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Fig. 6. Esr10 proneural enhancer activity requires intact E-boxes. HeLa
cells were transfected with expression vectors for ICD, Xngnr1 and
E47 (N/E), or both, and with luciferase vectors driven by wild-type
(Esr10/Dra) or mutated (Esr10/DramE1E2) elements, shown
schematically above (A). In the case of Esr10/Dra, ICD and N/E
synergistically activate transcription approximately three times more over ICD
alone (A, left). Synergy was lost when E-boxes were mutant (A, right). E-box
motifs were also required for GFP expression driven by
Esr10/Dra in transgenic frogs (compare C with B). (D,E) Injection of
Xngnr1 mRNA with a lacZ tracer into Esr10/DramE1E2
transgenic embryos (E) could not activate enhancer activity as was seen with
controls.(F) EMSA showing that Xngnr1 (N) and E47 (E) proteins shift an E2
oligo; shifts were competed by 10x and 100x cold competitor (WT)
but not by similar increases mutant E2 oligos (Mut) or oligos corresponding to
a binding site of a heterologous activator (Vax)
(Mui et al., 2005). O, oligo;
R, reticulocyte lysate; N/E, Xngnr1 plus E47. Complexes formed by E47
homodimers (Ex2) are of higher mobility than those formed by Xngnr1/E47 (N/E)
heterodimers. ns, nonspecific complexes attributable to reticulocyte
proteins.
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