Fig. 7. SRF and GATA4 can simultaneously bind XMLC2 promoter in vitro, and can synergistically activate an XMLC2-luciferase reporter in animal pole explants. (A) The CArG-like site in –122/–85 promoter fragment binds SRF only weakly (complex labelled S); GATA4 protein binds efficiently to the GATA#2 site present in the same probe (labelled G); simultaneous binding of SRF and GATA4 can be detected (arrow). (B) Overexpression of SRF activates an XMLC2(–123/–41) cyt.actin-luciferase reporter only weakly in animal pole explants, while ectopic GATA4 gives strong activation. Co-expression of both factors leads to synergistic activation. Animal poles were excised at stage 9 and collected after three hours of incubation at 23°C. Firefly luciferase activity was normalised using coinjected tk-Renilla luciferase as a control and was set at 1 for XMLC2(–123/–41) cyt.actin-luciferase in the absence of SRF or GATA4. Values shown represent a single experiment; a second experiment gave similar results.