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.

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 K below 50 µM. One candidate, WEN05-03, with a K=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 K=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.

Recombination activating gene-2 (RAG-2) and NWC are strongly evolutionarily conserved overlapping genes which are convergently transcribed. In non-lymphoid cells the NWC promoter is active whereas in lymphocytes it is inactive due to the DNA methylation. Analysing the mechanism responsible for lymphocyte-specific methylation and inactivation of NWC promoter we found that Ikaros, a lymphocyte-specific transcription factor, acts as a repressor of NWC promoter - thus identifying a new Ikaros target - but is insufficient for inducing its methylation which depends on the antisense transcription driven by RAG-2 promoter. Possible implications of these observations for understanding evolutionary mechanisms leading to lymphocyte specific expression of RAG genes are discussed.