Fig. 2. Identification of a Notch-regulated endoderm/mesoderm enhancer. (A) The ref-1 promoter regions shown were fused to GFP and assayed for expression in Notch-activated endodermal cells (green, expressed; red, not expressed). Transgenes were generated as plasmids (e.g. pAN20) or PCR products (e.g. ANPCR); pAN2 and ANPCR have been described previously (Neves and Priess, 2005). Numbers indicate distance from the initiator ATG (position 0). Inverted black and white triangles represent CSL- and GATA-binding sites, respectively. (B) Diagram of C. elegans enh^A. Cyan and yellow arrows indicate orientation of CSL and GATA sites, respectively. The green box represents a possible NK-binding site. Bold line indicates region used for EMSA experiments (see Fig. 4). (C) Sequence alignment of the enh^A element from C. elegans (C.e), C. remanei (C.r) C. briggsae (C.bri) and C. brenneri (C.bre). Black lettering indicates that the nucleotide is conserved in at least three species. Note that the 3-CSL sequence in C. brenneri (TCTGGGAA) differs from a consensus CSL binding sites (YRTGRGAA) (Yoo et al., 2004); this sequence was confirmed by PCR amplification and analysis of genomic DNA from C. brenneri.