One base pair change abolishes the T cell-restricted activity of a kB-like proto-enhancer element from the interleukin 2 promoter

Abstract

The inducible, T cell-specific enhancers of murine and human Interleukin 2 (Il-2) genes contain the kB-like sequence GGGATTTCACC as an essential cis-acting enhancer motif. When cloned in multiple copies this so-called TCEd (distal T cell element) acts as an inducible proto-enhancer element in E14 T lymphoma cells, but not in HeLa cells. In extracts of induced, Il-2 secreting El4 cells three individual protein factors bind to TCEd DNA. The binding of the most prominent factor, named TCF-1 (T cell factor 1), is correlated with the proto-enhancer activity of TCEd. TCF-1 consists of two polypeptides of about 50 kD and 105 kD; the former seems to be related to the 50 kD polypeptide of NF-kB. Purified NF-kB is also able to bind to the TCEd, but TCF-1 binds stronger than NF-kB to TCEd DNA. The conversion of the TCEd to a 'perfect' NF-kB binding site leads to a tighter binding of NF-kB to TCEd DNA and, as a functional consequence, to the activity of the 'converted' TCEd motifs in HeLa cells. Thus, the substitution of the underlined A residue to a C within the GGGATTTCACC motif abolishes its T cell-restricted activity and leads to its functioning in both El4 cells and HeLa cells. These results indicate that lymphocyte-specific factors binding to the TCEd are involved in the control of T cell specific-transcription of the Il-2 gene.

Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site.

“…The latter are binding sites for positive-acting transacting factors. When these sites are cloned in multiple copies in front of an indicator gene, they exhibit an inducible, T-cell-restricted activity like that of the entire IL-2 enhancer (4,5,35,42). Previously, we and others showed that CsA and FK506 selectively inhibit the activity of purine boxes, the binding site of the lymphoid-specific factor NFAT-1, and of the UPS (4, 10, 35) when they are added to the cells at the same time as (or after) the inducers.…”

“…All recombinant DNA work was done by standard procedures (38). The construction of the protoenhancer constructs 5 xUPS, 5 xTCEd, and 4xPu-bd, containing multimers of the protein-binding sites of the IL-2 enhancer in front of the thymidine kinase (tk) promoter of CAT vector pBLCAT2, has been described before (4,5,35,42). Construct 5XUPS carries five copies of the UPS, spanning nucleotides from positions -64 to -94 of the IL-2 enhancer (4) (see Fig.…”

Calcineurin acts in synergy with PMA to inactivate I kappa B/MAD3, an inhibitor of NF-kappa B.

“…This transcription factor plays an important role in activating the IL-2 promoter in T cells (Hoyos et al, 1989;Shibuya et al, 1989;Baumann et al, 1991;Briegel et al, 1991) (and Figure 3) as well as numerous other promoters, in many cell types (Baeuerle, 1991). Therefore understanding how calcineurin activates NF-xB might provide generalizable insights into the mechanisms by which this transcription factor is controlled.…”

“…DNA sequences recognized by these factors are found within the 300 base pairs preceding the IL-2 gene transcription start site. There are minimally five factors, uniquely defined by nucleotide sequence recognition, essential for (ionomycin+PMA)-induced transcription from the IL-2 gene promoter: NF-AT, which binds the IL-2E element (Durand et al, 1988), NF-xB, which binds the IL-2C element (Baumann et al, 1991;Briegel et al, 1991;Hoyos et al, 1989;Shibuya et al, 1989), which recognizes a sequence within the IL-2B element (Jain et al, 1992a,b;Northrop et al, 1992), and OAP (Ullman et al, 1991) and Oct-I (Kamps et al, 1990), which function together, each recognizing an independent sequence within the IL-2A element (Durand et al, 1988 (Mattila et al, 1990). (ii) An NF-xB-dependent enhancer is responsive to PMA, unresponsive to ionomycin, but very responsive to ionomycin in the presence of PMA.…”

Characterization of the mouse dynamin I gene promoter and identification of sequences that direct expression in neuronal cells

“…Several factors other than NF-κB have been shown to interact with NF-κB-like motifs, including AP-3, MP-1, PRDII-BF1, HIV-EP1 and MBPII [35][36][37][38][39]. Briegel et al [40] showed that TCF-1 binds more strongly than NF-κB to an NF-κB-like motif (TCEd) in the interleukin 2 promoter. They demonstrated that the conversion of TCEd to a perfect NF-κB-binding site leads to a tighter binding of NF-κB to TCEd DNA and abolishes its T-cellrestricted activity.…”