Highly restricted GATA factor occupancy at endogenous Lyl1 and Rgs18 domains. (A) Comparison of transcript levels in G1E cells expressing wild-type ER–GATA-1 or ER–GATA-1(V205G) following β-estradiol treatment (1 μM) for various times up to 40 hours. Transcript levels were normalized to GAPDH transcript levels and, for each clone, the mean value for untreated samples was set to 1 (mean ± SE from 3 independent experiments). GATA factor occupancy was measured at (B) the Lyl1 locus and (C) the Rgs18 locus by quantitative real-time ChIP analysis in G1E–ER–GATA-1 cells. For GATA-2 and GATA-1, ChIPs were conducted using untreated or β-estradiol–treated (1 μM, 24 h) cells, respectively (mean ± SD, 2 to 4 independent ChIP experiments). Mean preimmune control signals did not exceed 0.0026 for Lyl1 or 0.0015 for Rgs18 and are not shown. The positions of conserved (mouse to man) WGATAR, NGATAR, and WGATAN sites in each locus are shown above the graph. All nonconserved WGATAR motifs in the Lyl1 locus were also analyzed by ChIP. Coordinates are based upon distance from the conserved WGATAR motif (TTATCA) in the Lyl1 promoter and the transcriptional start site of the Rgs18 locus. (D) Multiple modes of GATA-1–mediated transcriptional regulation. An example of a gene regulated via each mode is indicated in parentheses.