Bidirectional activity of the NWC promoter is responsible for RAG-2 transcription in non-lymphoid cells.

The recombination-activating genes (RAG-1 and RAG-2) encode a V(D)J recombinase responsible for rearrangements of antigen-receptor genes during T and B cell development, and RAG expression is known to correlate strictly with the process of rearrangement. In contrast to RAG-1, the expression of RAG-2 was not previously detected during any other stage of lymphopoiesis or in any other normal tissue. Here we report that the CpG island-associated promoter of the NWC gene (the third evolutionarily conserved gene in the RAG locus), which is located in the second intron of RAG-2, has bidirectional activity and is responsible for the detectable transcription of RAG-2 in some non-lymphoid tissues. We also identify evolutionarily conserved promoter fragments responsible for this bidirectional activity, and show that it is activated by transcription factor ZFP143. The possible implications of our findings are briefly discussed

Design of small molecule inhibitors of type III secretion system ATPase EscN from enteropathogenic Escherichia coli

Enteropathogenic E. coli (EPEC) is a human pathogen using type III secretion system for delivery of proteins directly into the human host. The system contains a single ATPase, EscN, which is essential for uncoupling of proteins from their complexes with chaperones before the delivery. The structure of EscN ATPase (PDB code: 2obm) was used to screen computationally for small molecule inhibitors blocking its active site. Two lead candidates were examined but only one, Compound 54, was selected for further optimization. After extended QSAR optimization, two derivatives were found to be competitive inhibitors of EscN capable of blocking ATPase activity with a Ki below 50 µM. One candidate, WEN05-03, with a Ki=16±2 µM, was also minimally toxic to mammalian cells as determined by other assays. In the cell infection model of HeLa cells with EPEC, Compound WEN05-03 completely blocked actin cluster formation at 100 µM concentration, when analyzed by confocal microscopy. The second best inhibitor of EscN ATPase activity was WEN04-34 with a Ki=46±2 µM. However, the compound was highly toxic to the BALB/3T3 cell line. In summary, the work identifies a compound blocking bacterial ATPase in its active site without causing cellular toxicity to the host cells. It is the first report showing feasibility of using bacterial virulence system ATPase as a target for safe, non-toxic compounds and offering a proof-of-concept for non-antibiotic alternatives

Characterization of the bidirectional activity of the <i>NWC</i> promoter by Dual-Luciferase Reporter (DLR) assays.

<p>A) Genomic fragments (represented schematically with open boxes at the center of the graph) were cloned into a firefly luciferase reporter vector in either the sense (<i>NWC</i>) or antisense (<i>RAG-2</i>) direction, as indicated with horizontal solids arrows. Numbers indicate positions relative to the <i>NWC</i> transcription start site. The activities of the promoter constructs were tested in NIH3T3 cells. The relative promoter activities are presented as a percent of the activity of the <i>NWC</i>-oriented +125/−258 fragment, which was taken as 100%. The results shown are the means of three to five experiments, with error bars representing ± 1 SD. Asterisks indicate significant differences (p < 0.05) in promoter activity between full-length (+125/−258) and truncated fragments. B) Activities of internal deletion mutants (M1–M6) of the −119/+125 and +28/−258 fragments cloned into a firefly luciferase reporter vector in the <i>NWC</i> and <i>RAG-2</i> orientations, respectively. The deletion ranges and their schematic representations (black boxes) are presented in the center of the graph. Numbers indicate positions relative to the <i>NWC</i> transcription start site. The relative promoter activities are presented as a percent of the activity of the +125/−119 (for <i>NWC</i>-oriented constructs) or +28/−258 (for <i>RAG-2</i>-oriented constructs) fragments, which were taken as 100%. The results shown are the means of three experiments, with error bars representing ± 1 SD. Asterisks indicate significant differences (p < 0.05) in promoter activity between the full-length constructs (+125/−119 or +28/−258) and the corresponding deletion mutants.</p

RT-PCR analysis of <i>RAG-2</i>

<p><b>expression.</b> RT-PCR analysis of <i>RAG-2</i> expression in (A) murine tissues; (C) and (D) testis of wild-type and NWC^tmpro1 mice; (B) and (E) thymus and testis of wild-type mice. The positions of the primers (solid horizontal arrows) used for each analysis are indicated. Primers were designed to distinguish <i>RAG-2</i> lymphoid-specific transcripts (B) from total <i>RAG-2</i> expression including non-lymphoid transcripts (A, C, E) and the full-length <i>RAG-2</i> open reading frame (D). Numbers indicate the utilized cDNA -fold dilution. Amplification of the <i>HPRT</i> gene was used for normalization of the cDNA quantity. The same cDNA was used in (C) and (D) and corresponding <i>HPRT</i> amplification is shown in panel (C).</p

The ZFP143 transcription factor activates the <i>NWC</i> promoter.

<p>Comparison of vertebrate <i>NWC</i> promoter sequences. The ranges of the analyzed internal deletion mutants M1–M6 (rounded rectangles), transcription start sites of <i>RAG-2</i> and <i>NWC</i> murine transcripts (horizontal lines), and regions of 80% and 100% identity among analyzed species (light and dark gray shadings, respectively) are shown. The promoter sequence is aligned with ZFP143 sequence motifs representing the ZFP143 consensus binding site <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044807#pone.0044807-Anno1" target="_blank">[19]</a>. The positions and nature of the point mutations introduced into the putative ZFP143 binding sites of the <i>NWC</i> promoter are shown. EMSA experiments demonstrating binding of the ZFP143 protein to the <i>NWC</i> promoter. The left panel shows binding of purified recombinant ZFP143-DBD (wild-type and mutant) to non-mutated and mutated probes. The mutant of the ZFP143 DNA-binding domain was obtained by substituting the cysteines of the third and seventh zinc finger domains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044807#pone.0044807-Schaub1" target="_blank">[20]</a> with alanines. The right panel shows binding of the non-mutated probe to nuclear lysates obtained from cells expressing His-tagged full length ZFP143. Horizontal arrows indicate unbound probe (1), shifts consistent with binding to one (2) or both (3) ZFP143 binding sites, and the supershift obtained using an anti-His antibody (4). Abbreviations: spec<b>.</b> comp. – specific competitor; mutA, mutT and mutAT – mutant probes (see panel A), anti-His – anti-His antibody. C) Activity of <i>NWC</i> promoter fragments containing point mutations that disrupt the ZFP143 binding sites. Genomic fragments spanning nucleotides −119/+125 (for <i>NWC</i>-oriented constructs) or +28/−258 (for <i>RAG-2</i>-oriented constructs) relative to the <i>NWC</i> transcription start site containing one (mutA or mutT) or both (mutAT) mutations were cloned into a firefly luciferase reporter vector and tested in NIH3T3 cells. The relative promoter activities are presented as a percent of the activity of the intact (−) +28/−258 fragment, which was taken as 100%. Asterisks indicate significant differences (p < 0.05) in promoter activity between non-mutated and mutated fragments. D) Activity of <i>NWC</i> promoter fragments in the presence of a dominant-negative form of ZFP143. Firefly luciferase reporter vectors containing genomic fragments spanning nucleotides −119/+125 (<i>NWC</i>-oriented) or +28/−258 (<i>RAG-2</i>-oriented) relative to the <i>NWC</i> transcription start were co-transfected with ZFP143 DNA binding domain (DBD), mutant ZFP143 DNA binding domain (DBDm) or empty (–) expression vector. A reporter vector containing the SV40 promoter (pGL3-Prom) and a promoter-less vector (pGL3-Basic) were used as controls. The relative promoter activities are presented as a percent of the activity of pGL3-Basic co-transfected with the appropriate expression vector, which was taken as 1%. The results shown are means of three to four experiments with error bars representing ± 1 SD. Asterisks indicate significant differences (p < 0.05) in promoter activity in cells transfected with empty vector and the ZFP143-DBD expression vector.</p

Organization of the mouse <i>RAG/NWC</i> locus and detailed structure of the region containing the <i>NWC</i> promoter.

<p>The relative positions of the exons encoding <i>RAG-1</i> (black boxes), <i>RAG-2</i> (open boxes), and <i>NWC</i> (gray boxes) are shown. Horizontal arrows indicate transcription start sites and orientations.</p