Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells.
Identification of a novel enhancer that binds Sp1 and contributes to induction of cold-inducible RNA-binding protein (cirp) expression in mammalian cells.
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DOI：
10.1186/1472-6750-12-72
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发表时间：
2012-10-10
影响因子：
3.5
通讯作者：
Fujita J
中科院分区：
文献类型：
--
作者：
Sumitomo Y;Higashitsuji H;Higashitsuji H;Liu Y;Fujita T;Sakurai T;Candeias MM;Itoh K;Chiba T;Fujita J
关于哺乳动物细胞亚生理温度培养以增加重组蛋白产量的报道越来越多。然而，效果各不相同，并且增强的原因尚未完全阐明。已知在哺乳动物细胞中，冷诱导性 RNA 结合蛋白（cirp，也称为 cirbp 或 hnRNP A18）的表达会因轻微而非严重的低温而被诱导。为了阐明诱导背后的分子机制并利用它来提高重组蛋白的生产力，我们尝试鉴定小鼠 cirp 基因 5' 侧翼区域的调控序列。通过用表达氯霉素乙酰转移酶的质粒作为报告基因瞬时转染HEK293细胞，我们发现cirp 5'侧翼区八核苷酸5'-TCCCCGCC-3'是一个温和的冷反应元件（MCRE）。当将 3 个拷贝的 MCRE 置于 CMV 启动子上游并用于瞬时转染时，在各种细胞系（包括 HEK293、U-2 OS、NIH/3T3、BALB/3T3 和 CHO-K1 细胞）中，报告基因表达在 32°C 下相对于 37°C 增加了 3 至 7 倍。在稳定转染子中，MCRE 还增强了 32°C 下报告基因的表达，尽管需要更多的 MCRE 拷贝数。 Sp1 转录因子在体外与 MCRE 结合。免疫组织化学和染色质免疫沉淀分析表明，与 37°C 相比，32°C 时更多的 Sp1（而非 Sp3）定位于细胞核中与含有 MCRE 的 cirp 调节区结合。 Sp1蛋白的过度表达增加了内源性Cirp以及由cirp基因5'侧翼区域驱动的报告基因的表达，而Sp1蛋白的下调则具有相反的效果。 5' 侧翼区域 MCRE 序列内的突变消除了 Sp1 在 37°C 和 32°C 下对报告基因表达的影响。冷诱导的 cirp 表达以及组成型表达至少部分依赖于 MCRE 和 Sp1。本新型增强子允许在适度低的培养温度下有条件地高水平基因表达，并且可用于增加哺乳动物细胞中重组蛋白的产量。
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. 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. 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.
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富含甘氨酸的RNA结合蛋白，介导了对哺乳动物细胞生长的冷诱导抑制。
影响因子：
7.8
作者：
Nishiyama, H;Itoh, K;Kaneko, Y;Kishishita, M;Yoshida, O;Fujita, J
通讯作者：
Fujita, J
影响因子：
3.7
作者：
De Leeuw, Frederic;Zhang, Tong;Gueydan, Cyril
通讯作者：
Gueydan, Cyril
影响因子：
4.8
作者：
Discher, DJ;Bishopric, NH;Webster, KA
通讯作者：
Webster, KA
DOI：
10.1016/j.bbamcr.2006.02.007
发表时间：
2006-03-01
影响因子：
5.1
作者：
Sakurai, Toshiharu;Itoh, Katsuhiko;Fujita, Jun
通讯作者：
Fujita, Jun
影响因子：
4.8
作者：
Iwata, T;Minucci, S;Carper, D
通讯作者：
Carper, D