The POZ domain is a protein-protein interaction motif that is found in many transcription factors, which are important for development, oncogenesis, apoptosis, and transcription repression. We cloned the POZ domain transcription factor, FBI-1, that recognizes the cis-element (bp ؊38 to ؊22) located just upstream of the core Sp1 binding sites (bp ؊22 to ؉22) of the ADH5/FDH minimal promoter (bp ؊38 to ؉61) in vitro and in vivo, as revealed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. The ADH5/FDH minimal promoter is potently repressed by the FBI-1. Glutathione S-transferase fusion protein pull-down showed that the POZ domains of FBI-1, Plzf, and Bcl-6 directly interact with the zinc finger DNA binding domain of Sp1. DNase I footprinting assays showed that the interaction prevents binding of Sp1 to the GC boxes of the ADH5/FDH promoter. Gal4-POZ domain fusions targeted proximal to the GC boxes repress transcription of the Gal4 upstream activator sequence-Sp1-adenovirus major late promoter. Our data suggest that POZ domain represses transcription by interacting with Sp1 zinc fingers and by interfering with the DNA binding activity of Sp1.

The POZ domain is a highly conserved protein-protein interaction motif found in many regulatory proteins. Nuclear factor-B (NF-B) plays a key role in the expression of a variety of genes in response to infection, inflammation, and stressful conditions. We found that the POZ domain of FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) interacted with the Rel homology domain of the p65 subunit of NF-B in both in vivo and in vitro proteinprotein interaction assays. FBI-1 enhanced NF-B-mediated transcription of E-selectin genes in HeLa cells upon phorbol 12-myristate 13-acetate stimulation and overcame gene repression by IB␣ or IB␤. In contrast, the POZ domain of FBI-1, which is a dominant-negative form of FBI-1, repressed NF-B-mediated transcription, and the repression was cooperative with IB␣ or IB␤. In contrast, the POZ domain tagged with a nuclear localization sequence polypeptide of FBI-1 enhanced NF-B-responsive gene transcription, suggesting that the molecular interaction between the POZ domain and the Rel homology domain of p65 and the nuclear localization by the nuclear localization sequence are important in the transcription enhancement mediated by FBI-1. Confocal microscopy showed that FBI-1 increased NF-B movement into the nucleus and increased the stability of NF-B in the nucleus, which enhanced NF-B-mediated transcription of the E-selectin gene. FBI-1 also interacted with IB␣ and IB␤.The BTB/POZ (broad complex, Tramtrack, and bric-a-brac/ poxvirus and zinc finger) domain is an evolutionarily conserved protein-protein interaction domain that is found at the N terminus of various cellular and viral regulatory proteins. The proteins containing the BTB/POZ domain have several C-terminal structures important in their biological functions, such as the zinc finger, actin-binding repeats, and ion channel motifs (1-3). The POZ domains of PLZF (promyelocytic leukemia zinc finger) and Bcl-6 (B-cell lymphoma-6) have been shown to interact with SMRT (silencing mediator for retinoid and thyroid hormone receptors)/N-CoR (nuclear receptor co-repressor), mSin3A, and histone deacetylases (4, 5).FBI-1 (factor that binds to the inducer of short transcripts of human immunodeficiency virus-1) was purified as a cellular factor that binds specifically to the wild-type IST (inducer of short transcripts) elements of human immunodeficiency virus-1 long terminal repeats and the proximal promoter of the ADH5/FDH gene, and its cDNA was cloned (6 -9). FBI-1 is a ubiquitous transcription factor that contains a BTB/POZ domain at its N terminus and Krü ppel-like zinc fingers at its C terminus. There have been several recent reports on the function of FBI-1. FBI-1 stimulates Tat (transactivator of transcription) activity on the human immunodeficiency virus-1 long terminal repeat (8) and represses human ADH5/FDH gene expression by interacting with Sp1 zinc fingers (9). The mouse counterpart of FBI-1, LRF (leukemia/lymphoma-related factor), is co-immunoprecipitated and co-localized with Bcl-6 (...

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