Figure 2.

NuRD binding is required for transcriptional activation by FOG-1. Transient transfection of 3T3 cells with constructs expressing GATA-1, FOG-1 and a luciferase reporter gene driven by the αIIb promoter. FOG-1 mutants bearing triple point mutations (tri-mut) or a deletion of the NuRD-binding domain (Δ45) fail to substantially activate GATA-1-dependent transcription (A). Bars denote averages of five independent experiments. Error bars represent s.e.m. ^*P<0.05; NS: not significant. (B) Western blots of cells co-transfected with siRNAs against MTA-1 and MTA-2. NS, non-specific bands. (C) Transient transfections as in (A), but 24 h before transfection with GATA-1, FOG-1 and the reporter gene, cells were additionally transfected with MTA-1, MTA-2 and MTA-3 siRNAs, no siRNAs (mock), or two different control siRNAs (ctr1 and ctr2). Note that the exposure to an additional round of transfection leads to non-specific reduction in GATA-1/FOG-1 activity. The results are averages of 3–12 independent experiments. Error bars represent s.e.m. ^*P<0.05; NS: not significant. (D) 3T3 cells were treated with control siRNA (siRNA ctr2) or siRNA against MTA-1 and MTA-2 (siRNA/MTA) as in (C); 24 h later, GAL4–VP16 or GAL4 alone were co-transfected with a reporter gene driven by the thymidine kinase promoter and containing 5 GAL4-binding sites; n=3 (error bars denote s.e.m.; NS: not significant).