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. 2007 Apr 9;27(12):4261–4272. doi: 10.1128/MCB.02212-06

FIG. 4.

FIG. 4.

GATA-1 activates the Bcl-x promoter and mediates Gfi-1B repression. (A) K562 cells were transfected with the Bcl-x reporter constructs pGL2-0.6 and pGL2-0.6mut1 (mut1; carrying a mutation at −401 in the GATT sequence) and pGL2-0.6mut2 (mut2; carrying a mutation at −404 in the GATT sequence), and luciferase activities were determined and expressed as described in the legend to Fig. 3D, with the activity in cells transfected with full-length pGL2-3.2 and the control vector set at 1. The error bars represent standard deviations. wt, wild type. (B) NIH 3T3 cells were transfected with the minimal Bcl-x reporter construct pGL2-0.6 or pGL2-0.6mut2 along with pCMV2-GATA-1 in combination with different amounts of pCS2-Gfi-1B as indicated. The luciferase activities were normalized and expressed relative to that in cells transfected with pGL2-0.6 and control vectors, which was set arbitrarily at 1. Values are the averages of three independent determinations. The error bars represent standard deviations. −, absent. (C) K562 cells with pS2-Gfi-1B-Flag retroviral integration were electroporated with GATA-1 siRNA or control siRNA for 48 h. Whole-cell lysates were subjected to Western blot analysis using anti-GATA-1, Gfi-1B, Bcl-xL, and β-tubulin antibodies (Ab). (D) Cells were treated with formaldehyde for ChIP assays using anti-Gfi-1B antibody or normal mouse IgG. DNA isolated from immunoprecipitates was amplified by PCR with primers specific for the Bcl-x (G-I [−445 to −196] and G-II [−1745 to −1536]), Gfi-1B, or c-jun promoter. +, present. (E) Results from gel shift assays of the human Bcl-x promoter region from −396 to −415. The 32P-labeled double-stranded oligonucleotide containing the sequence from −396 to −415 of the Bcl-x promoter was used for the assays in the absence or presence of an unlabeled competitor (at a 10-fold excess) with 2.5 μg of nuclear extracts from 293T cells overexpressing Flag-GATA-1, as indicated. The sequence of each competitor is shown below. Mutated and corresponding wild-type sequences are underlined. Control IgG and GATA-1(C-20) antibodies were added to the reaction mixtures where indicated. mut, mutant.