The E3 E box within the immunoglobulin heavy-chain (IgH) enhancer binds several proteins of the basic helix-loop-helix-leucine zipper (bHLHzip) class, including TFE3, USF1, and Max. Both TFE3 and USF have been described as transcriptional activators, and so we investigated their possible roles in activating the IgH enhancer in vivo. Although TFE3 activated various enhancer-based reporters, both USF1 and Max effectively inhibited transcription. Inhibition by USF correlated with the lack of a strong activation domain and was the result of the protein neutralizing the E3 site. The effects of dominant-negative derivatives of TFE3 and USF1 confirmed that TFE3, or a TFE3-like protein, is the primary cellular bHLHzip protein that activates the IgH enhancer. In addition to providing a physiological role for TFE3, our results call into question the traditional view of USF1 as an obligate transcriptional activator.B-cell-specific transcription of the immunoglobin heavychain (IgH) locus is directed primarily by an enhancer located within the intron separating the J and C regions of the gene. Transfection experiments and in vitro binding studies have defined numerous sites within an approximately 250-bp region critical for enhancer activity. Among the proteins that bind these sites are two cell-restricted proteins (Oct-2 and PU.1) and several additional transcription factors of the basic helixloop-helix (bHLH), bHLH-leucine zipper (bHLHzip), ets, zinc finger, and POU-homeodomain families (for a review, see reference 24). Previous work in this laboratory has identified a 60-bp region containing three E boxes (E5, E2, and E3) that generates B-cell-specific activity in transfected cells (35). Two of the E boxes, E5 and E2, bind members of the bHLH family of transcription factors, including the products of the E2A (E12 and E47 [32-34]), E2-2 (22), and HEB (23) genes. The E2A proteins are major determinants of lymphoid development, as deletion of the E2A gene leads to a complete loss of the B-cell lineage in mice (4, 39). Although the E47 protein is widely expressed, DNA binding by E47 homodimers is restricted to B cells (36). DNA binding by E47 in certain other cell types requires dimerization with distinct cell-restricted bHLH partners such as MyoD (28,33).Transfection of E47 expression plasmids results in the activation of various E-box-containing reporters and, additionally, the chromosomal IgH locus in non-B cells (12). E47 homodimers have been shown to competitively displace a repressor protein from the E5 site (18). In the absence of this E47-mediated derepression, the repressor inhibits activity that would otherwise be generated through widely expressed proteins that bind other enhancer sites, including the neighboring E3 site (35). The E3-binding proteins identified thus far are members of the bHLHzip family and include TFE3 (5), USF1 (20), TFEB (10), Myc:Max heterodimers (8, 27), and Max homodimers (7,8). Although TFE3, USF1, and Myc:Max heterodimers have all been described as activators at E3-like elements, it is uncl...

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