Global control of rhythmic gene expression by the transcription factor LHY

转录因子 LHY 对节律基因表达的全局控制

• 批准号: BB/F021143/1
• 负责人: Peter Stockley
• 金额: $ 0.49万
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF021143%2F1
• 关键词: Global; control; rhythmic; gene; expression

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.

真核生物如植物和动物中基因表达的调节是复杂的，因为聚集到单个基因启动子上以调节转录的空间和时间信号的多样性。影响mRNA降解的信号可以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对转录的作用，或者多个转录因子的作用是否以相加或协同的方式结合联合收割机，我们知之甚少。为了解决这些问题，该项目旨在分析一种称为LHY的已知转录因子与调节LHY靶基因表达的其他调节蛋白之间的相互作用逻辑。LHY转录因子是高等植物24小时生物钟的组成部分，也被称为昼夜节律钟。已知LHY的表达的节律模式是以各种相位表达的靶基因的振荡表达的基础。这表明LHY对不同靶启动子的不同亲和力（即结合和解离速率）可能影响转录激活的时间。或者，或另外，LHY对单个靶基因表达的影响可以通过与其他调节蛋白的相互作用而改变。mRNA降解的速率可进一步调节mRNA积累的时间。为了检验这些假设，我们建议首先确定全基因组范围的LHY结合位点。LHY对不同类型结合位点的亲和力将使用生物信息学和实验方法的组合来确定。来自不同物种的一个子集的orthopathic LHY靶基因的启动子序列的比较将确定保守的元素，可能会发挥作用，以调节LHY对转录激活的影响。在拟南芥中，通过测试这些元件是否在相似的表达时间模式内富集在LHY靶基因的特定簇内，将证实在调节基因表达时间方面的作用。为了产生用于转录激活和mRNA积累的定量数学建模的数据，我们将在昼夜节律周期中量化以下参数：（i）LHY蛋白水平的变化;（ii）结合到选定的靶启动子组的转录因子的量的变化;（iii）同源基因的转录激活水平的变化;（iv）转录因子的量的变化。和（iv）相应mRNA水平的变化。然后将开发一组数学工具来搜索最可能的调控逻辑，以解释数据，预测mRNA降解速率的贡献，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与荧光素酶报告基因融合的一组启动子构建体进行体内测试。