title: Alternative promoters regulate cold inducible RNA-binding (CIRP) gene expression and enhance transgene expression in mammalian cells.
abstract: The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well characterized; however, the equivalent cold stress responses are not. In particular, the transcriptional mechanisms that lead to enhanced gene-specific expression upon cold stress have yet to be elucidated. We report here in silico and experimental identification and characterization of transcriptional control elements that regulate cold inducible RNA-binding (CIRP) gene expression and demonstrate these can be used for enhanced transgene expression. In silico analysis identified the core CIRP promoter and a number of conserved transcription factor-binding sites across mammalian species. The core promoter was confirmed by experimental studies that located the basal transcriptional regulatory elements of CIRP within 264 nucleotides upstream of the transcription start site. Deletion analysis of a fragment from -264 to -64 that contained two putative CAAT-binding sites abolished promoter activity. A second promoter was identified in the region -452 to -264 of the transcription start site which was able to drive transcription independent of the core promoter. As the two CIRP promoters were transcriptionally active and possibly cold responsive, we used electrophoretic mobility shift assays to show that both promoter regions are able to bind factors within a nuclear extract in a dose-dependent manner and that the formation of these complexes was specific to the promoter regions. Finally, we successfully demonstrate using a reporter gene approach that enhanced transgene expression can be achieved using the identified CIRP promoter.
authors.affiliation: School of Biosciences, University of Kent, Canterbury, Kent, UK.
authors.forename: Mohamed B
authors.initials: MB
authors.lastname: Al-Fageeh
authors.forename: C Mark
authors.initials: CM
authors.lastname: Smales
mesh_terms: D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003080:Cold Temperature; D015870:Gene Expression / Q000235:genetics*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012313:RNA / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000235:genetics; D019076:Transgenes
chemical_list: D011994:Recombinant Proteins; D014157:Transcription Factors; D012313:RNA