The C. elegans Twist target gene, arg-1, is regulated by distinct E box promoter elements

Proper metazoan mesoderm development requires the function of a basic helix-loop-helix (bHLH) transcription factor, Twist. Twist-containing dimers regulate the expression of target genes by binding to E box promoter elements containing the site CANNTG. In Caenorhabditis elegans, CeTwist functions in a subset of mesodermal cells. Our study focuses on how CeTwist controls the expression of its target gene, arg-1. We find that a 385 bp promoter region of arg-1, which contains three different E box elements, is sufficient for maintaining the full CeTwist-dependent expression pattern. Interestingly, the expression of arg-1 in different tissues is regulated distinctly, and each of the three E boxes plays a unique role in the regulation. The first and the third E boxes (E1 and E3) are required for expression in a distinct subset of the mesodermal tissues where arg-1 is normally expressed, and the second E box (E2) is required for expression in the full set of those tissues. The essential role of E2 in arg-1 regulation is correlated with the finding that E2 binds with greater affinity than E1 or E3 to CeTwist dimers. A potential role for additional transcription factors in mesodermal gene regulation is suggested by the discovery of a novel site that is also required for arg-1 expression in a subset of the tissues but is not bound in vitro by CeTwist. On the basis of these results, we propose a model of CeTwist gene regulation in which expression is controlled by tissue-specific binding of distinct sets of E boxes.