Gfi-1 Encodes a Nuclear Zinc Finger Protein That Binds DNA and Functions as a Transcriptional Repressor

Abstract

The Gfi-1 proto-oncogene encodes a zinc finger protein with six C 2 H 2 -type, C-terminal zinc finger motifs and is activated by provirus integration in T-cell lymphoma lines selected for interleukin-2 independence in culture and in primary retrovirus-induced thymomas. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis and is enhanced in mitogen-stimulated splenocytes. In this report, we show 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 defined via random oligonucleotide selection utilizing a bacterially expressed glutathione S-transferase-Gfi-1 fusion protein. Binding to this site was confirmed by electrophoretic mobility shift assays and DNase I footprinting. Methylation interference analysis and electrophoretic mobility shift assays with mutant oligonucleotides defined the relative importance 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 binding sites were detected in a large number of eukaryotic promoter-enhancers, including the enhancers of several proto-oncogenes and cytokine genes and the enhancer of the human cytomegalovirus (HCMV) major immediate-early promoter, which contains two such sites. HCMV major immediate-early-chloramphenicol acetyltransferase reporter constructs, transfected into NIH 3T3 fibroblasts, were repressed by Gfi-1, and the repression 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 HCMV biology and may contribute to oncogenesis and T-cell activation by repressing the expression of genes that inhibit these processes.

Tpl-2 induces IL-2 expression in T-cell lines by triggering multiple signaling pathways that activate NFAT and NF-κB

“…Each transfection was carried out in quadruplicate and every experiment was repeated at least twice. Chloramphenicol acetyl transferase (CAT) assays were performed as previously described (Zweidler et al, 1996). The acetyl transferase reaction was carried out for 3 h. The acetylated and the non-acetylated forms of 14 C-chloramphenicol (Amersham) were separated on TLC plates (Sigma).…”

The transcription factor Gfi1 regulates G-CSF signaling and neutrophil development through the Ras activator RasGRP1

“…14 Derepression of these genes explains important aspects of defective neutrophil differentiation in Gfi1-null mice and patients with Gfi1 mutations. Gfi1 can bind consensus DNA target sequences by a C-terminal zinc-finger domain and repress transcription by its N-terminal SNAG domain, 15 in part by recruiting corepressors. [16][17][18][19] Besides acting as a transcriptional repressor, Gfi1 interacts with protein inhibitor of activated signal transducer and activator of transcription (STAT), which can bind to and inhibit STAT3 signaling.…”

Gfi1 and Gfi1b: key regulators of hematopoiesis

“…5 At the C terminus both proteins contain six C 2 H 2 -type zinc fingers of which zinc fingers 3-5 are essential for DNA binding. 2,6,7 Between the SNAG and zinc finger domains, there is an intermediate domain that binds proteins implicated in transcriptional regulation, RNA splicing and protein modifications ( Figure 1). More than 89% of the SNAG and zinc finger domains are identical in Gfi1 and Gfi1b, whereas the intermediate region only shows low similarity (39%) at the protein level.…”

“…At the N terminus they contain a small conserved SNAG (Snail/Gfi1) domain that is also present in other transcriptional repressors (Figure 1). [1][2][3][4] The SNAG domain serves as an interaction domain to recruit proteins that modify histones. 5 At the C terminus both proteins contain six C 2 H 2 -type zinc fingers of which zinc fingers 3-5 are essential for DNA binding.…”