Fig. 4.

Rapid GATA-1-dependent displacement of GATA-2 from the –2.8-kb site. G1E-ER-GATA-1 cells were incubated for 0, 0.5, 2, 4, or 10 h with 1 μM tamoxifen. (A) Quantitative ChIP analysis was used to measure the binding of GATA-1 and -2, as well as H3 acetylation at the –2.8-kb site and at the 1S and 1G promoters. Samples were analyzed quantitatively relative to a standard curve generated from input chromatin. The plots depict relative levels of binding and acetylation (means from four independent experiments). (B) RT-PCR was used to measure primary GATA-2 transcripts arising from use of the 1S promoter (Exon1S/Intron1S), the 1G promoter (Exon1G/Intron2), or both promoters (Intron2/Exon2). GAPDH mRNA was measured as a control. Relative GATA-2 primary transcript levels were normalized by the levels of GAPDH transcripts. The plots depict the mean GATA-2/GAPDH ratios from four independent experiments. (C) Time course for GATA-1-dependent reduction in GATA-2 protein levels. Whole-cell lysates isolated from the same samples as those analyzed by ChIP and RT-PCR were subjected to Western blot analysis. A representative Western blot of GATA-2 and α-tubulin is shown. (D) GATA-2 and α-tubulin levels were quantitated by means of densitometric analysis. The plot depicts GATA-2/α-tubulin ratios at various times after tamoxifen treatment. Note that GATA-2 protein levels are not reduced upon 30 min of tamoxifen treatment, despite GATA-1 binding, GATA-2 displacement, and reduced primary transcripts.