Hsiao Hung-Liang, Su Yeu
Institute of Pharmacology, College of Medicine, National Yang-Ming University, Taipei, Taiwan, Republic of China.
Mol Cell Biochem. 2005 Apr;272(1-2):75-84. doi: 10.1007/s11010-005-7638-0.
We previously showed that the -278 to +410 region of mouse thymosin beta4 (mT,beta4) gene supports high levels of reporter gene expression in NIH3T3 cells. This region contains part of the 5'-flanking sequences (-278 to -1), the intact first exon (+1 to +133), and portion of the first intron (+134 to +410). However, the size of this exon is much longer than those of its rat and human counterparts. To resolve the question regarding this size discrepancy, transcription start site for the mTbeta4 gene was re-examined by primer extension and bioinformatics analyses. We found that the first exon of mTbeta4 gene spans 56 bp with its cap site situated in a putative initiator highly similar to the consensus mammalian sequence. In addition, a TATA box-like motif and two consecutive downstream promoter elements were also found. To delineate the cis-elements involved in modulating the constitutive expression of mTbeta4 gene, transient transfection assay was performed. Interestingly, expression level of the reporter gene driven by the -117 to +56 region of mTbeta4 gene was approximately 8-fold higher than that directed by the SV40 promoter and significant promoter activity was found to be associated with the smaller (-56 to +56) fragment. A nuclear protein-bound silencer was located in the region between the -167 and -118 and an enhancer whose effect did not seem to be dependent on protein binding was identified in the downstream (-117 to -88) region. However, neither of these cis-elements affected reporter expression driven by a SV40 promoter. Intriguingly, mTbeta4 promoter functioned well in human colorectal (SW480) and cervical (HeLa) carcinoma cells. Taken together, our findings not only provide crucial information for further elucidation of the transcriptional regulation of mTbeta4 gene but also raise the possibility of utilizing its promoter to produce large quantity of recombinant proteins in mammalian cells.
我们先前表明，小鼠胸腺素β4（mTβ4）基因的-278至+410区域在NIH3T3细胞中支持高水平的报告基因表达。该区域包含部分5'侧翼序列（-278至-1）、完整的第一个外显子（+1至+133）以及第一个内含子的部分（+134至+410）。然而，这个外显子的大小比其大鼠和人类对应物的外显子长得多。为了解决关于这种大小差异的问题，通过引物延伸和生物信息学分析重新检查了mTβ4基因的转录起始位点。我们发现mTβ4基因的第一个外显子跨度为56 bp，其帽位点位于一个与哺乳动物共有序列高度相似的推定起始子中。此外，还发现了一个类似TATA盒的基序和两个连续的下游启动子元件。为了确定参与调节mTβ4基因组成型表达的顺式元件，进行了瞬时转染实验。有趣的是，由mTβ4基因的-117至+56区域驱动的报告基因的表达水平比由SV40启动子驱动的表达水平高约8倍，并且发现显著的启动子活性与较小的（-56至+56）片段相关。一个核蛋白结合沉默子位于-167和-118之间的区域，并且在下游（-117至-88）区域鉴定出一个其作用似乎不依赖于蛋白结合的增强子。然而，这些顺式元件均未影响由SV40启动子驱动的报告基因表达。有趣的是，mTβ4启动子在人结肠（SW480）和宫颈（HeLa）癌细胞中功能良好。综上所述，我们的发现不仅为进一步阐明mTβ4基因的转录调控提供了关键信息，还提高了利用其启动子在哺乳动物细胞中大量生产重组蛋白的可能性。
