Analysis of evolution within the bHLH transcription factor family based on a complete set of DNA binding interaction specificities. Hannah Pham, Jianhong Ou, Scot Wolfe, Michael Brodsky. Program in Gene Function and Expression, University of Massachusetts Medical School, Worcester, MA.

   We describe a comprehensive analysis of DNA binding specificities and protein-protein interactions within the bHLH family of transcription factors in D. melanogaster. bHLH proteins typically bind DNA as homo- or hetero-dimeric complexes to E-box related sequences, with some variant of CANNTG. We identify homo- or heterodimeric binding partners for all but one of the 56 bHLHs. The lone exception is Her, which binds to a unique, non-E-box sequence as a monomer, the first identified example of a monomeric, DNA binding bHLH protein. DNA binding specificities were also determined for all bHLHs, revealing highly diverse E-box related motifs that can vary at any position within the E-box and have different prefences flanking the E-box. This set includes the Tai/dSRC, a known coactivator protein; we find that Tai can directly binds DNA as a homodimer or a heterodimer with the JH receptors Met and gce. Analysis of bHLH sequences in 15 insect species reveals a core set of 52 bHLH proteins. D mel has 2 losses and 6 gains relative to the core set. The losses are in bHLHs conserved in mammals; our motif data combined with mammalian expression data reveal that other bHLHs present in D.mel have DNA binding and development expression related to the missing bHLH, suggesting a mechanism to compensate for loss of a core bHLH. 5 of 6 bHLH gains represent duplications that generate multiple proteins with similar binding specificities. However, the remaining gain is Her, a very recent derivative of the E(spl) family that acquired a new DNA binding specificity reflecting a novel DNA binding mechanism. Using the D.mel dataset to predict bHLH specificities in other insects, we find that most bHLH gains and losses are unlikely to result in the loss or gain of a particular DNA binding specificity. However, in rare cases, bHLH proteins evolve with substantial alterations in specificity determining residues, providing opportunities to regulate novel gene sets.