Shuttle Vector-Based Transformation System for Pyrococcus furiosus▿

Pyrococcus furiosus is a model organism for analyses of molecular biology and biochemistry of archaea, but so far no useful genetic tools for this species have been described. We report here a genetic transformation system for P. furiosus based on the shuttle vector system pYS2 from Pyrococcus abyssi. In the redesigned vector, the pyrE gene from Sulfolobus was replaced as a selectable marker by the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene (HMG-CoA) conferring resistance of transformants to the antibiotic simvastatin. Use of this modified plasmid resulted in the overexpression of the HMG-CoA reductase in P. furiosus, allowing the selection of strains by growth in the presence of simvastatin. The modified shuttle vector replicated in P. furiosus, but the copy number was only one to two per chromosome. This system was used for overexpression of His6-tagged subunit D of the RNA polymerase (RNAP) in Pyrococcus cells. Functional RNAP was purified from transformed cells in two steps by Ni-NTA and gel filtration chromatography. Our data provide evidence that expression of transformed genes can be controlled from a regulated gluconeogenetic promoter

Transcriptional activation of endogenous viral and cellular target genes by END domain mutants.

<p>1x10⁷ Eli-BL cells were transfected with expression constructs for EBNA-2 wt, N-terminal deletion mutants, END domain mutants or the corresponding control vectors (pSG5). Relative transcript levels of the viral LMP1 and LMP2A gene or the cellular CD23 or CCL3 genes were determined by real-time RT-PCR. Transcript levels were normalized to actin transcript levels. EBNA-2 activation was set to 100% and the data are shown as mean values of four independent experiments. Error bars indicate the standard deviations.</p

Structure of the EBNA-2 N-terminal dimerization (END) domain.

<p>Schematic representation of important features of the EBNA-2 protein: two dimerization motifs (Dim1/Dim2), N-terminal and C-terminal transactivation domains (N-TAD, C-TAD), repetitive primary sequence motifs like the poly-proline (polyP) and the poly arginine-glycine (polyRG) stretch, the nuclear localization signals (NLS),and the adapter region of EBNA-2, which interacts with CBF1/CSL, are illustrated. (B) NMR solution structure of the END (EBNA-2 N-terminal Dimerization) domain. Left: β-strands are shown in blue, helices in orange, and loops in gray. Right: Monomers highlighted in gray and blue. (C) Dimerization of monomers is stabilized by hydrophobic interactions. The inside of each monomer is lined with numerous hydrophobic residues (left; sticks). A subset of these residues is located at the dimer interface (blue/bold labels). Panels (right) show side views of the END domain and highlight the interface residues of each monomer.</p

LMP1 activation by EBNA-2 requires dimerization, the surface residue His15, and the protruding α1-helix.

<p>1x10⁷ EBV positive but EBNA-2 negative Eli-BL cells were transfected with 5 μg expression constructs for EBNA-2 wt, N-terminal deletion mutants (A), END interface (B) or END surface (C) mutants or the corresponding vector controls (pSG5). 30 μg of whole cell lysates of transfected cells were analyzed on western blots using EBNA-2, LMP1, EBNA-1 and GAPDH specific antibodies. Staining for EBNA-1 and GAPDH was used as loading controls. EBV negative (DG75: 30 μg of total cell lysate) and EBV infected LMP1 positive B cells (721: 5 μg total cell lysate) were used as controls. (D) The chemilumiscence signals were quantified by digital imaging using the Fusion Fx7 and the data are shown as % signal intensity relative to EBNA2 wt (100%). The bars represent the mean values of 4 independent experiments. Standard deviations are shown as error bars.</p

Dimerization analysis of wild-type and mutant END domains by SEC/SLS and NMR.

<p>ND—Not determined. Protein sample not stable and/or not suitable for NMR analysis.</p><p>^A Molecular weights were calculated from refractive index (RI) and right angle light scattering (RALS) data (Fig D in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004910#ppat.1004910.s001" target="_blank">S1 Text</a>).</p><p>^B For NMR, proteins without a Z-tag were analyzed.</p><p>^C 2D ¹H,¹⁵N-HSQC spectrum indicates the presence of two populations, interpreted as an equilibrium between a folded dimer and the unfolded monomer of the END domain (Fig D in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004910#ppat.1004910.s001" target="_blank">S1 Text</a>).</p><p>Dimerization analysis of wild-type and mutant END domains by SEC/SLS and NMR.</p

EBF1 binds to EBNA2 and promotes the assembly of EBNA2 chromatin complexes in B cells.

Epstein-Barr virus (EBV) infection converts resting human B cells into permanently proliferating lymphoblastoid cell lines (LCLs). The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a key role in this process. It preferentially binds to B cell enhancers and establishes a specific viral and cellular gene expression program in LCLs. The cellular DNA binding factor CBF1/CSL serves as a sequence specific chromatin anchor for EBNA2. The ubiquitous expression of this highly conserved protein raises the question whether additional cellular factors might determine EBNA2 chromatin binding selectively in B cells. Here we used CBF1 deficient B cells to identify cellular genes up or downregulated by EBNA2 as well as CBF1 independent EBNA2 chromatin binding sites. Apparently, CBF1 independent EBNA2 target genes and chromatin binding sites can be identified but are less frequent than CBF1 dependent EBNA2 functions. CBF1 independent EBNA2 binding sites are highly enriched for EBF1 binding motifs. We show that EBNA2 binds to EBF1 via its N-terminal domain. CBF1 proficient and deficient B cells require EBF1 to bind to CBF1 independent binding sites. Our results identify EBF1 as a co-factor of EBNA2 which conveys B cell specificity to EBNA2

Comparative transcript profiling of EBNA2 target gene expression in CBF1 proficient and deficient DG75 cells.

<p>DG75 cells expressing ER/EBNA2 were cultivated in estrogen supplemented medium for 24 h or were left untreated. Total cellular RNA was isolated and submitted to gene expression analysis using the Human Gene 2.0 ST array. All probe sets represent single transcripts (trxs). For each condition, 3 biological replicates were examined. Each vertical column represents the results obtained after hybridizing a single microarray. Horizontal rows represent data obtained for a particular probe set across all cell lines and conditions adjusted to a scale ranging from -2.0 to + 2.0. The relative high, medium and low expression values are represented by red, white and blue color, respectively. Vertical columns are ranked according to fold changes from highest induction levels on top to highest repression levels at the bottom. (A) Expression levels of 136 transcripts which change expression levels at least 4-fold (p ≤ 0.001) in response to EBNA2 in CBF1 proficient DG75 (DG75^ER/EBNA2 CBF1 wt) cells are displayed. The transcript cluster ID and the assigned genes/transcripts, including non-coding RNAs, are annotated. (B) 21 transcripts regulated at least 4-fold (p ≤ 0.001) in CBF1 deficient DG75 (DG75^ER/EBNA2 CBF1 ko). (C) Boxplots depicting the fold change distribution of EBNA2 induced and repressed transcripts for the subset of target genes changed at least 2-fold (p ≤ 0.05) in CBF1 wt and ko cells, respectively. EBNA2 induced (D) and repressed (E) transcripts are shown to illustrate the dynamic range of each system. Boxplot whiskers extend to 1.5x interquartile range. Dotted lines mark the 2-fold change chosen as cut-off. (F) Expression levels of EBNA2 (prior to and after estrogen treatment) and CBF1 proteins were monitored by Western blot analysis. Equal amounts of total protein lysates were applied and GAPDH served as an internal loading control. One representative experiment (n = 3) is shown.</p

EBNA2 can access more than 15% of its chromatin binding sites in CBF1 deficient DG75 B cells.

<p>(A) Intersection of EBNA2 binding sites identified in CBF1 proficient or deficient cells 24 h post doxycycline induction. 1,546 peaks that were identified in CBF1 proficient but not in CBF1 deficient cells were defined as "CBF1 dependent" EBNA2 peaks. 243 EBNA2 peaks identified in CBF1 deficient and proficient DG75 cells were defined as "CBF1 independent". (B-E) Comparison of EBNA2 ChIP-seq signal distributions at CBF1 independent or dependent peaks. (B) Anchor and (C) scatter plots (mean + 95% CI) depicting ChIP-seq signal distributions at EBNA2 peak subsets. Regions flanking the peak center for 2 kb in each direction were analyzed (Data underlying panel B). Absolute means and SEMs are indicated below. (D) Anchor and (E) scatter plots (mean + 95% CI) as shown in B and C but depicting EBNA2 ChIP-seq signal intensities for the two different subsets of EBNA2 peaks as defined in A. Statistical significance for differences of all means were assessed applying unpaired two-tailed t-test for log values with Welch’s correction (**** p < 0.0001); absolute means and SEMs are indicated below. (F) List of EBNA2 mean ChIP-seq signal intensities at CBF1 independent and dependent peaks.</p