E2A Antagonizes PU.1 Activity through Inhibition of DNA Binding

Figure 5

E47 inhibits PU.1 binding to DNA. (a) Transient transfections of 293 T cells with a GAL4 responsive luciferase reporter construct. Cells were cotransfected with either GAL4 full-length PU.1 expression plasmid or GAL4-PU.1 activation domain in the presence or absence of MigR1-E47 plasmid. Luciferase activity is the mean standard deviation of three independent transfections and luciferase activity is reported as fold-induction above the activity seen in 293 T cells transfected with the GAL4 DNA binding domain expression plasmid. (b) Immunoblot of nuclear extracts prepared from 293 T cells transfected with empty expression plasmids (NE1), PU.1 expression plasmid (NE2), and PU.1 and E47 expression plasmids (NE3, NE4). NE4 was prepared from cells transfected with an increased amount of E47 expression plasmid (++). Immunoblots were probed with anti-PU.1 or anti-E47 antibody as indicated. (c) EMSA performed with nuclear extracts from (b) and a ³²P-labeled MCSFR probe. The PU.1 containing complex was identified by performing competitions with unlabeled wildtype and mutant MCSFR probes as indicated. Additionally the PU.1 containing complex could be ablated by inclusion of anti-PU.1 antibody in the binding mix.

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