FIG. 10.

Runx perturbations abolish silencing activity in immature T cells (Adh.2C2). (A) Cells were nucleofected with PU.1 reporters, plus 1 to 2 μg of the indicated plasmid. Only cotransfection with the Runx dominant negative expression vector relieved silencing (red bars). (B) Runx1 overexpression in RAW 264.7 cells enhances CE5 to CE3 activity and does not lead to repression. *, P < 0.03. Data are from three experiments performed in duplicate and are reported relative to L1 activity. (C) Western blot of whole-cell lysates from Adh.2C2 cells transfected with or without Runx1 morpholino, showing Runx1 loss. (D) Antisense morpholino knockdown of endogenous Runx1 blocks silencing (red bar). Data from two independent experiments are shown as the L98+4/L98 reporter activity ratio. *, P < 0.02 compared to the no-morpholino data set. (E) Runx sites are essential for silencing in a chromatin context in stably transfected Adh.2C2 cells. With L93 mRunx, all predicted Runx sites in the CE4A-B region mutated. L93 M5, L93 with the M5 mutation; L93 mRunx M5, mutations combined. Data are plotted as for Fig. 4D with data from two independent experiments, each with four to six independent mixed pools of stable cells. *, P < 0.001 compared to L96; #, P < 0.001 compared to L9-3.