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

Yasuhiko Sumitomo⁵  Hiroaki Higashitsuji¹  Hisako Higashitsuji¹  Yu Liu¹  Takanori Fujita¹  Toshiharu Sakurai²  Marco M Candeias³  Katsuhiko Itoh¹  Tsutomu Chiba⁴  Jun Fujita¹ 

[1] Department of Clinical Molecular Biology, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawaharacho, Sakyo-ku, Kyoto, 606-8507, Japan

[2] Department of Gastroenterology and Hepatology, Kinki University School of Medicine, Osaka, 589-8511, Japan

[3] Current address: Department of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Kyoto, 606-8501, Japan

[4] Department of Gastroenterology and Hepatology, Graduate School of Medicine, Kyoto University, Kyoto, 606–8507, Japan

[5] Current address: Department of Gastroenterology and Hepatology, Kobe City Medical, Center West Hospital, Kobe, 653-0013, Japan

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

Others  :  1134762
DOI  :  10.1186/1472-6750-12-72

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

【 摘 要 】

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.

【 授权许可】

   
2012 Sumitomo et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Fujita J: Cold shock response in mammalian cells. J Mol Microbiol Biotechnol 1999, 1:243-255.
• [2]Nishiyama H, Itoh K, Kaneko Y, Kishishita M, Yoshida O, Fujita J: A glycine-rich RNA-binding protein mediating cold-inducible suppression of mammalian cell growth. J Cell Biol 1979, 137:899-908.
• [3]Yang C, Carrier F: The UV-inducible RNA-binding protein A18 (A18 hnRNP) plays a protective role in the genotoxic stress response. J Biol Chem 2001, 276:47277-47284.
• [4]Wellmann S, Bührer C, Moderegger E, Zelmer A, Kirschner R, Koehne P, Fujita J, Seeger K: Oxygen-regulated expression of the RNA-binding proteins RBM3 and CIRP by a HIF-1-independent mechanism. J Cell Sci 2004, 117:1785-1794.
• [5]De Leeuw F, Zhang T, Wauquier C, Huez G, Kruys V, Gueydan C: The cold-inducible RNA-binding protein migrates from the nucleus to cytoplasmic stress granules by a methylation-dependent mechanism and acts as a translational repressor. Exp Cell Res 2007, 313:4130-4144.
• [6]Sakurai T, Itoh K, Higashitsuji H, Nonoguchi K, Liu Y, Watanabe H, Nakano T, Fukumoto M, Chiba T, Fujita J: Cirp protects against tumor necrosis factor-alpha-induced apoptosis via activation of extracellular signal-regulated kinase. Biochim Biophys Acta 2006, 1763:290-295.
• [7]Zeng Y, Kulkarni P, Inoue T, Getzenberg RH: Down-regulating cold shock protein genes impairs cancer cell survival and enhances chemosensitivity. J Cell Biochem 2009, 107:179-188.
• [8]Artero-Castro A, Callejas FB, Castellvi J, Kondoh H, Carnero A, Fernández-Marcos PJ, Serrano M, Ramón y Cajal S, Lleonart ME: Cold-inducible RNA-binding protein bypasses replicative senescence in primary cells through extracellular signal-regulated kinase 1 and 2 activation. Mol Cell Biol 2009, 29:1855-1868.
• [9]Masuda T, Itoh K, Higashitsuji H, Higashitsuji H, Nakazawa N, Sakurai T, Liu Y, Tokuchi H, Fujita T, Zhao Y, Nishiyama H, Tanaka T, Fukumoto M, Ikawa M, Okabe M, Fujita J: The cold-inducible RNA-binding protein Cirp interacts with Dyrk1b/Mirk and promotes proliferation of immature male germ cells in mice. Proc Natl Acad Sci U S A 2012, 109:10885-10890.
• [10]Archer MC: Role of sp transcription factors in the regulation of cancer cell metabolism. Genes Cancer 2011, 2:712-719.
• [11]Wierstra I: Sp1: emerging roles–beyond constitutive activation of TATA-less housekeeping genes. Biochem Biophys Res Commun 2008, 372:1-13.
• [12]Al-Fageeh MB, Marchant RJ, Carden MJ, Smales CM: The cold-shock response in cultured mammalian cells: harnessing the response for the improvement of recombinant protein production. Biotechnol Bioeng 2006, 93:829-835.
• [13]Mohan C, Kim YG, Koo J, Lee GM: Assessment of cell engineering strategies for improved therapeutic protein production in CHO cells. Biotechnol J 2008, 3:624-630.
• [14]Yoon SK, Song JY, Lee GM: Effect of low culture temperature on specific productivity, transcription level, and heterogeneity of erythropoietin in Chinese hamster ovary cells. Biotechnol Bioeng 2003, 82:289-298.
• [15]Park KS, Seol W, Yang HY, Lee SI, Kim SK, Kwon RJ, Kim EJ, Roh YH, Seong BL, Kim JS: Identification and use of zinc finger transcription factors that increase production of recombinant proteins in yeast and mammalian cells. Biotechnol Prog 2005, 21:664-670.
• [16]Becerra-Arteaga A, Mason HS, Shuler ML: Production, secretion, and stability of human secreted alkaline phosphatase in tobacco NT1 cell suspension cultures. Biotechnol Prog 2006, 22:1643-1649.
• [17]TESS: Transcription Element Search System. [http://www.cbil.upenn.edu/cgi-bin/tess/tess webcite]
• [18]Al-Fageeh MB, Smales CM: Cold-inducible RNA binding protein (CIRP) expression is modulated by alternative mRNAs. RNA 2009, 15:1164-1176.
• [19]Iwata T, Minucci S, McGowan M, Carper D: Identification of a novel cis-element required for the constitutive activity and osmotic response of the rat aldose reductase promoter. J Biol Chem 1997, 272:32500-32506.
• [20]Bulger M, Groudine M: Functional and mechanistic diversity of distal transcription enhancers. Cell 2011, 144:327-339.
• [21]Thaisuchat H, Baumann M, Pontiller J, Hesse F, Ernst W: Identification of a novel temperature sensitive promoter in CHO cells. BMC Biotechnol 2011, 11:51. BioMed Central Full Text
• [22]Tan NY, Khachigian LM: Sp1 phosphorylation and its regulation of gene transcription. Mol Cell Biol 2009, 29:2483-2488.
• [23]Discher DJ, Bishopric NH, Wu X, Peterson CA, Webster KA: Hypoxia regulates beta-enolase and pyruvate kinase-M promoters by modulating Sp1/Sp3 binding to a conserved GC element. J Biol Chem 1998, 273:26087-26093.
• [24]Iwahori S, Yasui Y, Kudoh A, Sato Y, Nakayama S, Murata T, Isomura H, Tsurumi T: Identification of phosphorylation sites on transcription factor Sp1 in response to DNA damage and its accumulation at damaged sites. Cell Signal 2008, 20:1795-1803.
• [25]Higashitsuji H, Higashitsuji H, Nagao T, Nonoguchi K, Fujii S, Itoh K, Fujita J: A novel protein overexpressed in hepatoma accelerates export of NF-kappa B from the nucleus and inhibits p53-dependent apoptosis. Cancer Cell 2002, 2:335-346.
• [26]Cullen BR, Malim MH: Secreted placental alkaline phosphatase as a eukaryotic reporter gene. Methods Enzymol 1992, 216:362-368.
• [27]Sharova LV, Sharov AA, Nedorezov T, Piao Y, Shaik N, Ko MS: Database for mRNA half-life of 19977 genes obtained by DNA microarray analysis of pluripotent and differentiating mouse embryonic stem cells. DNA Res 2009, 16:45-58.