Direct interaction between FOG-1 and GATA-1 is required for suppression of the β-chain promoter. (A) CV-1 cells were transfected with 500 ng each of reporter plasmids (pGL3-Basic; β-69/pGL3-Basic; or β M1234/pGL3-Basic [β-chain mutant promoter lacking 4 GATA motifs by nucleotide replacement]) and 100 ng each of expression plasmids (pCR-GATA-1; pCR-GATA-1 V205G [GATA-1 mutant lacking FOG-1 binding activity]; and/or pCR-FOG-1). Total amount of expression plasmids was adjusted to 200 ng by addition of the empty plasmid pCR3.1. The ratio of luciferase activity of each expression plasmid in the presence of reporter plasmid to that of the empty expression plasmid was represented as fold activation. (B) Transcriptional level of endogenously produced GATA-1 and FOG-1 in CV-1 cells. RT-PCR was performed with total RNA prepared from each transfectant. The expression level of GATA-1 V205G was similar to that of wild-type GATA-1 (data not shown). (C) Dose dependency of the FOG-1 effect on β-chain promoter activity. CV-1 cells were transfected with 100 ng of reporter plasmid β-69/pGL3-Basic with or without 100 ng of pCR-3.1 (empty vector), pCR-GATA-1, or various amounts of pCR-FOG-1 (10, 30, 100, or 300 ng). The ratio of luciferase activity of each construct in the absence of expression plasmid to that of pGL3-Basic was represented as relative luciferase activity. Protein levels of exogenously expressed GATA-1 and FOG-1 and control YY1 are analyzed by Western blotting (bottom).