Identification of an essential site for transcriptional activation within the human T-cell receptor delta enhancer

Abstract

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 microE3-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.