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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

Regulation of miR clusters by EBNA3A and EBNA3C.(A) MiR-221 and miR-222 expression in four independent LCLs EBNA3A-KO and EBNA3A-REV (D1, D2, D3 and D4) as well as two p16- LCL 3CHT (A2 and C1) cultured for 29 days with (+HT) or without 4HT (Washed) were determined by real time quantitative RT-PCR (qPCR). MiR-221/miR-222 expression was normalized to RNU6B and is shown relative to each “wild type” cell LCL EBNA3A-REV (3A-REV) or p16- 3CHT cultured with 4HT (+HT). (B) As in (A) but analysing miR-143 and miR-145 expression.
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ppat.1005031.g001: Regulation of miR clusters by EBNA3A and EBNA3C.(A) MiR-221 and miR-222 expression in four independent LCLs EBNA3A-KO and EBNA3A-REV (D1, D2, D3 and D4) as well as two p16- LCL 3CHT (A2 and C1) cultured for 29 days with (+HT) or without 4HT (Washed) were determined by real time quantitative RT-PCR (qPCR). MiR-221/miR-222 expression was normalized to RNU6B and is shown relative to each “wild type” cell LCL EBNA3A-REV (3A-REV) or p16- 3CHT cultured with 4HT (+HT). (B) As in (A) but analysing miR-143 and miR-145 expression.

Mentions: Fig 1A shows the results of qPCR assays for miR-221/miR-222 in extracts from four independent EBNA3A-KO LCLs and four LCLs established with revertant viruses (and therefore expressing all the latency-associated EBV proteins). Consistently, failure to express EBNA3A resulted in a large reduction in miR-221 and miR-222 expression (Fig 1A and S1 Fig). Similarly using two independent LCLs conditional for EBNA3C function (3CHT, established in a p16- B cell background in order to allow the cells to proliferate in the absence of EBNA3C, as described in [30]), it was shown that removal of the activating ligand (4HT) resulted in a less substantial, but clearly significant reduction in both miR-221 and miR-222 expression (Fig 1A and S1 Fig). Analysis of the same lines for expression of miR-143 and miR-145 confirmed the TLDA result showing that in the absence of EBNA3A or functional EBNA3C (by washing out 4HT) there was an increase in the expression of miR-143 and miR-145 (Fig 1B). When EBNA3A was deleted there was particularly robust expression of both miR-143 and miR-145. When conditional EBNA3C was inactivated there was a rather modest, but still significant and reproducible increase in both miRs. Consistent with this, when 4HT was added to a p16- EBNA3C-conditional LCL that had been established in its absence (never HT), there was a substantial repression of the miR-143/miR-145 cluster (Fig 2A and S1 Fig). The differential expression of miRs are not due to the 4HT treatment since no significant change in miR expression was detected in two wild-type LCLs (D11 and D13 LCL WT) treated with 4HT for 30 days (S2 Fig). Control RNAs RNU48 and ALAS1 were unaffected by the EBNA3A or EBNA3C status of the LCLs (S3 Fig). Expression of protein-encoding gene clusters previously reported to be regulated by EBNA3A and EBNA3C (eg CXCL9/CXCL10 and ADAM28/ADAMDEC1) was as expected from previous reports ([20,21,28], Fig 2B and S3 Fig). Although in some knockout and revertant LCL pairs, EBNA3B expression appeared to influence the levels of these miR clusters, the changes were very slight and/or inconsistent (S1 and S4 Figs).


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)

Regulation of miR clusters by EBNA3A and EBNA3C.(A) MiR-221 and miR-222 expression in four independent LCLs EBNA3A-KO and EBNA3A-REV (D1, D2, D3 and D4) as well as two p16- LCL 3CHT (A2 and C1) cultured for 29 days with (+HT) or without 4HT (Washed) were determined by real time quantitative RT-PCR (qPCR). MiR-221/miR-222 expression was normalized to RNU6B and is shown relative to each “wild type” cell LCL EBNA3A-REV (3A-REV) or p16- 3CHT cultured with 4HT (+HT). (B) As in (A) but analysing miR-143 and miR-145 expression.
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Related In: Results  -  Collection

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ppat.1005031.g001: Regulation of miR clusters by EBNA3A and EBNA3C.(A) MiR-221 and miR-222 expression in four independent LCLs EBNA3A-KO and EBNA3A-REV (D1, D2, D3 and D4) as well as two p16- LCL 3CHT (A2 and C1) cultured for 29 days with (+HT) or without 4HT (Washed) were determined by real time quantitative RT-PCR (qPCR). MiR-221/miR-222 expression was normalized to RNU6B and is shown relative to each “wild type” cell LCL EBNA3A-REV (3A-REV) or p16- 3CHT cultured with 4HT (+HT). (B) As in (A) but analysing miR-143 and miR-145 expression.
Mentions: Fig 1A shows the results of qPCR assays for miR-221/miR-222 in extracts from four independent EBNA3A-KO LCLs and four LCLs established with revertant viruses (and therefore expressing all the latency-associated EBV proteins). Consistently, failure to express EBNA3A resulted in a large reduction in miR-221 and miR-222 expression (Fig 1A and S1 Fig). Similarly using two independent LCLs conditional for EBNA3C function (3CHT, established in a p16- B cell background in order to allow the cells to proliferate in the absence of EBNA3C, as described in [30]), it was shown that removal of the activating ligand (4HT) resulted in a less substantial, but clearly significant reduction in both miR-221 and miR-222 expression (Fig 1A and S1 Fig). Analysis of the same lines for expression of miR-143 and miR-145 confirmed the TLDA result showing that in the absence of EBNA3A or functional EBNA3C (by washing out 4HT) there was an increase in the expression of miR-143 and miR-145 (Fig 1B). When EBNA3A was deleted there was particularly robust expression of both miR-143 and miR-145. When conditional EBNA3C was inactivated there was a rather modest, but still significant and reproducible increase in both miRs. Consistent with this, when 4HT was added to a p16- EBNA3C-conditional LCL that had been established in its absence (never HT), there was a substantial repression of the miR-143/miR-145 cluster (Fig 2A and S1 Fig). The differential expression of miRs are not due to the 4HT treatment since no significant change in miR expression was detected in two wild-type LCLs (D11 and D13 LCL WT) treated with 4HT for 30 days (S2 Fig). Control RNAs RNU48 and ALAS1 were unaffected by the EBNA3A or EBNA3C status of the LCLs (S3 Fig). Expression of protein-encoding gene clusters previously reported to be regulated by EBNA3A and EBNA3C (eg CXCL9/CXCL10 and ADAM28/ADAMDEC1) was as expected from previous reports ([20,21,28], Fig 2B and S3 Fig). Although in some knockout and revertant LCL pairs, EBNA3B expression appeared to influence the levels of these miR clusters, the changes were very slight and/or inconsistent (S1 and S4 Figs).

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