GFI-1 ENCODES A NUCLEAR ZINC-FINGER PROTEIN THAT BINDS DNA AND FUNCTIONS AS A TRANSCRIPTIONAL REPRESSOR

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C2H2-t ype, C-terminal zinc finger motifs and is activated by provirus integr ation in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas, Gfi-1 expressi on in adult animals is restricted to the thymus, spleen, and testis an d is enhanced in mitogen-stimulated splenocytes. In this report, we sh ow that Gfi-1 is a 55-kDa nuclear protein that binds DNA in a sequence -specific manner, The Gfi-1 binding site, TAAATCAC(A/T)GCA, was define d via random oligonucleotide selection utilizing a bacterially express ed glutathione S-transferase-Gfi-1 fusion protein, Binding to this sit e was confirmed by electrophoretic mobility shift assays and DNase I f ootprinting. Methylation interference analysis and electrophoretic mob ility shift assays with mutant oligonucleotides defined the relative i mportance of specific bases at the consensus binding site, Deletion of individual zinc fingers demonstrated that only zinc fingers 3, 4, and 5 are required for sequence-specific DNA binding, Potential Gfi-1 bin ding sites were detected in a large number of eukaryotic promoter-enha ncers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major imme diate-early promoter, which contains two such sites, HCMV major immedi ate-early-chloramphenicol acetyltransferase reporter constructs, trans fected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repr ession was abrogated by mutation of critical residues in the two Gfi-1 binding sites, These results suggest that Gfi-1 may play a role in HC MV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.