The Role of SIRT6 in Modulating Circadian Gene Expression

描述（由申请人提供）：昼夜节律控制24小时内许多生理过程的时间，包括睡眠-觉醒周期、体温调节、摄食、代谢调节和激素产生。构成生物钟的分子网络的核心是异源二聚化的核心转录因子clock和BMAL1，它们与转录机制一起，直接节律性地表达生物钟控制基因（CCGs）。昼夜节律基因表达的一个关键组成部分与CLOCK有关，CLOCK是一种已知的组蛋白乙酰转移酶，它指导组蛋白H3赖氨酸9/14和BMAL1的节律性乙酰化。在昼夜节律转录方面，我们对与CLOCK和BMAL1协同作用指导昼夜节律基因表达的转录机制知之甚少。本研究的目的是阐明哺乳动物sirtuins III类组蛋白去乙酰化酶（hdac）对体内昼夜节律基因表达和生物节律的调控机制。SIRT6是一种已知的染色质相关HDAC，可在相关基因启动子处指导组蛋白H3赖氨酸9的去乙酰化，但SIRT6与昼夜生物学无关。特别重要的是，SIRT1负责BMAL1的去乙酰化和调控，以及H3赖氨酸9/14在昼夜节律基因启动子上的去乙酰化。令人惊讶的是，SIRT1是一种主要定位于核质中的核sirtuin，因此尚不清楚SIRT1的效率是适合组蛋白靶点，还是更适合非组蛋白如BMAL1。假设SIRT6和SIRT1在调节昼夜基因表达方面存在功能相互作用，并且这些sirtuins的亚细胞定位可能决定了脱乙酰酶对组蛋白和非组蛋白靶标的活性效率。假设HDAC SIRT6与核心昼夜节律转录因子CLOCK和BMAL1相关，并可能通过使昼夜节律基因启动子上的H3K9去乙酰化来调节昼夜节律基因的表达，从而导致转录抑制和随后的CCG表达振荡。为了验证这一假设，将使用野生型和SIRT6敲除小鼠以及小鼠胚胎成纤维细胞（mef）来确定SIRT6介导的对体外和体内昼夜节律基因表达的影响，以及这些作用与SIRT1有何不同。

DESCRIPTION (provided by applicant): Circadian rhythms control the timing of numerous physiological processes over a 24-hour period, including sleep-wake cycles, thermoregulation, feeding, metabolic regulation and hormone production. At the heart of the molecular network that constitutes the circadian clock are the core transcription factors CLOCK and BMAL1 that heterodimerize, and in conjunction with the transcriptional machinery, direct rhythmic expression of clock-controlled genes (CCGs). A critical component of circadian gene expression is linked to CLOCK, a known histone acetyltransferase, which directs rhythmic acetylation of histone H3 lysine 9/14 as well as BMAL1. In terms of circadian transcription, little is known about the transcriptional machinery that works in concert with CLOCK and BMAL1 to direct circadian gene expression. The goal of this proposal is to elucidate the mechanisms of regulation of the mammalian sirtuins, class III histone deacetylases (HDACs), on circadian gene expression and biological rhythmicity in vivo. SIRT6 is a known chromatin-associated HDAC that directs deacetylation of histone H3 lysine 9 at relevant gene promoters, but SIRT6 has not been implicated in circadian biology. Of particular importance, SIRT1 is responsible for deacetylation and regulation of BMAL1, as well as deacetylation of H3 lysine 9/14 at circadian gene promoters. Surprisingly, SIRT1 is a nuclear sirtuin that is mostly localized in the nucleoplasm, therefore it in unclear whether the efficiency of SIRT1 is suited for histone targets or better directed towards non-histone proteins such as BMAL1. It is hypothesized that a functional interplay exists between SIRT6 and SIRT1 in modulating circadian gene expression, and that the subcellular localization of these sirtuins may dictate efficiency of deacetylase activity towards histone versus non-histone targets. It is hypothesized that the HDAC SIRT6 is associated with the core circadian transcription factors, CLOCK and BMAL1, and may be involved in modulating circadian gene expression by deacetylating H3K9 at circadian gene promoters, resulting in transcriptional repression and subsequent oscillation of CCG expression. To test this hypothesis, the use of wild-type and SIRT6 knockout mice as well as mouse embryo fibroblasts (MEFs) will be utilized to determine SIRT6-mediated effects on circadian gene expression in vitro and in vivo, and how these actions differ from SIRT1. PUBLIC HEALTH RELEVANCE: Circadian rhythms are inherent biological timekeeping mechanisms that regulate our daily physiology, and moreover, disruptions in the biological circadian clock can lead to numerous diseases including sleep disorders, depression, metabolic syndrome, cardiovascular disturbances and tumorigenesis. The goal of this proposal is to elucidate the mechanisms of regulation of circadian gene expression and ultimately the regulatory events in place that modulate circadian rhythms in vivo.