A T cell nuclear factor resembling NF-AT binds to an NF-kappa B site and to the conserved lymphokine promoter sequence "cytokine-1".

Nuclear extracts from a nontransformed murine T lymphocyte clone contained two inducible factors that bound to a nuclear factor kappa B (NF-kappa B) site. One factor was NF-kappa B, and the other was differentiated from NF-kappa B by its mobility in the electrophoretic mobility shift assay and its lack of sensitivity to protein kinase C depletion. Competition and methylation interference assays showed that the binding site for the novel factor was limited to nucleotides in the 3' half of the kappa B site. This part of the kappa B site resembled sequences in the binding site for a second inducible nuclear factor of T cells, NF-AT, as well as a conserved sequence found in several lymphokine genes, termed "cytokine-1" (CK-1). Competition and methylation interference analysis showed that both NF-AT and CK-1 sequences bound a factor similar to the novel kappa B-binding factor and that binding involved a four-nucleotide sequence (TTCC) that the kappa B, CK-1, and NF-AT sites have in common. The complexes that form with each site have characteristics of NF-AT: they are induced upon T cell receptor stimulation, are sensitive to protein synthesis inhibitors and cyclosporin A, and are not sensitive to protein kinase C depletion. Thus, a factor or factors similar to NF-AT can bind to three distinct promoter sequences which occur commonly in several T cell activation genes. These results raise the possibility that related factors binding to kappa B, CK-1, and NF-AT sequences could play a role in the coordinate induction of T cell activation genes. In addition, our results suggest that kappa B and CK-1 sites represent potential cyclosporin-sensitive promoter elements by virtue of their ability to bind an NF-AT-like factor.     

Pi 1 binding sites are negative regulators of bcl-2 expression in pre-B cells.

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.