The hLEF/TCF-1 alpha HMG protein contains a context-dependent transcriptional activation domain that induces the TCR alpha enhancer in T cells.

Abstract

hLEF/TCF-Iot is a lymphoid cell-specific HMG protein that activates the distal enhancer of the gene encoding the a-subunit of the T-cell receptor (TCRot). We have shown previously that transcriptional activation by hLEF is highly dependent on the context of its binding site within the TCRa enhancer. Here, we demonstrate that hLEF contains a potent transcriptional activation domain that is separate from the HMG motif and is preferentially active in T cells. We find that hLEF/GAL4 fusion proteins can activate a GAL4-substituted TCRcz enhancer up to 50-fold in T-cell lines and are as active as GAL4/VP16 in this context. Unlike GAL4/VP16, however, hLEF/GAL4 could not activate heterologous promoters bearing only GAL4 DNA-binding sites. Thus, activation by hLEF/GAL4, like that noted previously for the native hLEF activator, was strongly influenced by the context of its DNA-binding site within the TCRc~ enhancer. Inspection of enhancer mutants suggests that trans-activation by hLEF/GAL4 is especially dependent on TCF-2, a distinct T-cell-enriched protein that binds to sequences flanking the hLEF-binding site in the enhancer. Analysis of small deletion or clustered amino acid substitution mutants in the hLEF-coding sequences identified a minimal activation region between amino acids 80 and 256 that appears to be bipartite in structure. The hLEF activation domain is not notably acid or glutamine-rich but is proline-rich and includes a motif rich in tyrosine and serine residues. We conclude that sequences outside of the hLEF HMG box mediate cell-and context-specific activation of the TCRoL enhancer and may facilitate interactions between hLEF and other T-cell-specific factors recruited to the enhancer.

Sequence-specific HMG box factors recognize 10-12 base pair minor groove motifs

“…This region coincides with a stretch of basic amino acids located C-terminal to the HMG box proper, which has been shown to contribute to the affinity of binding in several studies (4,40,41), and more recently has been found to increase the bending angle of a DNA motif complexed to the Lef-1 HMG box (10). Our deletion and footprinting data on the Ste11 HMG box indicate that the C-terminal basic region mediates recognition of the A 11 A 12 3' flank sequence.…”

Wnt-signalling pathway in ovarian epithelial tumours: increased expression of β-catenin and GSK3β

“…Proteins were transferred onto a polyvinyldifluoride (PVDF) membrane (Amersham, Buckinghamshire, UK) using a blotting system (NOVEX). The membrane was then incubated with antibodies against b-catenin (monoclonal, 1 : 1000), GSK3b, (monoclonal, 1 : 500) (Transduction Laboratories, Nottingham, UK), a-catenin (monoclonal, 1 : 1000; Alexis, Lausen, Switzerland) APC-COOH terminus or APC-NH 2 -terminus (polyclonal, 1 : 500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Tcf-Lef (polyclonal) and Lef-1 (monoclonal, 1 : 500; Kamiya Biomedical Company, Seattle, WA, USA) or Lef-1 antiserum (1 : 10000) (Carlsson et al, 1993). Prestained standards (SeeBlue; NOVEX) were used as weight markers.…”

Three habits of highly effective signaling pathways: principles of transcriptional control by developmental cell signaling

“…However, a more minimal reporter construct in transgenic flies, containing Lef/Tcf binding sites comparable to those in TOPFLASH, is not expressed in any embryonic cells, even under conditions of ubiquitous expression of the Wingless ligand ). More significantly, the fact that natural Lef/Tcf-activated enhancers require additional activators for proper expression (Carlsson et al 1993;Giese et al 1995;Brannon et al 1997;McKendry et al 1997;Szuts et al 1998;Nishita et al 2000;Darken and Wilson 2001) indicates that Lef/Tcf binding sites are insufficient for normal target gene activation in their native cis-regulatory contexts.…”