Figure 3.

FIAT represses ATF4-mediated transcription and binding of ATF4 to DNA. (A) COS-7 cells were transfected with a luciferase reporter gene under the control of three canonical ATF4 binding sites, together with expression vectors for human ATF4 and human FIAT. (B) MC3T3-E1 osteoblastic cells were transfected with a reporter construct in which the luciferase gene was regulated by six copies of the wild-type (OSE1-luc) or mutated (mut OSE1) OSE1 sequence from the mouse osteocalcin gene promoter. Cotransfected plasmids included expression vectors for ATF4, RSK2, or FIAT (2× signifies that twice the amount of plasmid was used). Reporter gene activity was measured with a luminometer 24 h after transfection. The activity measured in cells transfected with the reporter construct and empty expression vectors was arbitrarily ascribed a value of 1. FIAT inhibited ATF4-induced transcription from both templates (A and B), even in the presence of an expression vector for the RSK2 kinase (B). FIAT expression by itself does not affect transcription from the reporter templates. (C) EMSA with an OSE1 oligonucleotide probe, ROS 17/2.8 nuclear extract, and recombinant FIAT. The ATF4 binding complex was identified using specific anti-ATF4 antibodies (lanes 3–5). FIAT did not bind the probe but inhibited binding of ATF4 to the DNA.