of clonal selection as an explanation for the changes in expression seen in the EBNA3A-KO lines. EBNA3A-ERT2 function was further validated by qPCR for expression of previously characterised EBNA3A target genes (CXCL9 and CXCL10 [21], Fig 5B and S5 Fig).


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)

Activation of EBNA3A represses miR-143/miR-145 expression.(A) MiR-143/miR-145 and RNU48 expression were determined by qPCR in EBNA3A-ERT2 LCL established without the presence of 4HT (never HT) and 28 days after addition of 4HT to culture medium (+HT). (B) As in (A) but analysing the expression of ALAS1, CXCL9 and CXCL10 as a controls for activation of EBNA3A following the addition of 4HT.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005031.g005: Activation of EBNA3A represses miR-143/miR-145 expression.(A) MiR-143/miR-145 and RNU48 expression were determined by qPCR in EBNA3A-ERT2 LCL established without the presence of 4HT (never HT) and 28 days after addition of 4HT to culture medium (+HT). (B) As in (A) but analysing the expression of ALAS1, CXCL9 and CXCL10 as a controls for activation of EBNA3A following the addition of 4HT.
Mentions: Several EBNA3A-ERT2 LCLs produced from two different individual B cell donors (D11 for LCLs number 1–2 and D13 for cell lines number 3–4) and a mixed donor population of B cells (LCL number 5) were established in the presence of 4HT and then analyzed ~30 days after removal (-4HT) or leaving in 4HT (+4HT) (Fig 4A). Consistently on removal of 4HT (washed), miR-221 and miR-222 were expressed at a lower level (Fig 4B), whereas miR-143 and miR-145 were modestly induced (Fig 4C). As with the experiments using conditional EBNA3C, when EBNA3A-conditional cells that had been grown into LCLs in the absence of 4HT (never HT), there was a substantial repression of miR-143 and miR-145 when 4HT was added to the culture medium (Fig 5A and S1 Fig). All these experiments showed that—like EBNA3C –active EBNA3A is necessary for the regulation of both miR clusters, ruling out the possibility of clonal selection as an explanation for the changes in expression seen in the EBNA3A-KO lines. EBNA3A-ERT2 function was further validated by qPCR for expression of previously characterised EBNA3A target genes (CXCL9 and CXCL10 [21], Fig 5B and S5 Fig).

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