Effect of PU.1 on GATA-1 DNA binding and the transactivation domain.

PU.1 inhibits GATA-1 DNA binding but not the transactivation domain. (A) Left panel: EMSA was performed using a³²P-α-dCTP–labeled GATA-binding site17oligonucleotide as a probe. GATA-1 protein was synthesized with the use of the Promega in vitro transcription and translation kit. GST-PU.1 fusion proteins were purified with the use of glutathione agarose. PU.1: GST-PU.1 full-length fusion protein. ETS: GST-PU.1 ETS-domain fusion protein (PU.1 amino acids 171-272). ΔN70: GST-PU.1 N-terminal 70–amino acid fusion protein. Δ2: deletion mutant of both the N-terminal 70–amino acid and β3/β4 (amino acids 243 to 254) region of the PU.1-GST fusion protein. GST: GST alone. Right panel: EMSA was performed by means of a ³²P-[γ]ATP–labeled PU.1 binding site from the murine PU.1 promoter as a probe.35The same amount of GST-ETS or GST protein was used for this experiment as was in the left panel. (B) Left panel: 0.2 μg of a luciferase reporter under the control of a promoter consisting of 3 GATA sites proximal to a minimal thymidine kinase (TK) promoter was cotransfected with 40 ng of N + C–VP16 with or without 0.25 μg of PU.1 or PU.1 ΔN70 with the use of the lipofectamine method in CV-1 cells. N + C–VP16 is the fusion protein consisting of the VP16 activation domain fused to the GATA-1 N + C finger region (DNA-binding domain). ΔN70: PU.1 with a deletion of the N-terminal 70 amino acids. Right panel: Whole-cell lysates from transfected CV-1 cells were analyzed by Western blots and detected with anti-PU.1 antibody. Lanes 1 and 2 are nuclear extracts from a PU.1^−/− cell line transfected with PU.1 and the nontransfected parental PU.1^−/− cell line41 as positive and negative controls, respectively. Lanes 3 and 4 contain whole-cell lysates from CV-1 cells transfected with wild-type and ΔN70, respectively. The asterisk indicates the position of an N-terminal degradation product of PU.1, which has been previously described.42