The impact of intragenic CpG content on gene expression.
The impact of intragenic CpG content on gene expression.
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DOI：
10.1093/nar/gkq115
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发表时间：
2010-07
影响因子：
14.9
通讯作者：
Wagner R
中科院分区：
文献类型：
--
作者：
Bauer AP;Leikam D;Krinner S;Notka F;Ludwig C;Längst G;Wagner R
疫苗组分或重组治疗剂的开发关键取决于相应转基因的持续表达。本研究旨在确定基因内CpG含量对瞬时和稳定转染哺乳动物细胞表达效率的贡献。基于在其编码序列内含有60个CpG的绿色荧光蛋白（GFP）的人源化版本，通过仔细调节密码子使用来建立GFP报告基因的CpG缺失变体。有趣的是，稳定转染的CHO和293细胞中的GFP报告基因活性和可检测的蛋白量在CpG耗尽后显著降低，并且与启动子使用无关（CMV，EF1α）。与CpG耗竭相关的蛋白质表达的减少同样在其他无关的报告基因中观察到，并且通过mRNA拷贝数而不是翻译效率的下降清楚地反映出来。此外，mRNA水平下降既不是由于核输出限制，也不是由于选择性剪接或mRNA不稳定性。相反，基因内CpG含量影响从头转录活性，从而暗示了通过CpG进行基因调控的常见的基于转录的机制。增加高CpG转录与核小体的位置在体外的变化，虽然在这两个基因的组蛋白密度在体内没有变化，通过ChIP监测。
The development of vaccine components or recombinant therapeutics critically depends on sustained expression of the corresponding transgene. This study aimed to determine the contribution of intragenic CpG content to expression efficiency in transiently and stably transfected mammalian cells. Based upon a humanized version of green fluorescent protein (GFP) containing 60 CpGs within its coding sequence, a CpG-depleted variant of the GFP reporter was established by carefully modulating the codon usage. Interestingly, GFP reporter activity and detectable protein amounts in stably transfected CHO and 293 cells were significantly decreased upon CpG depletion and independent from promoter usage (CMV, EF1α). The reduction in protein expression associated with CpG depletion was likewise observed for other unrelated reporter genes and was clearly reflected by a decline in mRNA copy numbers rather than translational efficiency. Moreover, decreased mRNA levels were neither due to nuclear export restrictions nor alternative splicing or mRNA instability. Rather, the intragenic CpG content influenced de novo transcriptional activity thus implying a common transcription-based mechanism of gene regulation via CpGs. Increased high CpG transcription correlated with changed nucleosomal positions in vitro albeit histone density at the two genes did not change in vivo as monitored by ChIP.
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