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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

Active chromatin markers and RNA polymerase (Pol) II occupancy on the miR-221/miR-222 cluster genomic locus.(A) ChIP-seq data at miR-221/miR-222 cluster genomic locus generated from LCL 3A-TAP and LCL 3C-TAP were displayed using UCSC Genome Browser as in Fig 7A. A schematic representation of the 2kb and 28kb pri-miR-221/222 is shown with grey and red arrows representing each transcription start site (TSS). Positions of primer pairs used for qPCR to analyse precipitated DNA from ChIP are indicated along with the position of previously described enhancers (grey squares A and B) [47]. (B) ChIP was performed on extracts from EBNA3A-KO and EBNA3A-REV LCL (D3) and antibodies specific for phospho-Ser5 Pol II, H3K4me3, H3K9Ac and H3K27Ac were used. As a control for antibodies and cell lines, a primer pair for CXCL10 TSS was used. Values represent ratio of chromatin precipitated, after correction for IgG, relative to 2.5% of input. (C) As in (B) but using p16- LCL 3CHT (LCL 3CHT never HT) cultured for 30 days with (LCL 3CHT +HT) or without 4HT. For the ChIP using LCL 3CHT, ADAM28 TSS primer pair was used as control.
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ppat.1005031.g008: Active chromatin markers and RNA polymerase (Pol) II occupancy on the miR-221/miR-222 cluster genomic locus.(A) ChIP-seq data at miR-221/miR-222 cluster genomic locus generated from LCL 3A-TAP and LCL 3C-TAP were displayed using UCSC Genome Browser as in Fig 7A. A schematic representation of the 2kb and 28kb pri-miR-221/222 is shown with grey and red arrows representing each transcription start site (TSS). Positions of primer pairs used for qPCR to analyse precipitated DNA from ChIP are indicated along with the position of previously described enhancers (grey squares A and B) [47]. (B) ChIP was performed on extracts from EBNA3A-KO and EBNA3A-REV LCL (D3) and antibodies specific for phospho-Ser5 Pol II, H3K4me3, H3K9Ac and H3K27Ac were used. As a control for antibodies and cell lines, a primer pair for CXCL10 TSS was used. Values represent ratio of chromatin precipitated, after correction for IgG, relative to 2.5% of input. (C) As in (B) but using p16- LCL 3CHT (LCL 3CHT never HT) cultured for 30 days with (LCL 3CHT +HT) or without 4HT. For the ChIP using LCL 3CHT, ADAM28 TSS primer pair was used as control.

Mentions: Next, in order to determine whether the up-regulation of pri-miR-221/222 by EBNA3A and EBNA3C correlates with histone modification and chromatin remodeling, ChIP analyses were performed on EBNA3A-KO and EBNA3A-REV LCLs as well as EBNA3C-HT LCLs (never HT) or treated with 4HT (Fig 8). Initially the phosphorylation of the RNA polymerase II at serine-5 (Ser5)–that indicates transcriptional initiation and activation [77]–was investigated (Fig 8B and 8C). This revealed that the level of phospho-Ser5 Pol II is elevated around the TSS of 28kb pri-miR-221/222 only when both EBNA3A and EBNA3C are expressed and functional (in EBNA3A-REV and EBNA3C-HT cultured with 4HT). No binding was seen at the putative 2kb TSS. Primers that amplify CXCL10 TSS and ADAM28 TSS were used as controls for EBNA3A and EBNA3C repressed genes respectively and found, as expected, a higher level of phospho-Ser5 Pol II only when EBNA3A or EBNA3C were absent/non-functional. We then performed ChIP analysis for marks of active chromatin (H3K4me3, H3K9ac and H3K27ac) across the miR-221/miR-222 cluster locus (Fig 8B and 8C). As expected, a higher level of activation marks was found around the 28kb pri-miR-221/222 TSS only when functional EBNA3A and EBNA3C were expressed. Again there were no changes around the putative 2kb TSS that suggest it is regulated. Interestingly, histone activation marks where higher (in particular H3K9ac and H3K27ac) at BS2a and BS2b sites, that is the region previously described as an enhancer for miR-221/miR-222, only when both EBV proteins are functional. Taken together these data are consistent with increased miR-221/miR-222 expression occurring when EBNA3A and EBNA3C are expressed in an active form, bind chromatin at specific sites and alter the epigenetic profile of the locus.


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)

Active chromatin markers and RNA polymerase (Pol) II occupancy on the miR-221/miR-222 cluster genomic locus.(A) ChIP-seq data at miR-221/miR-222 cluster genomic locus generated from LCL 3A-TAP and LCL 3C-TAP were displayed using UCSC Genome Browser as in Fig 7A. A schematic representation of the 2kb and 28kb pri-miR-221/222 is shown with grey and red arrows representing each transcription start site (TSS). Positions of primer pairs used for qPCR to analyse precipitated DNA from ChIP are indicated along with the position of previously described enhancers (grey squares A and B) [47]. (B) ChIP was performed on extracts from EBNA3A-KO and EBNA3A-REV LCL (D3) and antibodies specific for phospho-Ser5 Pol II, H3K4me3, H3K9Ac and H3K27Ac were used. As a control for antibodies and cell lines, a primer pair for CXCL10 TSS was used. Values represent ratio of chromatin precipitated, after correction for IgG, relative to 2.5% of input. (C) As in (B) but using p16- LCL 3CHT (LCL 3CHT never HT) cultured for 30 days with (LCL 3CHT +HT) or without 4HT. For the ChIP using LCL 3CHT, ADAM28 TSS primer pair was used as control.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1005031.g008: Active chromatin markers and RNA polymerase (Pol) II occupancy on the miR-221/miR-222 cluster genomic locus.(A) ChIP-seq data at miR-221/miR-222 cluster genomic locus generated from LCL 3A-TAP and LCL 3C-TAP were displayed using UCSC Genome Browser as in Fig 7A. A schematic representation of the 2kb and 28kb pri-miR-221/222 is shown with grey and red arrows representing each transcription start site (TSS). Positions of primer pairs used for qPCR to analyse precipitated DNA from ChIP are indicated along with the position of previously described enhancers (grey squares A and B) [47]. (B) ChIP was performed on extracts from EBNA3A-KO and EBNA3A-REV LCL (D3) and antibodies specific for phospho-Ser5 Pol II, H3K4me3, H3K9Ac and H3K27Ac were used. As a control for antibodies and cell lines, a primer pair for CXCL10 TSS was used. Values represent ratio of chromatin precipitated, after correction for IgG, relative to 2.5% of input. (C) As in (B) but using p16- LCL 3CHT (LCL 3CHT never HT) cultured for 30 days with (LCL 3CHT +HT) or without 4HT. For the ChIP using LCL 3CHT, ADAM28 TSS primer pair was used as control.
Mentions: Next, in order to determine whether the up-regulation of pri-miR-221/222 by EBNA3A and EBNA3C correlates with histone modification and chromatin remodeling, ChIP analyses were performed on EBNA3A-KO and EBNA3A-REV LCLs as well as EBNA3C-HT LCLs (never HT) or treated with 4HT (Fig 8). Initially the phosphorylation of the RNA polymerase II at serine-5 (Ser5)–that indicates transcriptional initiation and activation [77]–was investigated (Fig 8B and 8C). This revealed that the level of phospho-Ser5 Pol II is elevated around the TSS of 28kb pri-miR-221/222 only when both EBNA3A and EBNA3C are expressed and functional (in EBNA3A-REV and EBNA3C-HT cultured with 4HT). No binding was seen at the putative 2kb TSS. Primers that amplify CXCL10 TSS and ADAM28 TSS were used as controls for EBNA3A and EBNA3C repressed genes respectively and found, as expected, a higher level of phospho-Ser5 Pol II only when EBNA3A or EBNA3C were absent/non-functional. We then performed ChIP analysis for marks of active chromatin (H3K4me3, H3K9ac and H3K27ac) across the miR-221/miR-222 cluster locus (Fig 8B and 8C). As expected, a higher level of activation marks was found around the 28kb pri-miR-221/222 TSS only when functional EBNA3A and EBNA3C were expressed. Again there were no changes around the putative 2kb TSS that suggest it is regulated. Interestingly, histone activation marks where higher (in particular H3K9ac and H3K27ac) at BS2a and BS2b sites, that is the region previously described as an enhancer for miR-221/miR-222, only when both EBV proteins are functional. Taken together these data are consistent with increased miR-221/miR-222 expression occurring when EBNA3A and EBNA3C are expressed in an active form, bind chromatin at specific sites and alter the epigenetic profile of the locus.

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