Global control of rhythmic gene expression by the transcription factor LHY

真核生物如植物和动物的基因表达调控是复杂的，因为空间和时间信号的多样性汇聚到单个基因启动子来调节转录。影响mRNA降解的信号可能进一步调节转录物积累的模式。对于转录因子对给定结合位点的亲和力如何影响其转录作用，以及多个转录因子的作用是否以相加或协同的方式结合，我们所知甚少。为了解决这些问题，本项目旨在分析已知转录因子LHY与其他调节LHY靶基因表达的调节蛋白之间相互作用的逻辑。LHY转录因子是高等植物24小时生物钟的一个组成部分，也被称为昼夜节律钟。已知LHY表达的节律模式是靶基因振荡表达的基础，靶基因在不同阶段表达。这表明LHY对不同目标启动子的不同亲和力（即结合的开启率和关闭率）可能影响转录激活的时间。另外，LHY对单个靶基因表达的影响可能通过与其他调节蛋白的相互作用而改变。mRNA降解的速率可能进一步调节mRNA积累的时间。为了验证这些假设，我们建议首先确定LHY结合位点的全基因组范围。LHY对不同类型结合位点的亲和力将通过生物信息学和实验方法的结合来确定。比较来自不同物种的同源LHY靶基因子集的启动子序列将发现可能调节LHY对转录激活作用的保守元件。拟南芥中调节基因表达时间的作用将通过测试这些元素是否在相似时间表达模式的LHY靶基因的特定簇中富集来证实。为了生成转录激活和mRNA积累的定量数学模型数据，我们将在昼夜周期中量化以下参数：(i) LHY蛋白水平的变化；（ii）与选定的一组目标启动子结合的转录因子数量的变化；（iii）同源基因转录激活水平的变化；（iv）相应mrna水平的变化。然后将开发一套数学工具，以搜索最可能的调控逻辑来解释数据，预测mRNA降解率的贡献，并预测该逻辑中任何改变的影响。这些预测将在转基因植物中使用一组与荧光素酶报告基因融合的启动子结构进行体内测试。

The regulation of gene expression in eukaryotic organisms such as plants and animals is complex because of the multiplicity of spatial and temporal signals that converge onto individual gene promoters to regulate transcription. Signals affecting mRNA degradation may further modulate the pattern of transcript accumulation. Not much is known about how the affinity of a transcription factor for a given binding site influences its effect on transcription, or whether the effects of multiple transcription factors combine in an additive or synergistic manner. In order to address these questions, this project aims to analyse the logic of interactions between a known transcription factor called LHY and other regulatory proteins modulating the expression of LHY target genes. The LHY transcription factor functions as a component of higher plant's 24-biological clock, also known as the circadian clock. The rhythmic pattern of expression of LHY is known to underlie oscillatory expression of target genes that are expressed with a variety of phases. This suggests that the differential affinty of LHY for different target promoters (i.e, the on- and off-rate of binding) may affect the timing of transcriptional activation. Alternatively, or in addition, the effect of LHY on expression of individual target genes may be altered by interaction with other regulatory proteins. The rate of mRNA degradation may further modulate the timing of mRNA accumulation. In order to test these hypotheses, we propose first to identify the genome-wide range of LHY binding sites. The affinity of LHY for different classes of binding sites will be determined using a combination of bioinformatic and experimental approaches. Comparison of promoter sequences of a subset of orthologous LHY target genes from different species will identify conserved elements that may act to modulate the effect of LHY on transcriptional activation. A role in adjusting the timing of gene expression will be confirmed in Arabidopsis by testing whether these elements are enriched within specific clusters of LHY target genes within similar temporal patterns of expression. In order to generate data for the quantitative mathematical modelling of transcriptional activation and mRNA accumulation, we will quantify the following parameters across the circadian cycle: (i) changes in LHY protein levels; (ii) changes in the amount of transcription factor bound to a chosen set of target promoters; (iii) changes in the level of transcriptional activation of the cognate genes; and (iv) changes in the level of the corresponding mRNAs. A set of mathematical tools will then be developed to search for the most probable regulatory logic to account for the data, to predict the contribution of mRNA degradation rates and to predict the effects of any alterations in this logic. These predictions will be tested in vivo using a set of promoter constructs fused to a luciferase reporter gene in transgenic plants.