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、进食、代谢调节和激素产生。在构成生物钟的分子网络的核心是核心转录因子CLOCK和BMAL 1，其异二聚化，并与转录机制结合，指导时钟控制基因（CCG）的节律表达。昼夜节律基因表达的一个关键组成部分与CLOCK（一种已知的组蛋白乙酰转移酶）有关，CLOCK指导组蛋白H3赖氨酸9/14以及BMAL 1的节律性乙酰化。在昼夜节律转录方面，人们对与CLOCK和BMAL 1协同工作以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（HDACs），在体内的昼夜基因表达和生物节律的调节机制。SIRT 6是已知的染色质相关HDAC，其指导组蛋白H3赖氨酸9在相关基因启动子处的脱乙酰化，但SIRT 6尚未涉及昼夜节律生物学。特别重要的是，SIRT 1负责BMAL 1的脱乙酰化和调节，以及昼夜节律基因启动子处H3赖氨酸9/14的脱乙酰化。令人惊讶的是，SIRT 1是一种主要位于核质中的核沉默调节蛋白，因此尚不清楚SIRT 1的效率是否适合于组蛋白靶向或更好地针对非组蛋白如BMAL 1。假设SIRT 6和SIRT 1在调节昼夜节律基因表达中存在功能性相互作用，并且这些sirtuins的亚细胞定位可能决定了脱乙酰酶活性对组蛋白与非组蛋白靶标的效率。据推测，HDAC SIRT 6与核心昼夜节律转录因子CLOCK和BMAL 1相关，并且可能通过在昼夜节律基因启动子处脱乙酰化H3 K9而参与调节昼夜节律基因表达，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，将利用野生型和SIRT 6敲除小鼠以及小鼠胚胎成纤维细胞（MEF）来确定SIRT 6介导的体外和体内对昼夜节律基因表达的影响，以及这些作用与SIRT 1的不同之处。 公共卫生相关性：昼夜节律是调节我们日常生理的固有生物计时机制，此外，生物昼夜节律钟的中断可导致许多疾病，包括睡眠障碍、抑郁症、代谢综合征、心血管紊乱和肿瘤发生。这个建议的目的是阐明昼夜节律基因表达的调节机制，并最终在体内调节昼夜节律的调节事件。

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.