Gfi1b is directly regulated by E47. (A) 0531 (top graph) and 1.F9 (bottom graph) cells were infected with pCS retrovirus (▪) or pCS-E47ER (□) and treated with 4-OHT (1 μM) for 4 hours in the presence or absence of CHX (4 μM) starting 24 hours after infection. Gfi1b and HPRT mRNA were quantified by QPCR. The standard error of triplicate measurements is shown. (B) Schematic of the Gfi1b gene showing the approximate location of the conserved E-boxes (▾) within the first intron. (C) EMSA using labeled Gfi1b E-box1, E-box2, or μE5 oligos and protein extracts prepared from 0531 cells infected with S003 or S003-E47 retrovirus 24 hours after infection. Prior to addition of labeled probe the extracts were treated with either ddH20 (−), 100 × excess of competitor oligo (E-box1 or E-box2), or anti-E47 antibody for 15 minutes. (D) ChIP assay performed on 0531 cells infected with MigR1 or MigR1-E47 using polyclonal anti-E2A antibody (E2A), anti–acetyl-histone H3 (aH3), or no antibody (−).
