Genes for TNF, LTalpha and LTbeta encode related cytokines. We are investigating the molecular control of the differential expression of these three genes at human and mouse genomic loci. We have cloned, sequenced and characterized by in situ transcriptional analysis the murine LTbeta gene. This gene is predominantly expressed in the thymus and spleen, with distinct pattern of expression in the medulla of the thymus and splenic follicles. In human peripheral T cells the LTbeta gene is not markedly induced by anti-CD3 activation, contrary to TNF and LT genes. On the other hand, only the LTD gene is activated (or derepressed) after incubation of T cells in serum-free medium. Jurkat cells showed high level of PMA inducible LTbeta transcription. On the basis of promoter deletions, binding and transfection assays, PMA response is tentatively attributed to the factors bound at Ets-1, NF-kB and EGR-1 sites. Our data indicate two levels of transcriptional activation of TNF gene by LPS. Firstly, LPS increases the rate of initiation via NF-kB/Rel family members, such as p65 and c-Rel, binding to the kB sites at the 5'-flank and to the newly characterized enhancer at the 3'-flank. Secondly, as indicated by nuclear run-on assays with short DNA probes, LPS also increases the processivity of transcription, enabling the primary transcripts to come to completion. With regard to transcriptional regulation by NF-kB/Re1 we addressed a general problem of the effects of DNA conformation on the affinity of these factors to their cognate sites. Our data indicate that p52 (NF-kB2) is the only homodimer that introduces directed bend into DNA. We also find that NF-kB/Rel factors are differentially sensitive to the """"""""conformational context"""""""" of the kB site placed on the minicircle and pre- bent in various orientations. This suggests a novel determinant of the specificity of transcriptional regulation.
