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Fig. 3. NeuroD contributes to the activation of Hb9. (A) P19 cells were
transfected with a luciferase reporter containing reiterated E boxes and
increasing amounts of expression constructs encoding bHLH factors. Ngn2
strongly activated transcription, whereas NeuroM (NeuM) and NeuroD (NeuD)
exhibited markedly less inherent trans-activating function. (B) NeuroD
activates M250 enhancer-mediated transcription in a synergistic manner with
Isl1 and Lhx3 in P19 cells. (C) NeuroD/E47 dimer binding to the M250 enhancer
was examined using gel retardation assays. Myc-tagged NeuroD [NrD(Myc)] and
HA-tagged E47 were translated in vitro separately and incubated with the M50
oligonucleotide in the presence or absence of antibodies directed against each
epitope. This translation condition favored the formation of E47:E47
complexes. Similar results were seen with oligos containing the M100 E box
(not shown). (D) The binding of co-translated NeuroD/E47 on M50 was challenged
by 20- or 100-fold molar excess of wild type (self), E-box mutated (E-mt) or
unrelated (non-self) DNA. Co-translation of proteins favored the formation of
NeuroD:E47 heterodimers, which bind with high specificity to the E box sites
within the enhancer. (E-H) The co-expression of NeuroD with Isl1 and Lhx3
triggers the differentiation of Hb9+ motoneurons in >70% of the
transfected P19 cells. This activity is comparable with that seen with NeuroM
(Lee and Pfaff, 2003).
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