Fig. 1. Twist binds DNA and activates gene expression as a homodimer. (A)Mobility shift assays with rho E box (CATATG; Ip et al., 1992) were performed with full-length Twist (lanes 2, 3, 4), truncated Twist (bHLHTwist; lanes 8, 9, 10) or both (lanes 5, 6, 7). An ^* marks the full-length homodimer (lane 2) and the truncated Twist homodimer (lane 8). A mixture of full-length and the bHLH region of Twist produced an intermediate band shift (lanes 5, 7 ^*), corresponding to a full-length and bHLH Twist heterodimer. These bands are competed away with 200x unlabeled rho E box (lanes 3, 6, 9), but not with a mutated (mt) E box (AGTGTG) (lanes 4, 7, 10). The unprogrammed lysate is included in lane 1. Probe is in excess in all lanes in all shifts shown. (B) Schematic representation of two Twist proteins joined in-frame by a flexible 16 amino acid Gly/Ser rich linker (Markus, 2000; Neuhold and Wold, 1993). The boxes signify the basic, DNA-binding domain and the HLH dimerization domain. The linker sequence is given in single letter amino acid code. Numbers below the line refer to the nucleotide sequence of the twist cDNA (Thisse et al., 1988). (C) DNA binding properties of tethered Twist-Twist. In vitro translated products are assayed for binding to rho E box (CATATG). Twist-Twist linked dimers (^*, lane 3 and 5) have the same DNA binding specificity as Twist alone (lane 2). The upper band in lanes 3 and 5 is a dimer of the linked dimers. These higher order complexes can be competed away easily upon addition of a bHLH monomer, leaving only the forced homodimer (data not shown). We have no evidence that this complex nor other complexes (the tethered dimer and another bHLH) are found in tissue culture or in vivo, yet this possibility does exist. (D) Twist and Twist-Twist linked dimers activate a 175 bp Mef2 enhancer in Drosophila SL2 cells (Cripps et al., 1998). SL2 cells are transfected with indicated amounts of actin-lacZ plasmid, 175bp Mef2 enhancer-luciferase reporter plasmid, equimolar amounts of Twist or Twist-Twist expression vector and carrier DNA to equalize total DNA/transfection. Activation values are expressed relative to controls (see Materials and Methods). Error bars represent the standard error of means of triplicated experiments. Transfection of Twist-Twist linked dimers results in slightly better reporter gene transactivation as compared to Twist monomers alone.