title: Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells.
abstract: BACKGROUND
  There are a growing number of reports on the sub-physiological temperature culturing of mammalian cells for increased recombinant protein yields. However, the effect varies and the reasons for the enhancement are not fully elucidated. Expression of cold-inducible RNA-binding protein (cirp, also called cirbp or hnRNP A18) is known to be induced in response to mild, but not severe, hypothermia in mammalian cells. To clarify the molecular mechanism underlying the induction and to exploit this to improve the productivity of recombinant proteins, we tried to identify the regulatory sequence(s) in the 5' flanking region of the mouse cirp gene.
  
  
  RESULTS
  By transiently transfecting HEK293 cells with plasmids expressing chloramphenicol acetyltransferase as a reporter, we found that the cirp 5' flanking region octanucleotide 5'-TCCCCGCC-3' is a mild-cold responsive element (MCRE). When 3 copies of MCRE were placed upstream of the CMV promoter and used in transient transfection, reporter gene expression was increased 3- to 7-fold at 32°C relative to 37°C in various cell lines including HEK293, U-2 OS, NIH/3T3, BALB/3T3 and CHO-K1 cells. In stable transfectants, MCRE also enhanced the reporter gene expression at 32°C, although more copy numbers of MCRE were necessary. Sp1 transcription factor bound to MCRE in vitro. Immunohistochemistry and chromatin immunoprecipitation assays demonstrated that more Sp1, but not Sp3, was localized in the nucleus to bind to the cirp regulatory region containing MCRE at 32°C than 37°C. Overexpression of Sp1 protein increased the expression of endogenous Cirp as well as a reporter gene driven by the 5' flanking region of the cirp gene, and down-regulation of Sp1 had the opposite effect. Mutations within the MCRE sequence in the 5' flanking region abolished the effects of Sp1 on the reporter gene expression both at 37°C and 32°C.
  
  
  CONCLUSIONS
  Cold-induced, as well as constitutive, expression of cirp is dependent, at least partly, on MCRE and Sp1. The present novel enhancer permits conditional high-level gene expression at moderately low culture temperatures and could be utilized to increase the yield of recombinant proteins in mammalian cells.
authors.affiliation: Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Sumitomo
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Higashitsuji
authors.forename: Hisako
authors.initials: H
authors.lastname: Higashitsuji
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.forename: Takanori
authors.initials: T
authors.lastname: Fujita
authors.forename: Toshiharu
authors.initials: T
authors.lastname: Sakurai
authors.forename: Marco M
authors.initials: MM
authors.lastname: Candeias
authors.forename: Katsuhiko
authors.initials: K
authors.lastname: Itoh
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Chiba
authors.forename: Jun
authors.initials: J
authors.lastname: Fujita
mesh_terms: D024506:5' Flanking Region; D000818:Animals; D041702:BALB 3T3 Cells; D016466:CHO Cells; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation; D004742:Enhancer Elements, Genetic; D015870:Gene Expression; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D009154:Mutation; D041681:NIH 3T3 Cells; D011485:Protein Binding; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014162:Transfection
chemical_list: C106176:Cirbp protein, mouse; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor