Global control of rhythmic gene expression by the transcription factor LHY

在植物和动物等真核生物中，基因表达的调控是复杂的，因为空间和时间信号的多样性汇聚到单个基因启动子上来调节转录。影响信使核糖核酸降解的信号可能进一步调节转录积累的模式。关于转录因子与给定结合位点的亲和力如何影响其对转录的影响，或者多个转录因子的效应是以相加的方式还是以协同的方式结合在一起，目前还知之甚少。为了解决这些问题，本项目旨在分析已知的转录因子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-生物钟的一个组成部分，也被称为生物钟。众所周知，LHY表达的节律性模式是靶基因振荡表达的基础，靶基因的表达具有不同的时相。这表明LHY对不同靶启动子的不同亲和力(即结合的开启和关闭速率)可能影响转录激活的时间。此外，LHY对单个靶基因表达的影响可能会通过与其他调控蛋白的相互作用而改变。信使核糖核酸的降解速度可能进一步调节信使核糖核酸积累的时间。为了验证这些假设，我们建议首先确定全基因组范围的LHY结合位点。LHY对不同类别结合位点的亲和力将使用生物信息学和实验相结合的方法来确定。通过比较来自不同物种的一组同源LHY靶基因的启动子序列，可以确定可能调节LHY对转录激活的影响的保守元件。通过测试这些元件是否在类似表达时间模式的特定LHY靶基因簇中富含，将在拟南芥中确认调节基因表达时间的作用。为了建立转录激活和mRNA积累的定量数学模型，我们将量化下列参数：(I)LHY蛋白水平的变化；(Ii)与选定的一组靶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；以及(Iv)相应mRNAs水平的变化。然后将开发一套数学工具来搜索最可能的调控逻辑来解释数据，预测信使核糖核酸降解率的贡献，并预测该逻辑中任何变化的影响。这些预测将使用一组融合到转基因植物中的荧光素酶报告基因的启动子构建物在体内进行验证。

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.