Deregulation of the Cell Cycle by EBV Nuclear Antigens EBNA3A and EBNA3C

Cyclin-dependent kinase inhibitor p16INK4A is an important tumour suppressor and inducer of cellular senescence often inactivated during the development of cancer. I investigated the mechanism by which EBV latency-associated nuclear antigens EBNA3A and EBNA3C repress p16INK4A expression. Using lymphoblastoid cell lines (LCL) expressing a conditional EBNA3C, I demonstrate that EBNA3C inactivation resets the epigenetic status of p16INK4A to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16INK4A transcription and allows proliferation. LCL lacking EBNA3A express relatively high levels of p16INK4A and have a similar pattern of histone modifications on p16INK4A as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP was linked to the oncogenic activity of EBNA3C and EBNA3A, LCL with viruses encoding EBNA3C- and/or EBNA3A-mutants that no longer bind CtBP were established. These novel LCL revealed that the epigenetic repression of p16INK4A requires the interaction of both EBNA3C and EBNA3A with CtBP. Epigenetic repression of p16INK4A by latent EBV may facilitate p16INK4A DNA methylation during lymphomagenesis. Furthermore, by transforming the peripheral blood lymphocytes (PBL) from an individual homozygous for a deletion in CDKN2A locus with recombinant EBV viruses expressing conditional EBNA3C, we developed a system that allows inactivation of EBNA3C in LCL lacking functional p16INK4A protein (p16-null LCL 3CHT). EBNA3C inactivation has no impact on the proliferation rate of p16-null LCL, proving that the repression of p16INK4A is the main function of EBNA3C in EBV-driven LCL proliferation. The p16INK4A locus is epigenetically modified by EBNA3C despite the absence of functional p16INK4A protein. Since the selection pressure based on faster outgrowth of advantageously modified subset of cells is removed, the gradual and relatively slow kinetics of H3K27me3 restoration at p16INK4A following EBNA3C reactivation in p16-null LCL 3CHT seems to be genuinely related to the mechanism of EBNA3C-mediated p16INK4A regulation. The p16-null LCL 3CHT system further allows distinguishing genes regulated specifically by EBNA3C, rather than as a consequence of activation of p16INK4A/Rb/E2F1 axis. Lastly, new cellular targets of EBNA3C and/or EBNA3A from the group of microRNAs are identified in this work. Most notably, both EBNA3C and EBNA3A are shown to repress the tumour supressor miR-143/145 cluster and their precursor long non-coding RNAs in LCL

Chromatin signatures at Notch-regulated enhancers reveal large-scale changes in H3K56ac upon activation.

The conserved Notch pathway functions in diverse developmental and disease-related processes, requiring mechanisms to ensure appropriate target selection and gene activation in each context. To investigate the influence of chromatin organisation and dynamics on the response to Notch signalling, we partitioned Drosophila chromatin using histone modifications and established the preferred chromatin conditions for binding of Su(H), the Notch pathway transcription factor. By manipulating activity of a co-operating factor, Lozenge/Runx, we showed that it can help facilitate these conditions. While many histone modifications were unchanged by Su(H) binding or Notch activation, we detected rapid changes in acetylation of H3K56 at Notch-regulated enhancers. This modification extended over large regions, required the histone acetyl-transferase CBP and was independent of transcription. Such rapid changes in H3K56 acetylation appear to be a conserved indicator of enhancer activation as they also occurred at the mammalian Notch-regulated Hey1 gene and at Drosophila ecdysone-regulated genes. This intriguing example of a core histone modification increasing over short timescales may therefore underpin changes in chromatin accessibility needed to promote transcription following signalling activation.This work was supported by a BBSRC project grant [BB/J008842/1] to SJB, BA and SR and by a MRC programme grant [G0800034] to SJB. JL is the recipient of a scholarship from the China Scholarship Council Cambridge.This is the author accepted manuscript. The final version is available from Wiley via http://dx.doi.org/10.15252/embj.20148992

Role of co-repressor genomic landscapes in shaping the Notch response.

Repressors are frequently deployed to limit the transcriptional response to signalling pathways. For example, several co-repressors interact directly with the DNA-binding protein CSL and are proposed to keep target genes silenced in the absence of Notch activity. However, the scope of their contributions remains unclear. To investigate co-repressor activity in the context of this well defined signalling pathway, we have analysed the genome-wide binding profile of the best-characterized CSL co-repressor in Drosophila, Hairless, and of a second CSL interacting repressor, SMRTER. As predicted there was significant overlap between Hairless and its CSL DNA-binding partner, both in Kc cells and in wing discs, where they were predominantly found in chromatin with active enhancer marks. However, while the Hairless complex was widely present at some Notch regulated enhancers in the wing disc, no binding was detected at others, indicating that it is not essential for silencing per se. Further analysis of target enhancers confirmed differential requirements for Hairless. SMRTER binding significantly overlapped with Hairless, rather than complementing it, and many enhancers were apparently co-bound by both factors. Our analysis indicates that the actions of Hairless and SMRTER gate enhancers to Notch activity and to Ecdysone signalling respectively, to ensure that the appropriate levels and timing of target gene expression are achieved

The interaction of PRC2 with RNA or chromatin is mutually antagonistic

Polycomb repressive complex 2 (PRC2) modifies chromatin to maintain genes in a repressed state during development. PRC2 is primarily associated with CpG islands at repressed genes and also possesses RNA binding activity. However, the RNAs that bind PRC2 in cells, the subunits that mediate these interactions, and the role of RNA in PRC2 recruitment to chromatin all remain unclear. By performing iCLIP for PRC2 in comparison with other RNA binding proteins, we show here that PRC2 binds nascent RNA at essentially all active genes. Although interacting with RNA promiscuously, PRC2 binding is enriched at specific locations within RNAs, primarily exon-intron boundaries and the 3'UTR. Deletion of other PRC2 subunits reveals that SUZ12 is sufficient to establish this RNA binding profile. Contrary to prevailing models, we also demonstrate that the interaction of PRC2 with RNA or chromatin is mutually antagonistic in cells and in vitro. RNA degradation in cells triggers PRC2 recruitment to CpG islands at active genes. Correspondingly, release of PRC2 from chromatin in cells increases RNA binding. Consistent with this, RNA and nucleosomes compete for PRC2 binding in vitro. We propose that RNA prevents PRC2 recruitment to chromatin at active genes and that mutual antagonism between RNA and chromatin underlies the pattern of PRC2 chromatin association across the genome

Regulatory feedback from nascent RNA to chromatin and transcription

Transcription and chromatin function are regulated by proteins that bind to DNA, nucleosomes or RNA polymerase II, with specific non-coding RNAs (ncRNAs) functioning to modulate their recruitment or activity. Unlike ncRNAs, nascent pre-mRNA was considered to be primarily a passive player in these processes. In this Opinion article, we describe recently identified interactions between nascent pre-mRNAs and regulatory proteins, highlight commonalities between the functions of nascent pre-mRNA and nascent ncRNA, and propose that both types of RNA have an active role in transcription and chromatin regulation

Deregulation of the cell cycle by EBV nuclear antigens EBNA3A and EBNA3C

Cyclin-dependent kinase inhibitor p16INK4A is an important tumour suppressor and inducer of cellular senescence often inactivated during the development of cancer. I investigated the mechanism by which EBV latency-associated nuclear antigens EBNA3A and EBNA3C repress p16INK4A expression. Using lymphoblastoid cell lines (LCL) expressing a conditional EBNA3C, I demonstrate that EBNA3C inactivation resets the epigenetic status of p16INK4A to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16INK4A transcription and allows proliferation. LCL lacking EBNA3A express relatively high levels of p16INK4A and have a similar pattern of histone modifications on p16INK4A as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP was linked to the oncogenic activity of EBNA3C and EBNA3A, LCL with viruses encoding EBNA3C- and/or EBNA3A-mutants that no longer bind CtBP were established. These novel LCL revealed that the epigenetic repression of p16INK4A requires the interaction of both EBNA3C and EBNA3A with CtBP. Epigenetic repression of p16INK4A by latent EBV may facilitate p16INK4A DNA methylation during lymphomagenesis. Furthermore, by transforming the peripheral blood lymphocytes (PBL) from an individual homozygous for a deletion in CDKN2A locus with recombinant EBV viruses expressing conditional EBNA3C, we developed a system that allows inactivation of EBNA3C in LCL lacking functional p16INK4A protein (p16-null LCL 3CHT). EBNA3C inactivation has no impact on the proliferation rate of p16-null LCL, proving that the repression of p16INK4A is the main function of EBNA3C in EBV-driven LCL proliferation. The p16INK4A locus is epigenetically modified by EBNA3C despite the absence of functional p16INK4A protein. Since the selection pressure based on faster outgrowth of advantageously modified subset of cells is removed, the gradual and relatively slow kinetics of H3K27me3 restoration at p16INK4A following EBNA3C reactivation in p16-null LCL 3CHT seems to be genuinely related to the mechanism of EBNA3C-mediated p16INK4A regulation. The p16-null LCL 3CHT system further allows distinguishing genes regulated specifically by EBNA3C, rather than as a consequence of activation of p16INK4A/Rb/E2F1 axis. Lastly, new cellular targets of EBNA3C and/or EBNA3A from the group of microRNAs are identified in this work. Most notably, both EBNA3C and EBNA3A are shown to repress the tumour supressor miR-143/145 cluster and their precursor long non-coding RNAs in LCL.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Epigenetic repression of p16(INK4A) by latent Epstein-Barr virus requires the interaction of EBNA3A and EBNA3C with CtBP.

As an inhibitor of cyclin-dependent kinases, p16(INK4A) is an important tumour suppressor and inducer of cellular senescence that is often inactivated during the development of cancer by promoter DNA methylation. Using newly established lymphoblastoid cell lines (LCLs) expressing a conditional EBNA3C from recombinant EBV, we demonstrate that EBNA3C inactivation initiates chromatin remodelling that resets the epigenetic status of p16(INK4A) to permit transcriptional activation: the polycomb-associated repressive H3K27me3 histone modification is substantially reduced, while the activation-related mark H3K4me3 is modestly increased. Activation of EBNA3C reverses the distribution of these epigenetic marks, represses p16(INK4A) transcription and allows proliferation. LCLs lacking EBNA3A express relatively high levels of p16(INK4A) and have a similar pattern of histone modifications on p16(INK4A) as produced by the inactivation of EBNA3C. Since binding to the co-repressor of transcription CtBP has been linked to the oncogenic activity of EBNA3A and EBNA3C, we established LCLs with recombinant viruses encoding EBNA3A- and/or EBNA3C-mutants that no longer bind CtBP. These novel LCLs have revealed that the chromatin remodelling and epigenetic repression of p16(INK4A) requires the interaction of both EBNA3A and EBNA3C with CtBP. The repression of p16(INK4A) by latent EBV will not only overcome senescence in infected B cells, but may also pave the way for p16(INK4A) DNA methylation during B cell lymphomagenesis

EBNA3A and EBNA3C induce chromosome looping at the miR-221/miR-222 cluster locus.

<p>(<b>A</b>) Schematic of the miR-221/miR-222 cluster locus depicts the location of both miRs, the HindIII sites, the 28kb pri-miR-221/222 transcription start site and primers used for the chromosome conformation capture assay. (<b>B</b>) EBNA3A-KO and EBNA3A-Rev LCLs (D3) were used for chromosome conformation analysis. Interaction between the promoter (P) of the 28kb pri-miR-221/222 and EBNA3A/3C binding site 2 (BS2 –including BS2a and BS2b, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g008" target="_blank">Fig 8A</a>) or 3 (BS3) is dependent on the presence of EBNA3A. PCR primers (NC) corresponding to a site located downstream of the miR-221/miR-222 locus were used as a negative control. Positive control (+ve control) showed PCR reactions using a DNA control template. (<b>C</b>) Same as (B) but using p16-null LCL 3CHT (LCL 3CHT never HT) cultured for 30 days with (LCL 3CHT +HT) or without 4HT. Interaction between P to BS2 and P to BS3 occurred only when EBNA3C is active (LCL 3CHT +HT). (<b>D</b>). Loading control primers L1 and L2 amplify DNA contained in a single HindIII fragment and have been used as DNA loading control between the DNA samples used for chromosome conformation capture. (<b>E</b>) Schematic model of chromatin loop formation induced by EBNA3A and EBNA3C at miR-221/miR-222 cluster locus.</p

Inactivation of miR-221 and miR-222 in LCLs with corresponding anti-miRs.

<p>LCLs were electroporated with the anti-miR indicated; p57^KIP2, p27^KIP1 p21^CIP1 and PUMA expression have been analysed by western blot. The blot was probed for γ-tubulin as an additional control for loading and a non-targeted protein. There were increases in 57^KIP2 and p27^KIP1, but not p21^CIP1 or PUMA in cells transfected with the specific LNA anti-miRs.</p