IDENTIFICATION OF AN ESSENTIAL SITE FOR TRANSCRIPTIONAL ACTIVATION WITHIN THE HUMAN T-CELL RECEPTOR-DELTA ENHANCER

A T-cell-specific transcriptional enhancer was previously identified within the J-delta-3-C-delta intron of the human T-cell receptor (TCR) delta-gene, and seven distinct binding sites for nuclear factors (delta-E1 to delta-E7) were defined by DNase I footprinting. In this study, we conducted a detailed functional analysis of the various cis-acting DNA sequence elements of the enhancer and show that a 60-bp fragment encompassing delta-E3 and delta-E4 displays potent enhancer activity, as judged by its ability to activate transcription from the V-delta-1 promoter. We show that the interaction of nuclear factors with the delta-E3 site is essential for enhancer activity. This element displays significant activity in the absence of additional segments of the enhancer. Further, methylation interference and in vitro mutagenesis identify a site within delta-E3 that mediates the binding of two nuclear factors (NF-delta-E3A and NF-delta-E3C) and that is required for significant transcriptional activation by the enhancer. NF-delta-E3C is ubiquitous and may be identical to a previously characterized mu-E3-binding factor. NF-delta-E3A is preferentially expressed in T lymphocytes, and we suggest that this factor may play the dominant role in transcriptional activation through the delta-E3 site. This factor interacts with the sequence TGTGGTTT, a motif that is also found within the enhancers of additional TCR and CD3 genes. Nuclear factor binding to delta-E4 is also analyzed. One of three specific complexes formed with a delta-E4 probe appears to be T-cell specific.