title: Identification and functional characterization of a novel binding site on TNF-alpha promoter.
abstract: Transcription of the tumor necrosis factor (TNF) gene is rapidly and transiently induced by lipopolysaccharide in cells of monocytemacrophage lineage. Previous studies have suggested that in the mouse, multiple NF-kappaBRel-binding sites contribute to the TNF transcriptional response to LPS. But the role of these regulatory elements in transcriptional activation of the TNF-alpha gene in human monocytes remains unclear. Previously, a transcription factor, termed lipopolysaccharide-induced TNF-alpha factor (LITAF), was found to regulate TNF-alpha gene expression. However, the specific protein domain(s) of human (h)LITAF that interact with the hTNF-alpha promoter had not been identified. In this study, we identify by footprinting a sequence motif, CTCCC (-515 to -511), within the TNF-alpha promoter that binds to hLITAF. We also identify the region of hLITAF (amino acids 165-180) that was named peptide B and specifically mediates binding to the hTNF-alpha promoter. When THP-1 cells were stimulated with this peptide B, it was sufficient to induce TNF-alpha secretion. Induction of TNF-alpha transcription by LPS or peptide B depended on the presence of the -515 to -511 promoter region, which was found to be essential for hLITAF binding. Together, these findings help to clarify the mechanism of hLITAFhTNF-alpha interaction and the manner by which hLITAF contributes to hTNF-alpha regulation in an attempt to design new pharmacological interventions to address TNF-related diseases.
authors.affiliation: Department of Periodontology and Oral Biology, School of Dental Medicine, Boston University, Boston, MA 02118, USA.
authors.forename: Xiaoren
authors.initials: X
authors.lastname: Tang
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Fenton
authors.forename: Salomon
authors.initials: S
authors.lastname: Amar
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D018983:DNA Footprinting; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics / Q000276:immunology; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics*; D016385:TATA Box; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics*
chemical_list: D017931:DNA Primers; D008070:Lipopolysaccharides; D010446:Peptide Fragments; D014409:Tumor Necrosis Factor-alpha