Sharp Brandi, Paquet Eric, Naef Felix, Bafna Akanksha, Wijnen Herman
Department of Biology, University of Virginia, Charlottesville, VA 22903, USA.
Institute of Bioengineering, School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
Nucleic Acids Res. 2017 Jun 20;45(11):6459-6470. doi: 10.1093/nar/gkx268.
Circadian clocks are autonomous daily timekeeping mechanisms that allow organisms to adapt to environmental rhythms as well as temporally organize biological functions. Clock-controlled timekeeping involves extensive regulation of rhythmic gene expression. To date, relatively few clock-associated promoter elements have been identified and characterized. In an unbiased search of core clock gene promoters from 12 species of Drosophila, we discovered a 29-bp consensus sequence that we designated as the Clock-Associated Transcriptional Activation Cassette or 'CATAC'. To experimentally address the spatiotemporal expression information associated with this element, we generated constructs with four separate native CATAC elements upstream of a basal promoter driving expression of either the yeast Gal4 or firefly luciferase reporter genes. Reporter assays showed that presence of wild-type, but not mutated CATAC elements, imparted increased expression levels as well as rhythmic regulation. Part of the CATAC consensus sequence resembles the E-box binding site for the core circadian transcription factor CLOCK/CYCLE (CLK/CYC), and CATAC-mediated expression rhythms are lost in the presence of null mutations in either cyc or the gene encoding the CLK/CYC inhibitor, period (per). Nevertheless, our results indicate that CATAC's enhancer function persists in the absence of CLK/CYC. Thus, CATAC represents a novel cis-regulatory element encoding clock-controlled regulation.
昼夜节律时钟是自主的日常计时机制，使生物体能够适应环境节律，并在时间上组织生物功能。时钟控制的计时涉及对节律性基因表达的广泛调控。迄今为止，相对较少的与时钟相关的启动子元件已被鉴定和表征。在对12种果蝇的核心时钟基因启动子进行无偏搜索时，我们发现了一个29个碱基对的共有序列，我们将其命名为时钟相关转录激活盒或“CATAC”。为了通过实验确定与该元件相关的时空表达信息，我们构建了在驱动酵母Gal4或萤火虫荧光素酶报告基因表达的基础启动子上游带有四个单独的天然CATAC元件的构建体。报告基因检测表明，野生型而非突变型CATAC元件的存在会导致表达水平增加以及节律性调控。CATAC共有序列的一部分类似于核心昼夜节律转录因子CLOCK/CYCLE（CLK/CYC）的E-box结合位点，并且在cyc或编码CLK/CYC抑制剂period（per）的基因存在无效突变的情况下，CATAC介导的表达节律会丧失。然而，我们的结果表明，在没有CLK/CYC的情况下，CATAC的增强子功能仍然存在。因此，CATAC代表了一种编码时钟控制调控的新型顺式调控元件。
