Abstract
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.
Highlights
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
Chromatin Immunoprecipitation (ChIP) Assays—We investigated in vivo molecular interaction between FBI-1 and the cis-element on the ADH5/formaldehyde dehydrogenase (FDH) promoter using ChIP assay kit (Upstate Biotechnology, Inc., Lake Placid, NY) [37, 38]
Isolation of Human POZ Transcription Factor, FBI-1—In a yeast one-hybrid screen for factors that bind to the cis-acting element located between bp Ϫ38 and Ϫ22 of the human ADH5/ FDH promoter, six cDNAs that encode a human POZ domain, Kruppel-like zinc finger transcription factor were isolated
Summary
Ent formaldehyde dehydrogenase (FDH; EC 1.2.11) [26]. The gene is believed to be the ancestral ADH gene [27]. The ADH5/ FDH promoter is very different from those of other ADH genes, which are expressed in a different tissue-specific pattern. It is very rich in GC base pairs (75% up to Ϫ200 bp) and does not contain TATA or CCAAT box and has the characteristics of a housekeeping gene [24, 31, 32]. We found that the Sp1 multigene family recognizes the core cis-elements (bp Ϫ22 to ϩ22) and regulates transcription of the ADH5/FDH minimal promoter [33]. We investigated and discovered a novel molecular mechanism of transcription repression by the POZ domain of FBI-1 on the human ADH5/FDH promoter and the Gal UAS-Sp1-AdML promoter. The POZ domain interacts with the Sp1 zinc finger DNA binding domain, and represses transcription by interfering with GC box recognition by Sp1
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