Identification and characterization of two functional domains within the murine heavy-chain enhancer

We have investigated the effect of polymerizing defined segments of the immunoglobulin heavy-chain enhancer on the activity of a single, linked transcription unit. Transient assays in lymphoid cells have led to the following observations. First, polymerizing the entire enhancer led to an increase in overall transcription. Second, polymerizing defined DNA segments revealed two distinct functional domains within the enhancer. Although each domain alone possessed only partial enhancer activity, greater than wild-type levels of activity could be obtained upon polymerization. One of these domains contains three regions thought to be involved in protein binding in vivo and in vitro (E motifs E1, E2, and E3). The other domain contains the fourth E motif (E4) and the conserved octanucleotide, ATTTGCAT. We have tested the functional importance of these motifs by determining the effect of mutating these elements singly or in combination in the context of the isolated domains. Although E2, E3, E4, and the octanucleotide are clearly important for enhancer function, mutation of the E1 motif did not appear to have an effect on enhancer activity in our assay. Transient assays in mouse L cells indicate that nonlymphoid cells are able to use a distinct subset of these motifs.