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.

Growth factor independence-1 (GFI1) and GFI1B are closely related, yet differentially expressed transcriptional repressors with nearly identical DNA binding domains. GFI1 is upregulated in the earliest thymocyte precursors, while GFI1B expression is restricted to T lymphopoiesis stages coincident with activation. Transgenic expression of GFI1 potentiates T-cell activation, while forced GFI1B expression decreases activation. Both mice and humans with mutant Gfi1 display lymphoid abnormalities. Here we describe autoregulation of Gfi1 in primary mouse thymocytes and a human T-cell line. GFI1 binding to cis-element sequences conserved between rat, mouse and human Gfi1 mediates direct and potent transcriptional repression. In addition, dramatic regulation of Gfi1 can also be mediated by GFI1B. These data provide the first example of a gene directly targeted by GFI1 and GFI1B. Moreover, they support a role for auto- and trans-regulation of Gfi1 by GFI1 and GFI1B in maintaining the normal expression patterns of Gfi1, and suggest that GFI1B may indirectly affect T-cell activation through repression of Gfi1.

HL-60 cells, a human promyelocytic leukemia cell line, contain amplified c-myc DNA sequences and mRNA transcripts. These cells can be induced to undergo macrophage differentiation by phorbol esters, which results in suppression of c-myc expression and cessation of cell proliferation. The nuclear matrix (NM), a nuclear skeleton resistant to DNase I digestion and high salt extraction, is proposed to be involved in DNA replication, gene regulation, and the correct distribution of DNA at mitosis. We have previously identified a nuclear-matrix-associated region (MAR) of the c-myc protooncogene to reside in a 1.4-kb region between Cla I and Eco RI restriction sites at the 3'-end of the gene. A 172-bp Dra I/Dra I subfragment of the 1.4-kb region was shown to be a major component of the MAR (myc-MAR), and this subfragment was demonstrated to be recognized by a nuclear protein (p25). In this report we demonstrate that phi X174 DNA, or the synthetic copolymers poly[d(G.C)] and poly[d(A.T)], are not effective suppressors of the binding of the myc-MAR to isolated NM, indicating that the binding sequence(s) are unique. We find that the addition of partially purified protein p25 increases the relative affinity of the myc-MAR for HL-60 NM in an in vitro assay system. NM isolated from HL-60 macrophages induced by phorbol esters retains significantly more myc-MAR DNA fragment in the presence of an excess amount of competitor DNA than does NM from untreated HL-60 cells. These data suggest that a change of the myc-MAR association with the NM occurs after monocytic differentiation of HL-60 cells.

Cells and viruses. 4437A is an IL-2-dependent T-cell lymphoma line established from a Moloney murine leukemia virus (MoMuLV)-induced rat thymoma (22, 33). The uninfected and virus-infected Mus dunni cells were kindly provided by J. Coffin (Tufts University School of Medicine) and have been described previously (33). COS-1 cells were kindly provided by J. Chernoff (Fox Chase Cancer Center). The 4437A cells were maintained in RPMI medium supplemented with horse serum (10%), penicillin (50 U/ml), streptomycin (50 ,ug/ml), kanamycin (100 jig/ml), and IL-2 (50 U/ml). The cocultivation of 4437A cells with M. dunni cells was carried out in the same media (33). M. dunni and COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, penicillin (50 U/ml), streptomycin (50 ,ug/ml), and kanamycin (100 ig/ml). Southern and Northern (RNA) blotting. Cellular genomic DNA and RNA were prepared as previously described (14, 29, 33). Southern and Northern blots were prepared and analyzed by standard procedures as previously described (14, 29, 33).