The PU.1 ETS domain is required for repression of PU.1 by GATA-1.

(A) Fusion of PU.1 transactivation domain and PEST domain to, either C- or N-terminally, the GAL4 amino acids 1-147 generates GAL4-PU.1 and PU.1-GAL4 hybrid activators, respectively. (B) Repression of PU.1 by GATA-1: 0.25 μg of pCMV-MTPU.1 or empty pcDNAI expression vector was cotransfected with 100 ng of pPU3-TK-LUC, 100 of ng pRSVβgal, and the indicated amount of pSPCMV-GATA-1 expression vector. The total amount of DNA was kept constant by addition of empty pSPCMV vector. Results are expressed as luciferase activity normalized to β-galactosidase, setting the level obtained with empty expression vector to 1. Each column represents the average of 2 determinations. (C) Parallel experiment to (B), where pPU3-TK-LUC was replaced with the GAL4-responsive reporter pG5B-LUC (100 ng) and pCMV-MTPU.1 with pGD-PU 1-170 expressing GAL4-PU.1 (0.25 μg). (D) Similar experiment as in (C), except that the GAL4-PU.1 activator was replaced by PU.1-GAL4 encoded by pCMV-MTPU1-170GD.