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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

EBNA3A and EBNA3C up-regulate a pri-miR-221/222 of ~28kb.(A) Screen-shot of UCSC Genome Browser at miR-221/miR-222 cluster genomic locus shows the position of mature miR-221/miR-222 as well as RNA-seq data for GM12878 (LCL), h1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK and NHLF cell lines. These RNA-seq data from ENCODE indicate the presence of different sizes of pri-miR-221/222 between cell lines (approximately 2kb, 28kb and 108kb). Red arrows represent the proposed transcription start sites and the asterisk shows the position of primers used. The expression level of the 28kb pri-miR-221/222 was determined by qRT-PCR in (B) EBNA3A-KO and-REV LCLs as well as in p16- LCLs 3CHT cultured for 29 days with (+HT) or without 4HT (Washed); (C) in five EBNA3A-ERT2 LCLs cultured with (+HT) or without 4HT (Washed) for ~30 days; (D) in LCL EBNA3A-ERT2 (never HT) cultured for 28 days with (+HT) or without 4HT; (E) and in p16- LCL 3CHT (never HT) cultured for 30 days with (+HT) or without 4HT.
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ppat.1005031.g006: EBNA3A and EBNA3C up-regulate a pri-miR-221/222 of ~28kb.(A) Screen-shot of UCSC Genome Browser at miR-221/miR-222 cluster genomic locus shows the position of mature miR-221/miR-222 as well as RNA-seq data for GM12878 (LCL), h1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK and NHLF cell lines. These RNA-seq data from ENCODE indicate the presence of different sizes of pri-miR-221/222 between cell lines (approximately 2kb, 28kb and 108kb). Red arrows represent the proposed transcription start sites and the asterisk shows the position of primers used. The expression level of the 28kb pri-miR-221/222 was determined by qRT-PCR in (B) EBNA3A-KO and-REV LCLs as well as in p16- LCLs 3CHT cultured for 29 days with (+HT) or without 4HT (Washed); (C) in five EBNA3A-ERT2 LCLs cultured with (+HT) or without 4HT (Washed) for ~30 days; (D) in LCL EBNA3A-ERT2 (never HT) cultured for 28 days with (+HT) or without 4HT; (E) and in p16- LCL 3CHT (never HT) cultured for 30 days with (+HT) or without 4HT.

Mentions: MiR-221 and miR-222, which together form a cluster, are thought to both be processed from a common pri-miR. Interestingly, three different species of pri-miR-221/222, approximately 2kb, 28kb and 108kb, have been described [51]. The expression of these three pri-miR-221/222 differs in different cell lines, however, publically available RNA-seq data from ENCODE revealed that in GM12878 (an EBV-immortalised LCL) the major pri-miR-221/222 to be expressed is the 28kb species (Fig 6A). Using LCLs described above (Figs 1 and 4) it was established that both EBNA3A and EBNA3C are necessary to up-regulate the 28kb pri-miR-221/222 (Fig 6B and 6C). The level of pri-miR-221/222 in those cells echoes the level of the mature miR-221/miR-222 detected (compare Figs 1 and 4 with Fig 6). Consistent with this, when 4HT was added to EBNA3A-ERT2 LCLs or EBNA3C-HT LCLs never HT, there was a significant up-regulation of the pri-miR-221/222 (Fig 6D and 6E). The activation of both EBNA3A and EBNA3C through addition of 4HT not only up-regulates the pri-miR-221/222, but also increases the expression of the mature miR-221 and miR-222 in these cells (S6 Fig). Furthermore, it was possible to show that EBNA3A and EBNA3C repress the well-characterised pri-miR-143/145 in the same LCLs (S7 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)

EBNA3A and EBNA3C up-regulate a pri-miR-221/222 of ~28kb.(A) Screen-shot of UCSC Genome Browser at miR-221/miR-222 cluster genomic locus shows the position of mature miR-221/miR-222 as well as RNA-seq data for GM12878 (LCL), h1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK and NHLF cell lines. These RNA-seq data from ENCODE indicate the presence of different sizes of pri-miR-221/222 between cell lines (approximately 2kb, 28kb and 108kb). Red arrows represent the proposed transcription start sites and the asterisk shows the position of primers used. The expression level of the 28kb pri-miR-221/222 was determined by qRT-PCR in (B) EBNA3A-KO and-REV LCLs as well as in p16- LCLs 3CHT cultured for 29 days with (+HT) or without 4HT (Washed); (C) in five EBNA3A-ERT2 LCLs cultured with (+HT) or without 4HT (Washed) for ~30 days; (D) in LCL EBNA3A-ERT2 (never HT) cultured for 28 days with (+HT) or without 4HT; (E) and in p16- LCL 3CHT (never HT) cultured for 30 days with (+HT) or without 4HT.
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Related In: Results  -  Collection

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ppat.1005031.g006: EBNA3A and EBNA3C up-regulate a pri-miR-221/222 of ~28kb.(A) Screen-shot of UCSC Genome Browser at miR-221/miR-222 cluster genomic locus shows the position of mature miR-221/miR-222 as well as RNA-seq data for GM12878 (LCL), h1-hESC, HeLa-S3, HepG2, HSMM, HUVEC, K562, NHEK and NHLF cell lines. These RNA-seq data from ENCODE indicate the presence of different sizes of pri-miR-221/222 between cell lines (approximately 2kb, 28kb and 108kb). Red arrows represent the proposed transcription start sites and the asterisk shows the position of primers used. The expression level of the 28kb pri-miR-221/222 was determined by qRT-PCR in (B) EBNA3A-KO and-REV LCLs as well as in p16- LCLs 3CHT cultured for 29 days with (+HT) or without 4HT (Washed); (C) in five EBNA3A-ERT2 LCLs cultured with (+HT) or without 4HT (Washed) for ~30 days; (D) in LCL EBNA3A-ERT2 (never HT) cultured for 28 days with (+HT) or without 4HT; (E) and in p16- LCL 3CHT (never HT) cultured for 30 days with (+HT) or without 4HT.
Mentions: MiR-221 and miR-222, which together form a cluster, are thought to both be processed from a common pri-miR. Interestingly, three different species of pri-miR-221/222, approximately 2kb, 28kb and 108kb, have been described [51]. The expression of these three pri-miR-221/222 differs in different cell lines, however, publically available RNA-seq data from ENCODE revealed that in GM12878 (an EBV-immortalised LCL) the major pri-miR-221/222 to be expressed is the 28kb species (Fig 6A). Using LCLs described above (Figs 1 and 4) it was established that both EBNA3A and EBNA3C are necessary to up-regulate the 28kb pri-miR-221/222 (Fig 6B and 6C). The level of pri-miR-221/222 in those cells echoes the level of the mature miR-221/miR-222 detected (compare Figs 1 and 4 with Fig 6). Consistent with this, when 4HT was added to EBNA3A-ERT2 LCLs or EBNA3C-HT LCLs never HT, there was a significant up-regulation of the pri-miR-221/222 (Fig 6D and 6E). The activation of both EBNA3A and EBNA3C through addition of 4HT not only up-regulates the pri-miR-221/222, but also increases the expression of the mature miR-221 and miR-222 in these cells (S6 Fig). Furthermore, it was possible to show that EBNA3A and EBNA3C repress the well-characterised pri-miR-143/145 in the same LCLs (S7 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