Genome-wide epigenetic control of circadian metabolism by heme receptor Rev-erb

描述(由申请人提供)：本申请涉及广泛的挑战领域(08)基因组学和特定挑战主题08-DK-107：核受体介导的功能转录单位的组装。这个实验室的一个主要目标是了解核受体(NRs)调节昼夜节律和代谢生理的转录机制。血红素受体Rev-erb在1989年被PI发现，此后成为控制昼夜节律和葡萄糖代谢的关键基因的重要抑制因子。以前对REV-ERB的研究一直基于关注启动子的候选基因方法。因此，考虑到对其他核受体的全基因组定位分析显示，大多数结合发生在启动子之外，因此仅有大约10个REV-ERB靶基因已知并不令人惊讶。我们假设REV-ERB通过结合数百个基因发挥作用，其中许多基因是组织特异性的。为了解决这个问题，我们将对小鼠肝脏和白色脂肪组织中的REV-ERB结合进行全基因组分析。具体目标1是在不同的昼夜节律时间在全基因组范围内识别肝脏中的REV-ERB基因靶点，并了解它们的调控模式。我们假设，REV-ERB蛋白水平的昼夜波动将影响其获取基因组中识别序列的能力，这反过来将对昼夜节律和代谢过程产生下游影响。我们将通过使用染色质免疫沉淀(CHIP)进行全基因组定位分析，然后对分离的DNA进行测序(CHIP-SEQ)来测试这一点。具体目标2是在全基因组范围内从喂食和禁食小鼠以及限食小鼠的肝脏中鉴定Rev-erb基因靶点，并了解它们的调节模式。我们假设REV-ERB活性的代谢调节将改变REV-ERB在许多基因上的结合，并将使用从喂养和禁食小鼠的肝脏分离的CHIP-SEQ来验证这一点。初步数据显示，禁食小鼠肝脏中的REV-ERB蛋白水平高于喂食小鼠的肝脏，提示REV-ERB结合活性可能受代谢信号的调节。具体目标3是在全基因组范围内识别白色脂肪组织中的REV-ERB基因靶点，并了解它们的调控模式。我们假设Rev-erb介导了脂肪细胞基因的昼夜表达，并将通过在不同的昼夜时间和喂养方案下对脂肪Rev-erb进行CHIP-SEQ来测试这一点。这将扩大我们对Rev-erb在脂肪生物学中的功能的理解，当与肝脏数据集相结合时，它将确定Rev-erb在肝脏和脂肪组织中的组织特异性靶点和功能。这些研究可以在两年的时间框架内完成，因为我们已经在代谢组织中验证了内源性Rev-erb芯片的抗体，并且实验范式涉及短期研究。全基因组范围内对昼夜节律和代谢基因的体内组织特异性调控的洞察将是巨大的，并将极大地有助于我们理解这些生理过程是如何相互关联的，并可能在肥胖症和糖尿病中出现调控失调，这两个疾病在美国很普遍。 公共卫生相关性：糖尿病和肥胖症等代谢紊乱会受到睡眠模式和倒班工作的影响。因此，令人兴奋的是，这一提议的焦点，核受体REV-ERB，已经成为昼夜节律和代谢生理学之间的关键纽带。拟议的实验旨在确定REV-ERB对昼夜节律和新陈代谢过程的贡献甚至控制的程度，以及它们之间发生的串扰。最终，更深入地了解REV-ERB对昼夜节律和新陈代谢的调节将有助于我们理解这些生理过程是如何相互关联的，以及在肥胖和糖尿病这两种在美国流行的疾病中可能存在的调节失调。

DESCRIPTION (provided by applicant): This application addresses the broad Challenge Area (08) Genomics and specific Challenge Topic 08-DK-107: Nuclear Receptor mediated assembly of functional transcriptional units. A major goal of this laboratory is to understand the transcriptional mechanisms by which nuclear receptors (NRs) regulate circadian and metabolic physiology. The heme receptor Rev-erb was identified by the PI in 1989, and has since emerged as an important repressor of key genes controlling circadian rhythm and glucose metabolism. Prior studies of Rev- erb have been based on candidate gene approaches focused on promoters. Thus, it is not surprising that only about ten Rev-erb-target genes are known, given that genome-wide location analyses for other nuclear receptors have revealed that most binding occurs outside of promoters. We hypothesize that Rev-erb functions through binding of hundreds of genes, many tissue-specifically. To address this, we will perform a genome-wide analysis of Rev-erb binding in mouse liver and white adipose tissues. Specific Aim 1 is to identify Rev-erb gene targets on a genome-wide scale in liver at different circadian times, and understand their modes of regulation. We hypothesize that circadian fluctuation of Rev-erb protein level will affect its ability to access recognition sequences in the genome, which, in turn, will have downstream effects on circadian and metabolic processes. We will test this by genome-wide location analysis using chromatin immunoprecipitation (ChIP) followed by sequencing of the isolated DNA (ChIP-seq). Specific Aim 2 is to identify Rev-erb gene targets on a genome-wide scale in liver from fed versus fasted mice and from mice under restricted feeding, and understand their modes of regulation. We hypothesize that metabolic regulation of Rev-erb activity will alter Rev-erb binding at many genes, and will test this by ChIP- seq using liver isolated from fed and fasted mice. Preliminary data reveal that Rev-erb protein level is higher in liver from fasted mice than in liver from fed mice, suggesting that Rev-erb binding activity may be modulated by metabolic signals. Specific Aim 3 is to identify Rev-erb gene targets on a genome-wide scale in white adipose tissue, and understand their modes of regulation. We hypothesize that Rev-erb mediates circadian expression of adipocyte genes and will test this by performing ChIP-seq for adipose Rev- erb at different circadian times and feeding regimens. This will expand our understanding of Rev-erb function in adipose biology and, when combined with the liver data set, it will identify tissue-specific targets and functions for Rev-erb in liver and adipose tissues. These studies can be accomplished in a two-year time frame because we have validated antibodies for ChIP of endogenous Rev-erb in metabolic tissues, and the experimental paradigms involve short-term studies. The genome-wide insights into in vivo tissue-specific regulation of circadian and metabolic genes will be enormous, and contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States. PUBLIC HEALTH RELEVANCE: Metabolic disorders such as diabetes and obesity are affected by sleep patterns and shift work. Thus, it is exciting that the focus of this proposal, the nuclear receptor Rev-erb, has emerged as a critical link between circadian and metabolic physiology. The proposed experiments aim to determine the extent that Rev-erb contributes to, or even controls, circadian and metabolic processes and the crosstalk that occurs between them. Ultimately, greater insight into the regulation of circadian rhythm and metabolism by Rev-erb will contribute greatly to our understanding of how these physiological processes are interrelated, and potentially dysregulated in obesity and diabetes, which are epidemic in the United States.