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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

Validation of EBNA3A-ERT2 conditional LCLs.Expression of latency-associated EBV proteins EBNA1, EBNA2, EBNA3A-ERT2, EBNA3B, EBNA3C, EBNA-LP and LMP1 were analysed by Western blotting extracts from four EBNA3A-ERT2 LCLs (named 1, 2, 3 and 4) cultured in medium with 4HT (+) and 29 days without 4HT (-). The blot was probed for γ-tubulin as a control for loading.
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ppat.1005031.g003: Validation of EBNA3A-ERT2 conditional LCLs.Expression of latency-associated EBV proteins EBNA1, EBNA2, EBNA3A-ERT2, EBNA3B, EBNA3C, EBNA-LP and LMP1 were analysed by Western blotting extracts from four EBNA3A-ERT2 LCLs (named 1, 2, 3 and 4) cultured in medium with 4HT (+) and 29 days without 4HT (-). The blot was probed for γ-tubulin as a control for loading.

Mentions: We, and others, have found that when EBNA3A-KO LCLs are produced there is a tendency for the selection of changes in gene expression as the lines become more clonal (for example loss of/reduced retinoblastoma (Rb) expression has been reported in independent studies [11,12]). In order to establish that the changes in miR expression highlighted by the TLDA (and subsequently confirmed by qPCR) were due to direct regulation of transcription by EBNA3A –rather than the result of selection during clone development—it was necessary to construct and validate an EBV recombinant that is conditional for EBNA3A (EBNA3A-ERT2) and use this to produce new LCLs. EBNA3A-ERT2 is very similar to the EBNA3C conditional virus used in the initial assays (Fig 1 and [12,30]), but has a fusion of the C-terminus of EBNA3A with a slightly more modified estrogen-receptor that responds to 4HT but not estrogen (see Material and Methods). LCLs established with these viruses were validated for the expression of EBNA3A and other EBV latency-associated proteins by western blotting; in the absence of 4HT the EBNA3A-ERT2 fusion protein is almost completely degraded, but the expression of other latency-associated EBV proteins was unaltered (Fig 3).


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)

Validation of EBNA3A-ERT2 conditional LCLs.Expression of latency-associated EBV proteins EBNA1, EBNA2, EBNA3A-ERT2, EBNA3B, EBNA3C, EBNA-LP and LMP1 were analysed by Western blotting extracts from four EBNA3A-ERT2 LCLs (named 1, 2, 3 and 4) cultured in medium with 4HT (+) and 29 days without 4HT (-). The blot was probed for γ-tubulin as a control for loading.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005031.g003: Validation of EBNA3A-ERT2 conditional LCLs.Expression of latency-associated EBV proteins EBNA1, EBNA2, EBNA3A-ERT2, EBNA3B, EBNA3C, EBNA-LP and LMP1 were analysed by Western blotting extracts from four EBNA3A-ERT2 LCLs (named 1, 2, 3 and 4) cultured in medium with 4HT (+) and 29 days without 4HT (-). The blot was probed for γ-tubulin as a control for loading.
Mentions: We, and others, have found that when EBNA3A-KO LCLs are produced there is a tendency for the selection of changes in gene expression as the lines become more clonal (for example loss of/reduced retinoblastoma (Rb) expression has been reported in independent studies [11,12]). In order to establish that the changes in miR expression highlighted by the TLDA (and subsequently confirmed by qPCR) were due to direct regulation of transcription by EBNA3A –rather than the result of selection during clone development—it was necessary to construct and validate an EBV recombinant that is conditional for EBNA3A (EBNA3A-ERT2) and use this to produce new LCLs. EBNA3A-ERT2 is very similar to the EBNA3C conditional virus used in the initial assays (Fig 1 and [12,30]), but has a fusion of the C-terminus of EBNA3A with a slightly more modified estrogen-receptor that responds to 4HT but not estrogen (see Material and Methods). LCLs established with these viruses were validated for the expression of EBNA3A and other EBV latency-associated proteins by western blotting; in the absence of 4HT the EBNA3A-ERT2 fusion protein is almost completely degraded, but the expression of other latency-associated EBV proteins was unaltered (Fig 3).

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