Fig. 2. Mesp2 can directly bind to the enhancer element of the Ripply2 gene and activate its transcription. (A) Comparison of the genomic sequences around the Ripply2 gene in mouse (top line) with those in human, dog and chick using MultiPipMaker sequence alignment software. A conserved region (framed in red) is evident across these species. (B) Sequence alignment of the 171 bp region conserved among the Ripply2 genes, within which a highly conserved E-box is located. HC E-box, highly conserved E-box. (C) The genomic organization of the mouse Ripply2 gene and the corresponding construct used in the transgenic analyses. A 1.5 kb DNA fragment containing this highly conserved 171 bp stretch (shown in A) of the Ripply2 upstream region was ligated to a cassette composed of the hsp promoter and nlacZ (lacZ harboring a nuclear localization signal). E, EcoRI; B, BamHI; N, NcoI. (D) The Ripply2 enhancer drives lacZ reporter gene expression in somitic mesoderm cells at E11.0. The inset shows high magnification of the somitic region. (E) Luciferase reporter assay for Mesp2 activation, with or without E47, using constructs harboring either the 1.5 kb Ripply2 enhancer (left) or six repeats of the conserved 171 bp fragment (right). The addition of E47 had negative effects upon transactivation. The data represent the means±s.d. from four separate experiments. ^*P<0.01, ^**P<0.04. (F) EMSA analyses revealing that a DNA fragment containing the conserved E-box (Region B, light-blue shading) from the Ripply2 upstream region can bind Mesp2 in the absence of E47. This binding of Mesp2 thus appears to be different from its binding to the Epha4 enhancer, which is dependant upon E47. Non-specific bands are indicated by the asterisk. (G) The binding specificity of Mesp2 was confirmed by successful competition with cold probe, but not with an E-box mutant probe (shown in B).