Abstract
Basic helix-loop-helix (bHLH) transcription factors often function as heterodimeric complexes consisting of a tissue-specific factor such as SCL/tal or MyoD bound to a broadly expressed E protein. bHLH dimerization therefore appears to represent a key regulatory step in cell lineage determination and oncogenesis. Previous functional and structural studies have indicated that the well defined HLH domain is both necessary and sufficient for dimerization. Most of these studies, however, have employed in vitro systems for analysis of HLH dimerization, and their implications for the requirements for in vivo dimerization remain unclear. Using multiple approaches, we have analyzed bHLH dimerization in intact, living cells and have identified a novel domain in E proteins, domain C, which is required for in vivo dimerization. Domain C, which lies just carboxyl-terminal to helix 2 of the HLH domain, represents the most highly conserved region within E proteins and appears to influence the in vivo conformation of the adjacent HLH domain. These results suggest that HLH dimerization in vivo may represent a complex, regulated process that is distinct from HLH dimerization in vitro.
Highlights
For several classes of transcription factors, such as members of the basic helix-loop-helix, leucine zipper, and nuclear receptor families, dimerization represents a key, obligatory step prior to DNA binding and transcriptional activation
E2-2, but Not SCL/tal or MyoD, Requires Sequence Outside the Basic helix-loop-helix (bHLH) Domain for in Vivo Dimerization—The minimal bHLH domains of E2-2, SCL/tal, and MyoD have been well defined by alignment analysis of a broad range of bHLH proteins from a wide variety of organisms [28]
These data suggest that the structural requirements for dimerization in the yeast two-hybrid system differ for tissuespecific bHLH proteins as compared with E proteins, the former requiring only the minimal bHLH domain and the latter requiring additional sequence
Summary
For several classes of transcription factors, such as members of the basic helix-loop-helix, leucine zipper, and nuclear receptor families, dimerization represents a key, obligatory step prior to DNA binding and transcriptional activation. This dimerization permits the mixing and matching of factors with different DNA half-site binding specificities and expands the repetoire of potential target sequences that may be recognized. Tain an array of different cell lineage-specific bHLH factors all competing with one another, as well as with inhibitory HLH proteins, for dimerization with a common E protein partner. The role of domain C appears to be as an in vivo conformational determinant, maintaining the bHLH domain of E proteins in a “receptive” conformation for heterodimerization with tissue-specific bHLH proteins
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