Molecular analysis of circadian regulation in Arabidopsis

描述(申请人提供)：昼夜节律为生物体提供了一种适应优势，通过确保不同的生理过程在一天中最合适的时间发生，增强了个体的健康和体能。这项建议的长期目标是了解真核细胞昼夜节律的分子基础。我们在以前的研究中已经确定了生物钟的关键调节因子，现在将使用基因组、生化、遗传和数学建模方法来适当地将这些蛋白质放置在生物钟系统中。我们将在拟南芥中进行我们的研究，这是一种模式植物，由于其紧凑的基因组、广泛的遗传和基因组资源以及耐受染色质调节途径中的突变的能力，而这些突变对其他复杂的真核生物是致命的，因此它是唯一适合这些实验的模式植物。我们将首先使用基因组、生物化学和遗传学方法来表征一种在真核生物中保守的蛋白质(但其生化功能未知)在染色质调节中的作用。我们预计这项工作将对控制染色质组织的机制，从而在不同的真核生物中的基因表达产生深刻的见解。接下来，我们将使用遗传和生化技术来研究相关转录因子家族在昼夜节律中的作用。 振荡器。在这个过程中，我们将测试一个数学模型做出的预测，评估这个模型描述驱动时钟的监管关系的程度。最后，我们将修改现有的竞争性染色质免疫沉淀方案，以研究拮抗转录因子与染色质的结合动力学如何在体内塑造基因表达的动态调节。这些实验将揭示控制基因表达时间调控的一般原理，因此将适用于许多生物体和过程。我们探索在不同生物体中控制生物钟功能的共同机制，将增加我们对时钟在人类中如何发挥作用的理解，并阐明它们如何促进人类的健康和福祉。

DESCRIPTION (provided by applicant): Circadian rhythms provide organisms with an adaptive advantage, enhancing the health and fitness of individuals by ensuring that diverse physiological processes occur at the most appropriate times of day. The long-term objective of this proposal is to understand the molecular basis of circadian rhythms in eukaryotic cells. We have identified key regulators of the circadian clock in our previous studies, and will now use genomic, biochemical, genetic, and mathematical modeling approaches to appropriately place these proteins in the circadian system. We will conduct our studies in Arabidopsis thaliana, a model plant that is uniquely well-suited for these experiments due to its compact genome, extensive genetic and genomic resources, and ability to tolerate mutations in chromatin regulatory pathways that are lethal to other complex eukaryotes. We will first use genomic, biochemical, and genetic approaches to characterize the role of a protein conserved across eukaryotes (but of unknown biochemical function) in the regulation of chromatin. We anticipate this work will generate insights into mechanisms governing chromatin organization and thus gene expression in diverse eukaryotes. Next, we will use genetic and biochemical techniques to investigate the roles of a family of related transcription factors in the workings of the circadian oscillator. In the process, we will test predictions made by a mathematical model, evaluating how well this model describes the regulatory relationships that drive the clock. Finally, we will modify an existing competitive chromatin immunoprecipitation protocol to investigate how the binding dynamics of antagonistic transcription factors to chromatin shape the dynamic regulation of gene expression in vivo. These experiments will reveal general principles governing the temporal regulation of gene expression and thus will be applicable to many organisms and processes. Our exploration of shared mechanisms that control circadian clock function in diverse organisms will increase our understanding of how clocks function in humans and shed light on how they promote human health and welfare.