ETS-Mediated Cooperation between Basic Helix-Loop-Helix Motifs of the Immunoglobulin μ Heavy-Chain Gene Enhancer

Abstract

The E motifs of the immunoglobulin heavy-chain gene enhancer bind ubiquitously expressed proteins of the basic helix-loop-helix (bHLH) family. These elements work together with other, more tissue-restricted elements to produce B-cell-specific enhancer activity by presently undefined combinatorial mechanisms. We found that E2 contributed to transcription activation in B cells only when the E3 site was intact, providing the first evidence for functional interactions between bHLH proteins. In vitro assays showed that bHLH zipper proteins binding to E3 enhanced Ets-1 binding to A. One of the consequences of this protein-protein interaction was to facilitate binding of a second bHLH protein, E47, to the E2 site, thereby generating a three-protein-DNA complex. Furthermore, transcriptional synergy between bHLH and bHLH zipper factors also required an intermediate ETS protein, which may bridge the transcription activation domains of the bHLH factors. Our observations define an unusual form of cooperation between bHLH and ETS proteins and suggest mechanisms by which tissue-restricted and ubiquitous factors combine to generate tissue-specific enhancer activity.The immunoglobulin (Ig) heavy-chain gene enhancer is a cell-specific transcription regulatory sequence. Located in the J H -C intron, it is necessary for the expression of a rearranged IgH gene in transfection as well as transgenic assays. Moreover, when taken out of its normal context, the enhancer is sufficient to target a heterologous transgene (1,14,18,27) for expression at the appropriate differentiation stage in the Blymphocyte lineage. In addition to activating transcription from a V H gene promoter that has recombined into the C locus, this enhancer has also been implicated in the initiation of V(D)J recombination at the IgH locus. This is based on two experimental observations: transgenic recombination substrates were shown to be activated by the enhancer (7), and genetic deletion of the endogenous enhancer was shown to suppress IgH recombination (4, 31). At present, it is not clear whether the recombination activation and transcription activation properties of the enhancer are directly related; however, it is likely that enhancer-mediated chromatin reorganization resulting in increased accessibility of the IgH locus to polymerases and recombinases is important for both processes.B-cell-specific enhancer function is determined by multiple trans-acting nuclear proteins that bind to specific sites within the enhancer (6,19). Proteins that bind to the enhancer can be broadly classified into two categories: those that are more restricted in their tissue distribution, such as A, B, and octamer binding proteins, and those that are ubiquitously expressed in most cell types, such as the E1, E2, E3, and E5 binding proteins. However, no enhancer binding protein identified to date has an expression pattern that correlates perfectly with the cells in which the IgH gene is expressed. Based on the expression pattern of proteins binding to the A and B sites, we have previ...

Characterization of an E-box–Dependent cis Element in the Smooth Muscle α-Actin Promoter

“…For example, muscle enhancer factor 2 potentiates the transcriptional activity of the myogenic HLH factors, 30 and the intermediate Ets-1 binding site in the immunoglobulin Heavy-Chain enhancer is required for transcriptional synergy of the flanking E-boxes. 31 Our data demonstrating E-box-dependent activity of the TGTTTATC-CCCA further extends this observation. Moreover, these data provide the first evidence for E-box-dependent combinatorial interactions regulating a SM selective gene in SMC, although the specific mechanisms whereby the TGTTTATC-CCCA sequence interacts with the E-boxes are unclear.…”

Regulation of Ets function by protein–protein interactions

“…The binding of Ets1 in the absence of an EBS is another example of protein-tethered transactivation (PTT). The transcriptional synergy between the bHLH proteins E47 and TFE3 is dependent upon Ets1-mediated three protein-DNA complexes on the immunoglobulin m heavy-chain gene enhancer (Dang et al, 1998). The NID (Net inhibitory domain) of Net/SAP2, a member of the TCF subfamily, interacts with the bHLH protein E47 through its HLH-like domain.…”

Differential Regulation of Epithelial and Mesenchymal Markers by δEF1 Proteins in Epithelial–Mesenchymal Transition Induced by TGF-β

“…In addition, Ets1 activity on ␦EF1 promoter was partially repressed by transfection of E12/E47 siRNA (Supplementary Figure 2, A and B), indicating that Ets1 may act in cooperative manner with E47 to regulate expression of ␦EF1. However, it is currently unknown whether Ets1 directly interacts with E47 or binds with it indirectly through other molecule(s) in TGF-␤-induced EMT (Dang et al, 1998). Taken together, these findings suggest that collaboration of Ets1 with E47 may regulate the expression of SIP1 and ␦EF1, which may occur in a cell-and promoter-context manner and that downregulation of Id protein expression by TGF-␤ may thus enhance this cooperation in inducing expression of ␦EF1 family proteins, leading to EMT.…”