Fig. 2. A cluster of three forkhead-binding sites is sufficient for dauer-specific lag-2 expression in the IL2 neurons. (A) A 3-kb region upstream of the lag-2 translational start site was subjected to deletion analysis to determine the minimal fragment necessary for IL2/dauer-specific lag-2 expression in daf-7 animals. Enzyme sites used for the generation of the different promoter variants are indicated: Ns (NspI), A (AccI), E (EcoRI), Nc (NcoI) and H (HhaI). Solid lines represent fragments of the lag-2 promoter that were cloned upstream of the GFP-coding sequence; dashed lines represent deleted sequence. Asterisks represent the location of the predicted forkhead-binding sites in the lag-2 promoter. (B) Two potential forkhead-binding sites, named A and B, were identified in the minimal fragment required for IL2 neuron/dauer-specific expression. The consensus binding sites for the FoxC1 transcription factor are indicated in the grey box above the lag-2 sequence (A binding sites). Capital letters represent the core binding site and small letters indicate nucleotides required for efficient binding. (C) Smaller deletions of the 270 bp fragment were created to determine which forkhead-binding sites are required for IL2 neuron/dauer-specific expression. The white and grey boxes represent the identified forkhead-binding sites and the crosses indicate regions where the core binding site sequence was deleted. The relative intensity of GFP expression in the IL2 neurons is indicated as follows: +++, strong; ++, moderate; +, faint; -, no expression. For the consensus binding sites: w can be A or T; m can be A or C; and n can be A, T, C or G.