Abstract：
Regulation of gene expression in eukaryotes is controlled by cis-regulatory modules (CRMs). A major class of CRMs are enhancers which are composed of activating cis-regulatory elements (CREs) responsible for upregulating transcription. To date, most enhancers and activating CREs have been studied in angiosperms; in contrast, our knowledge about these key regulators of gene expression in green algae is limited. In this study, we aimed at characterizing putative activating CREs/CRMs from the histone genes of the unicellular model alga Chlamydomonas reinhardtii. To test the activity of four candidates, reporter constructs consisting of a tetramerized CRE, an established promoter, and a gene for the mCerulean3 fluorescent protein were incorporated into the nuclear genome of C. reinhardtii, and their activity was quantified by flow cytometry. Two tested candidates, Eupstr and Ehist cons, significantly upregulated gene expression and were characterized in detail. Eupstr, which originates from highly expressed genes of C. reinhardtii, is an orientation-independent CRE capable of activating both the RBCS2 and β2-tubulin promoters. Ehist cons, which is a CRM from histone genes of angiosperms, upregulates the β2-tubulin promoter in C. reinhardtii over a distance of at least 1.5 kb. The octamer motif present in Ehist cons was identified in C. reinhardtii and the related green algae Chlamydomonas incerta, Chlamydomonas schloesseri, and Edaphochlamys debaryana, demonstrating its high evolutionary conservation. The results of this investigation expand our knowledge about the regulation of gene expression in green algae. Furthermore, the characterized activating CREs/CRMs can be applied as valuable genetic tools.
摘要：
真核生物中基因表达的调节由顺式调节模块(CRM)控制。主要类型的CRM是增强子,其由负责上调转录的激活顺式调节元件(CREs)组成。迄今为止,大多数增强子和激活CREs已经在被子植物中进行了研究；相比之下，我们对绿藻基因表达的这些关键调控因子的了解是有限的。在这项研究中,我们旨在从单细胞模型藻类衣藻的组蛋白基因中表征推定的激活CREs/CRM。为了测试四个候选人的活动，由四聚CRE组成的报告构建体，一个成熟的推动者，mCerulean3荧光蛋白的基因被整合到莱茵衣原体的核基因组中，并通过流式细胞术对其活性进行定量。两个测试的候选人，Eupstr和Ehistcons，显着上调基因表达，并进行了详细的表征。Eupstr,它来源于莱茵衣原体的高表达基因，是能够激活RBCS2和β2-微管蛋白启动子的不依赖方向的CRE。Ehistcons，这是一种来自被子植物组蛋白基因的CRM，在至少1.5kb的距离上上调莱茵衣原体中的β2-微管蛋白启动子。Ehistcons中存在的八聚体基序在C.reinhardtii和相关的绿藻衣藻中得到了鉴定，Schloesseri衣藻,和Edaphochlamysdebaryana,证明了它的高度进化保守性。这项研究的结果扩展了我们对绿藻基因表达调控的认识。此外，表征的活化CREs/CRM可以用作有价值的遗传工具。
