CROC-1 encodes a protein which mediates transcriptional activation of the human FOS promoter

• Published in: Gene 1997-08, Vol.195 (2), p.141-149
• Main Authors: Rothofsky, Marnie L, Lin, Stanley L
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Amino Acid Sequence; Animals; Antigens, Viral, Tumor - genetics; Base Sequence; Cells, Cultured; Contingent replication; Direct repeat enhancers; Gene Library; Genes, fos; Genes, Reporter; Genetic Vectors; Humans; Mice; Molecular Sequence Data; Nuclear Proteins - genetics; Nuclear Proteins - metabolism; Plasmids - genetics; Polyomavirus - genetics; Promoter Regions, Genetic; Proto-Oncogene Proteins c-fos - genetics; RAF; Rats; Repetitive Sequences, Nucleic Acid; Sequence Analysis, DNA; Signal transduction; Signal Transduction - genetics; Trans-Activators - genetics; Trans-Activators - metabolism; Transcription Factors; Transcriptional Activation; Transformation, Genetic; Ubiquitin-conjugating enzyme; Ubiquitin-Conjugating Enzymes
• ISSN: 0378-1119; 1879-0038
• DOI: 10.1016/S0378-1119(97)00097-8

The cloning of signal transducing molecules capable of activating the human FOS proto-oncogene promoter was achieved by co-transfecting a modified human FOS promoter-driven polyomavirus large T antigen gene ( P fLAG-8 ) with a human brain cDNA library, incorporated into a replication-competent mammalian retroviral expression vector whose replication occurs in the presence of T antigen. In murine cells, transcriptional activation of the P fLAG-8 promoter by a biologically active, cDNA-encoded signalling molecule resulted in plasmid replication. Replicated plasmids, following selective cleavage of unreplicated plasmids by Dpn1, were recovered by transformation into competent bacteria. Successive P fLAG-8 /cDNA library co-transfections, using library plasmids resulting from prior transfections, ultimately resulted in the identification of individual plasmids capable of transcriptionally activating the FOS promoter. DNA sequencing revealed the first plasmid, denoted CROC-1, to contain a 1.8-kb cDNA encoding a 16.5-kDa nuclear protein possessing a bipartite structure comprised an amino-terminal acidic domain and a carboxy-terminal basic domain, and displaying partial homology to the HMG domain of the TAF II250 transcription cofactor. Co-transfection of CROC-1 with various FOS/CAT reporter genes revealed that the human FOS promoter region spanning −56 to −105, encompassing two identical 8-bp DR enhancer sequences, was necessary for CROC-1-mediated transcriptional activation. Results suggest that CROC-1 participates in intracellular signalling pathways involved in induction of the human FOS promoter.