Abstract
A T-cell-specific transcriptional enhancer was previously identified within the Jδ3-Cδ intron of the human T-cell receptor (TCR) δ gene, and seven distinct binding sites for nuclear factors (δE1 to δE7) 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 δE3 and δE4 displays potent enhancer activity, as judged by its ability to activate transcription from the Vδ1 promoter. We show that the interaction of nuclear factors with the δE3 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 8E3 that mediates the binding of two nuclear factors (NF-δE3A and NF-δE3C) and that is required for significant transcriptional activation by the enhancer. NF-δE3C is ubiquitous and may be identical to a previously characterized μE3-binding factor. NF-δE3A is preferentially expressed in T lymphocytes, and we suggest that this factor may play the dominant role in transcriptional activation through the δE3 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 δE4 is also analyzed. One of three specific complexes formed with a δE4 probe appears to be T-cell specific.