Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells

BMC Biotechnology

Research Article

Hiroaki Higashitsuji¹  Yu Liu¹  Katsuhiko Itoh¹  Jun Fujita¹  Takanori Fujita¹  Hisako Higashitsuji¹  Marco M Candeias²  Yasuhiko Sumitomo³  Tsutomu Chiba⁴  Toshiharu Sakurai⁵ 

[1] Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, 606-8507, Kyoto, Japan;Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, 606-8507, Kyoto, Japan;Department of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, 606-8501, Kyoto, Japan;Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, 606-8507, Kyoto, Japan;Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 606–8507, Kyoto, Japan;Department of Gastroenterology and Hepatology, Kobe City Medical, Center West Hospital, 653-0013, Kobe, Japan;Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, 606–8507, Kyoto, Japan;Department of Gastroenterology and Hepatology, Kinki University School of Medicine, 589-8511, Osaka, Japan;

关键词: Cold shock protein;    Stress response;    Enhancer;    Transcription factor;    Recombinant protein;   

DOI  :  10.1186/1472-6750-12-72

 received in 2012-06-22, accepted in 2012-10-09,  发布年份 2012

来源: Springer

【 摘 要 】

BackgroundThere 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.ResultsBy 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.ConclusionsCold-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.

【 授权许可】

Unknown   
© Sumitomo et al.; licensee BioMed Central Ltd. 2012. This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.