Epstein-Barr virus nuclear protein EBNA3C directly induces expression of AID and somatic mutations in B cells

Activation-induced cytidine deaminase (AID), the enzyme responsible for induction of sequence variation in immunoglobulins (Igs) during the process of somatic hypermutation (SHM) and also Ig class switching, can have a potent mutator phenotype in the development of lymphoma. Using various Epstein-Barr virus (EBV) recombinants, we provide definitive evidence that the viral nuclear protein EBNA3C is essential in EBV-infected primary B cells for the induction of AID mRNA and protein. Using lymphoblastoid cell lines (LCLs) established with EBV recombinants conditional for EBNA3C function, this was confirmed, and it was shown that transactivation of the AID gene (AICDA) is associated with EBNA3C binding to highly conserved regulatory elements located proximal to and upstream of the AICDA transcription start site. EBNA3C binding initiated epigenetic changes to chromatin at specific sites across the AICDA locus. Deep sequencing of cDNA corresponding to the IgH V-D-J region from the conditional LCL was used to formally show that SHM is activated by functional EBNA3C and induction of AID. These data, showing the direct targeting and induction of functional AID by EBNA3C, suggest a novel role for EBV in the etiology of B cell cancers, including endemic Burkitt lymphoma

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

EBNA3A and EBNA3C bind near the miR-221/miR-222 locus.

<p>(<b>A</b>) ChIP-seq data at miR-221/miR-222 cluster genomic locus generated from LCL 3A-TAP and LCL 3C-TAP [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.ref027" target="_blank">27</a>] were displayed using UCSC Genome Browser. EBNA3A and EBNA3C binding sites (BS1, BS2a, BS2b and BS3) are shown in black squares. The ChIP-seq data from ENCODE for H3K27ac in GM12878 is also displayed. Below it, a schematic representation of the 2kb and 28kb pri-miR-221/222 is shown. Grey and red arrows show the transcription start site of the 2kb and 28kb pri-miR-221/222 respectively. Positions of primer pairs used for qPCR to analyse precipitated DNA from ChIP are indicated, along with the positions of previously described enhancers (grey squares A and B) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.ref047" target="_blank">47</a>]. (<b>B</b>) ChIP qPCR analyses using anti-Flag antibody to precipitate 3C-TAP and chromatin associated with it in LCL 3C-TAP was performed. As a control for antibody specificity similar ChIP was performed using LCL infected with B95.8-BAC virus (LCL WT). No binding was detected at the enhancer A (EnhA) used as negative control. Values represent ratio of chromatin precipitated, after correction for IgG, relative to 2.5% of input. (<b>C</b>) As in (B) but using LCL 3A-TAP in order to precipitate 3A-TAP protein and associated chromatin. The ENCODE ChIP-seq data indicate a clear peak for RNA Pol II and TBP (<a href="https://genome.ucsc.edu" target="_blank">https://genome.ucsc.edu</a>; at coordinates ChrX: 45,629,468–45,629,838 GRCCh37/hg19) this is where the RNA-seq signal for pri-miR-221/222 (28kb) starts, so we have assumed this was the transcription start site (TSS) throughout our analysis.</p

Regulation of miR clusters by EBNA3A and EBNA3C.

<p>(<b>A</b>) 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-null 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-null 3CHT cultured with 4HT (+HT). (<b>B</b>) As in (A) but analysing miR-143 and miR-145 expression.</p

Zhodnocení vlivu výstavby Vysočina arény na rozvoj Nového Města na Moravě

This bachelor thesis is occupied by latest development of small city called Nové Město na Moravě. In 2011 there passed off a construction of sport centre called Vysočina Arena and it had a major influence on the town. The aim of this thesis is the evaluation of development judged from social economic and environmental view. Firstly the bachelor thesis is concerned with theoretical basics. It explains the fundamental concept and connections. Afterwards this thesis is focused to ascertainment individuals views of different researched sectors. During my research I used quantitative and qualitative survey. It was implemented by using web pages and straight interviews with Mayor his deputy and representative of sports club

The increase of p57^KIP2 level is associated with de-phosphorylation of Rb and reduced entry into S phase and proliferation.

<p>(<b>A</b>) Western blot analysis of extracts from LCL EBNA3A-ERT2 (line 5) cultured with (+) or without (-) 4HT for 30 days showing that after inactivation (and degradation) of the EBNA3A-ERT2 fusion protein, p57^KIP2 expression increases, and the amount of hyperphosphorylated Rb (ppRb) is dramatically reduced and is no longer detected using a phospho-Rb-specific antibody. The blot was probed for γ-tubulin as a control for loading. (<b>B</b>) Cell cycle distribution of LCL EBNA3A-ERT2 (line 5) culture with or without 4HT for four weeks was determined by flow cytometry following exposure to EdU for 2 hours (2h pulse). (<b>C</b>) A comparison of the population growth rate between these cells cultured with or without 4HT was analysed by counting the number of viable cells every 2–3 days. Total cell numbers were plotted at each time point. Data are representative of two independent experiments.</p

EBNA3A and EBNA3C regulate p57^KIP2 expression.

<p>(<b>A</b>) Western blot showing the level of p57^KIP2 expression in four EBNA3A-KO (KO) LCLs and their revertant equivalent (REV) (used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g001" target="_blank">Fig 1</a>). Below each Western blot is shown the level of p57^KIP2 mRNA determined by qPCR, normalized to GN2BL1 and relative to each “wild type” cell LCL EBNA3A-REV (3A-REV) of which the mRNA level is set to 1. (<b>B</b>) As in (A) but using five EBNA3A-ERT2 conditional LCLs cultured with (+) or without (-) 4HT (used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g004" target="_blank">Fig 4</a>). (<b>C</b>) Western blot showing the expression of p57^KIP2 protein (left panel) and qPCR showing the level of p57^KIP2 mRNA (right panel) in two p16-null LCL 3CHT cultured with (+) or without (-) 4HT (used in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005031#ppat.1005031.g001" target="_blank">Fig 1</a>).</p

Regulation of miRs in EBNA3A-conditional LCLs.

<p>(<b>A</b>) Five EBNA3A-ERT2 LCLs (established in the presence of 4HT and named from 1 to 5) were cultured for ~30 days with (+) or without (-) 4HT and EBNA3A-ERT2 protein expression was analysed by western blot. (<b>B</b>) MiR-221/miR-222 expression was determined by qPCR using total RNA extracted from the same five EBNA3A-ERT2 cell lines (LCLs 1, 2, 3, 4 and 5). (<b>C</b>) As in (B) but analysing miR-143 and miR-145 expression.</p

Activation of EBNA3C represses miR-143/miR-145 expression.

<p>(<b>A</b>) MiR-143, miR-145 and RNU48 expression were analysed by qPCR from p16-null LCL 3CHT established without the presence of 4HT (never HT) or 30 days after 4HT was added to culture medium (+HT). MiR levels in p16-null LCL 3CHT +HT are relative to the LCL never HT. (<b>B</b>) As in (A) but analysing the expression of ALAS1, ADAM28 and ADAMDEC1 by qPCR as controls for the activation of EBNA3C following the addition of 4HT.</p