Effect of overexpression of PU.1 on GATA-1 DNA binding and erythroid differentiation in an erythroid cell line dependent on GATA-1 for erythroid differentiation.

Overexpression of PU.1 blocks GATA-1 DNA binding and erythroid differentiation in an erythroid cell line dependent on GATA-1 for erythroid differentiation (A) G1ER cells were mock-infected (G1ER/M, top 2 pictures) or infected with a PU.1 retrovirus (G1ER/PU, bottom 2 pictures). Cells were stained with benzidine before (left side, EST) or after (right side, +β-EST) treatment with β-estradiol. (B) EMSA of GATA-1 DNA binding. Nuclear extracts were made from G1ER, G1ER/PU-1, and G1ER/PU-2 cells 0, 28, and 48 hours after β-estradiol induction as shown in the upper panel. The GATA-1 probe is the same as that used in Figure 3A. Oct-1 DNA binding was used as a control as shown on the lower panel. G1ER/PU-1, and G1ER/PU-2 are 2 individual clones of PU.1-virus–transduced G1ER cells. (C) Total RNA was prepared from G1ER/M and G1ER/PU cells at 0, 8, 24, and 48 hours after stimulation with β-estradiol. Then, 10 μg of each sample was loaded onto the gel and hybridized to a murine major-globin probe.31