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31. Dedicated to the memory of R. A. Zann, who inspired us through his work. Funded by NIH MH062656 (study section on Biobehavioral Regulation, Learning, and Ethology) to J.L.G. 31.为了纪念R。A. Zann通过他的工作激励了我们。由NIH MH 062656(生物行为调节、学习和行为学研究部分)资助,J.L.G.
The Transcriptional Repressor DEC2 Regulates Sleep Length in Mammals 转录抑制因子DEC 2调节哺乳动物的睡眠时间
Ying He, ^(1){ }^{1} Christopher R. Jones, ^(2){ }^{2} Nobuhiro Fujiki, ^(3){ }^{3} Ying Xu, ^(1**){ }^{1 *} Bin Guo, ^(4){ }^{4} Jimmy L. Holder Jr., ^(1)†{ }^{1} \dagger Moritz J. Rossner, ^(5){ }^{5} Seiji Nishino, ^(3){ }^{3} Ying-Hui Fu ^(1)‡{ }^{1} \ddagger 何英, ^(1){ }^{1} Christopher R.琼斯, ^(2){ }^{2} 藤木信弘, ^(3){ }^{3} 徐颖, ^(1**){ }^{1 *} 郭斌, ^(4){ }^{4} 吉米L。小保持器, ^(1)†{ }^{1} \dagger 莫里茨·J·罗斯纳(Moritz J. Rossner)、 ^(5){ }^{5} 西野征尔(Seiji Nishino)、 ^(3){ }^{3} 傅莹慧(Ying-Hui Fu) ^(1)‡{ }^{1} \ddagger
Sleep deprivation can impair human health and performance. Habitual total sleep time and homeostatic sleep response to sleep deprivation are quantitative traits in humans. Genetic loci for these traits have been identified in model organisms, but none of these potential animal models have a corresponding human genotype and phenotype. We have identified a mutation in a transcriptional repressor (hDEC2-P385R) that is associated with a human short sleep phenotype. Activity profiles and sleep recordings of transgenic mice carrying this mutation showed increased vigilance time and less sleep time than control mice in a zeitgeber time- and sleep deprivation-dependent manner. These mice represent a model of human sleep homeostasis that provides an opportunity to probe the effect of sleep on human physical and mental health. 睡眠不足会损害人类的健康和表现。习惯性总睡眠时间和对睡眠剥夺的稳态睡眠反应是人类的数量特征。这些性状的遗传基因座已在模式生物中得到鉴定,但这些潜在的动物模型都没有相应的人类基因型和表型。我们已经确定了一个突变的转录抑制因子(hDEC 2-P385 R),这是与人类短睡眠表型。携带这种突变的转基因小鼠的活动曲线和睡眠记录显示,与对照小鼠相比,警觉时间增加,睡眠时间减少,这与时间和睡眠剥夺有关。这些小鼠代表了人类睡眠稳态的模型,为探索睡眠对人类身心健康的影响提供了机会。
Although sleep is an essential process for life, the brain circuits regulating sleep and the cellular and/or molecular mechanisms involved in this complex process are still enigmatic (1-3). Sleep or a “sleeplike” behavior is present in virtually every animal species where it has been studied. Total sleep deprivation can be fatal, and partial deprivation of sleep has serious consequences on cognition, mood, and health (4-6). It is obvious that situational increases in behavioral drive can transiently delay sleep, but very little is known about chronic partial sleep curtailment as a possible consequence of a persistent elevation in waking behavioral drive. The latter trait, sometimes referred to as a “hyperthymic” temperament (7), is a theoretical third influence on sleep habits. 虽然睡眠是生命的一个基本过程,但调节睡眠的大脑回路以及参与这一复杂过程的细胞和/或分子机制仍然是个谜(1-3)。睡眠或类似睡眠的行为几乎存在于所有被研究过的动物物种中。完全剥夺睡眠可能是致命的,部分剥夺睡眠对认知,情绪和健康有严重后果(4-6)。很明显,行为驱动力的情境性增加可以短暂地延迟睡眠,但人们对清醒时行为驱动力持续升高可能导致的慢性部分睡眠缩短知之甚少。后一种特质,有时被称为“情绪亢奋”气质(7),是理论上对睡眠习惯的第三种影响。
Murine Dec2 (mDec2) is a negative component of the circadian clock (8-10). It belongs to a basic helix-loop-helix (bHLH) protein family in which members can dimerize with each other and 小鼠Dec 2(mDec 2)是昼夜节律钟的负分量(8-10)。它属于碱性螺旋-环-螺旋(bHLH)蛋白家族,其中成员可以彼此二聚化,
can affect gene transcription by binding to specific DNA sequences (11). While performing candidate gene resequencing in DNAs from human families, segregating alleles for extremely early wake up times, we identified an hDEC2\mathrm{h} D E C 2 point mutation in a small family with two affected individuals (Fig. 1A) (12). Subjects carrying this mutation had lifelong shorter daily sleep times than normal individuals (Table 1). The self-reported nonworkday habitual sleep-offset times of the mutation carriers were much earlier than those of the noncarriers (including noncarrier family members and general controls). However, these two individuals have sleep-onset times that are similar to that of conventional sleepers. The habitual selfreported total sleep time per 24 -hour day was much shorter in mutation carriers (average 6.25 hours) compared with the noncarriers (average 8.06 hours) in this family. Thus, they represent “natural short sleepers” who routinely sleep less than individuals with familial advanced sleep-phase syndrome (FASPS) or general controls (Table 1). The average total sleep time for American adults on nonworkdays is ∼7.4\sim 7.4 hours (www.sleepfoundation.org). The mutation changes a C to G in the DNA sequence of DEC2D E C 2, which is predicted to cause a proline-to-arginine alteration at amino acid position 385 of DEC2 (Fig. 1B). This change was not found in over 250 control DNA samples. The proline at position 385 of DEC2 (P385) is conserved in mammals but not invertebrates. P385 is located in a highly conserved region within a proline-rich domain of unknown function and is close to the C-terminal 可以通过与特定DNA序列结合来影响基因转录(11)。在对来自人类家族的DNA进行候选基因重测序时,分离了极早觉醒时间的等位基因,我们在一个有两个受影响个体的小家族中鉴定了 hDEC2\mathrm{h} D E C 2 点突变(图1A)(12)。携带这种突变的受试者终生的每日睡眠时间比正常人短(表1)。突变携带者自我报告的非工作日习惯性睡眠偏移时间远早于非携带者(包括非携带者家庭成员和一般对照)。然而,这两个人的睡眠开始时间与传统睡眠者相似。在这个家庭中,突变携带者(平均6.25小时)的习惯性自我报告的每天24小时的总睡眠时间比非携带者(平均8.06小时)短得多。 因此,他们代表了“自然短睡眠者”,他们的睡眠时间通常少于家族性晚期睡眠综合征(FASPS)或一般对照者(表1)。美国成年人在非工作日的平均总睡眠时间是 ∼7.4\sim 7.4 小时(www.sleepfoundation.org)。该突变将 DEC2D E C 2 的DNA序列中的C变为G,预测其导致DEC 2的氨基酸位置385处的脯氨酸变为精氨酸(图1B)。在超过250个对照DNA样品中未发现这种变化。DEC 2的385位脯氨酸(P385)在哺乳动物中是保守的,但在无脊椎动物中不是。 P385位于功能未知的富含脯氨酸的结构域内的高度保守区域,并且靠近C-末端。
histone deacetylase (HDAC)-interacting region of DEC2 (Fig. 1B). Activity-rest recording in one mutation carrier using 10 -day sleep logs with coincident wrist actigraphy demonstrated an extended active period each day (Fig. 1C). DEC 2的组蛋白脱乙酰酶(HDAC)相互作用区域(图1B)。在一个突变携带者中,使用10天睡眠日志与手腕活动记录仪同步记录的活动-休息记录显示每天活动时间延长(图1C)。
To examine the effect of the P385R mutation on Dec2 repressor activity, a wild-type (WT) or a P385R mDec2 construct was used in a luciferase assay, and the results showed that P385R attenuated Dec2 repressive activity of Clk//Bmal1-\mathrm{Clk} / \mathrm{Bmal1}- mediated transactivation (fig. S1A). The reduction in Dec2 repressive activity was moderate compared with that of the R57A/K mutations (in which arginine 57 was replaced by alanine or lysine) reported before (13). Dec2 was previously shown to preferentially bind to class B E-box elements (CACGTG) as a homodimer and to repress the transcription of target genes in an HDACdependent manner (13). The effect of HDAC on the mutant Dec 2 repression was then analyzed by monitoring mPer 2 promoter-driven luciferase activity with or without a general HDAC inhibitor trichostatin A (TSA) (fig. S1B). HDAC inhibition resulted in similar increases in luciferase activity for both WT and mutant Dec2. Coimmunoprecipitation was then performed for mDec 2 (WT or P385R) and human sirtuin-1 (hSIRT1). HEK293 cells were transiently cotransfected with green fluorescent protein (GFP)-tagged (WT or mutated) mDec 2 and FLAG-tagged hSIRT1, followed by FLAG-peptide pull-down and detection of GFP with antibodies on Western blots. The results showed similar physical interactions between WT or P385R mDec2 and hSIRT1 (fig. S1C). Taken together, these results suggest that the P385R mutation affects Dec2 transcriptional repression activity independently from its interaction with HDAC/SIRT. 为了检查P385 R突变对Dec 2阻遏物活性的影响,在荧光素酶测定中使用野生型(WT)或P385 R mDec 2构建体,结果显示P385 R减弱了 Clk//Bmal1-\mathrm{Clk} / \mathrm{Bmal1}- 介导的反式激活的Dec 2阻遏活性(图S1 A)。与之前报道的R57 A/K突变(其中精氨酸57被丙氨酸或赖氨酸取代)相比,Dec 2抑制活性的降低是中等的(13)。Dec 2先前显示优先结合B类E-box元件(CACGTG)作为同源二聚体,并以HDAC依赖性方式抑制靶基因的转录(13)。然后通过监测mPer 2启动子驱动的荧光素酶活性,在有或没有通用HDAC抑制剂阿司他丁A(TSA)的情况下,分析HDAC对突变体Dec 2阻遏的作用(图S1 B)。HDAC抑制导致WT和突变体Dec 2的荧光素酶活性的类似增加。 然后对mDec 2(WT或P385 R)和人沉默调节蛋白-1(hSIRT 1)进行免疫共沉淀。用绿色荧光蛋白(GFP)标记的(WT或突变的)mDec 2和FLAG标记的hSIRT 1瞬时共转染HEK 293细胞,然后进行FLAG肽下拉,并用抗体在Western印迹上检测GFP。结果显示WT或P385 R mDec 2与hSIRT 1之间的相似物理相互作用(图S1 C)。总之,这些结果表明,P385 R突变影响Dec 2转录抑制活性独立于其与HDAC/SIRT的相互作用。
Because there are only two human mutation carriers in this study, the question remained whether the natural short sleep phenotype was caused by the DEC2D E C 2 mutation. Thus, we generated WT and P385R DEC2 transgenic (Tg) mice using a human bacterial artificial chromosome (BAC) clone (RP11-288E19) carrying the entire hDEC2\mathrm{h} D E C 2 gene to test this hypothesis. As DEC2D E C 2 has been established as a component of circadian clock (9,14)(9,14), we first set out to determine the circadian period ( tau\tau ) of DEC2-P385R mice. Mice with Dec2 deleted [knockout (KO) mice] (10) and WT littermates were tested in parallel as controls. No significant differences in tau\tau were detected among mice of different genotypes (table S1). 因为在这项研究中只有两个人类突变携带者,所以问题仍然是自然的短睡眠表型是否是由 DEC2D E C 2 突变引起的。因此,我们使用携带完整 hDEC2\mathrm{h} D E C 2 基因的人细菌人工染色体(BAC)克隆(RP 11 - 288 E19)产生WT和P385 R DEC 2转基因(Tg)小鼠以测试该假设。由于 DEC2D E C 2 已被确立为生物钟 (9,14)(9,14) 的组成部分,我们首先着手确定DEC 2-P385 R小鼠的昼夜节律周期( tau\tau )。平行检测Dec 2缺失小鼠[敲除(KO)小鼠](10)和WT同窝小鼠作为对照。在不同基因型的小鼠中未检测到 tau\tau 的显著差异(表S1)。
Because the mutation was identified in human short sleepers who, presumably, have cor- 因为这种突变是在短睡眠者中发现的,据推测,他们有心脏病-
respondingly longer total daily activity periods, we next determined the duration of the activity period ( alpha\alpha ) for these mice. DEC2-P385R mice retained the WT pattern of rest and activity (running primarily during the dark phase). However, alpha\alpha was ∼1.2\sim 1.2 hours longer for DEC2-mutant transgenic mice (Fig. 2A) than for wild-type mice, DEC2-WTD E C 2-W T Tg mice, and Dec 2 KO mice, which suggests that the expression of the DEC2P385R allele leads to a dominant increase in the quantity of wakefulness in mice. In agreement with this notion, the alpha\alpha was lengthened further ( ∼2.5\sim 2.5 hours) when the endogenous Dec2 alleles were removed by crossing DEC2-P385R mice onto the Dec 2 KO background. 相应地,更长的总每日活动期,我们接下来确定这些小鼠的活动期( alpha\alpha )的持续时间。DEC 2-P385 R小鼠保留了休息和活动的WT模式(主要在黑暗期跑步)。然而,DEC 2突变转基因小鼠的 alpha\alpha 比野生型小鼠、 DEC2-WTD E C 2-W T Tg小鼠和Dec 2 KO小鼠长 ∼1.2\sim 1.2 小时(图2A),这表明DEC 2 P385 R等位基因的表达导致小鼠觉醒量的显性增加。与此观点一致,当通过将DEC 2-P385 R小鼠与Dec 2 KO背景杂交去除内源性Dec 2等位基因时, alpha\alpha 进一步延长( ∼2.5\sim 2.5 小时)。
To study sleep directly (versus activity rhythms) and to investigate a possible role for DEC2D E C 2 in sleep-quantity regulation, electroencephalography (EEG) and electromyography (EMG) were performed. Because we did not observe a change in alpha\alpha for DEC2-WTD E C 2-W T Tg mice (Fig. 2A) and because human mutation carriers have one normal allele with one mutant allele, we chose to perform EEG and EMG on DEC2-P385R mice and their WT littermates. Mice of both genders (female:male/ 1:1) were included in all EEG studies to exclude the possibility of sex differences noticed in other reports (15). DEC2-P385R mice were awake (as defined by EEG) ∼8%\sim 8 \% longer than WT mice in the light phase (Fig. 2B, table S2). The shortsleep phenotype of these mice was reflected in 为了直接研究睡眠(相对于活动节律)并研究 DEC2D E C 2 在睡眠量调节中的可能作用,进行了脑电图(EEG)和肌电图(EMG)。因为我们没有观察到 DEC2-WTD E C 2-W T Tg小鼠的 alpha\alpha 的变化(图2A),并且因为人突变携带者具有一个正常等位基因和一个突变等位基因,所以我们选择对DEC 2-P385 R小鼠及其WT同窝仔进行EEG和EMG。所有EEG研究均包括两种性别的小鼠(雌性:雄性/ 1:1),以排除其他报告中注意到的性别差异的可能性(15)。DEC 2-P385 R小鼠在光相比WT小鼠清醒(如EEG所定义)时间长 ∼8%\sim 8 \% (图2B,表S2)。这些小鼠的短睡眠表型反映在
the significant shortening of both non-rapid eye movement (NREM) and rapid eye movement (REM) during sleep in the light phase for DEC2D E C 2 P385R when compared with control mice (Fig. 2C and table S2). NREM sleep was ∼6%\sim 6 \% less and REM sleep was ∼2%\sim 2 \% less in DEC2-P385R versus WT mice during the light phase. Sleep architecture was further characterized by counting sleep and wakefulness episodes. Over a 12-hour period, DEC2-P385R mice showed more episodes of wakefulness than WT mice (193 +-12\pm 12 与对照小鼠相比, DEC2D E C 2 P385R在睡眠期间在光相的非快速眼动(NREM)和快速眼动(REM)均显著缩短(图2C和表S2)。与WT小鼠相比,DEC 2-P385 R小鼠在光相期间的NREM睡眠少 ∼6%\sim 6 \% ,REM睡眠少 ∼2%\sim 2 \% 。睡眠结构的进一步特点是计数睡眠和觉醒事件。在12小时的时间内,DEC 2-P385 R小鼠比WT小鼠表现出更多的觉醒发作(193
versus 133+-10,P < 0.05133 \pm 10, P<0.05 ), but the mean duration of each episode was slightly shorter during the light phase ( 97+-10s97 \pm 10 \mathrm{~s} versus 116+-12,P <116 \pm 12, P< 0.05 ) (Fig. 2D and table S3). Consistent with this, DEC2-P 385 RD E C 2-P 385 R mice also showed more NREM episodes during light periods (190 +-10\pm 10 versus 139+-9,P < 0.05139 \pm 9, P<0.05 ) but each episode was shorter ( 118+-3s118 \pm 3 \mathrm{~s} versus 184+-5,P < 0.05184 \pm 5, P<0.05 ) (Fig. 2 E and table S3). REM episodes were similar in abundance ( 41+-541 \pm 5 versus 53+-653 \pm 6 ) and duration ( 63+-3s63 \pm 3 \mathrm{~s} versus 64+-364 \pm 3 ) for DEC2-P 385 RD E C 2-P 385 R 与 133+-10,P < 0.05133 \pm 10, P<0.05 相比),但在光照期,每次发作的平均持续时间略短( 97+-10s97 \pm 10 \mathrm{~s} 与 116+-12,P <116 \pm 12, P< 0.05)(图2D和表S3)。与此一致, DEC2-P 385 RD E C 2-P 385 R 小鼠在光照期也显示出更多的NREM发作(190次, +-10\pm 10 对 139+-9,P < 0.05139 \pm 9, P<0.05 ),但每次发作较短( 118+-3s118 \pm 3 \mathrm{~s} 对 184+-5,P < 0.05184 \pm 5, P<0.05 )(图2 E和表S3)。对于 DEC2-P 385 RD E C 2-P 385 R ,REM发作在丰度( 41+-541 \pm 5 与 53+-653 \pm 6 )和持续时间( 63+-3s63 \pm 3 \mathrm{~s} 与 64+-364 \pm 3 )方面相似
Table 1. Sleep schedule comparison for human subjects. Age refers to when data were collected. Status: C, mutation carrier; NC, nonmutation carrier. Sleep offset is local standard clock time of “average” final morning awakening, and sleep onset is evening time of first falling asleep as stated by individuals recalling extended vacations based on structured interviews. Values are +-\pm SD. 表1.人类受试者的睡眠时间表比较。年龄是指收集数据的时间。状态:C,突变携带者; NC,非突变携带者。睡眠偏移是“平均”最后早晨醒来的当地标准时钟时间,而睡眠开始是根据结构化访谈回忆延长假期的个人所述的第一次入睡的晚上时间。数值为 +-\pm SD。
^(1){ }^{1} Department of Neurology, University of California at San Francisco, Mission Bay, 1550 Fourth Street, San Francisco, CA 94158, USA. ^(2){ }^{2} Department of Neurology, University of Utah, Salt Lake City, UT 84132, USA. ^(3){ }^{3} Sleep and Circadian Neurobiology Laboratory, Stanford University, 1201 Welch Road, P213, Palo Alto, CA 94304, USA. ^(4){ }^{4} Mechanical Engineering, University of California, Berkeley, Hesse Hall, Room 245, Berkeley, CA 94720, USA. ^(5){ }^{5} Max Planck Institute of Experimental Medicine, 37075 Göttingen, Germany. ^(1){ }^{1} 美国弗朗西斯科第四大街1550号使命湾,弗朗西斯科,加州大学神经病学系,邮编:94158。 ^(2){ }^{2} 美国湖城犹他州大学神经病学系,邮编84132。 ^(3){ }^{3} 睡眠和昼夜神经生物学实验室,斯坦福大学,1201韦尔奇路,P213,帕洛阿尔托,CA 94304,美国。 ^(4){ }^{4} 机械工程,加州大学伯克利分校,黑森厅,245室,伯克利,CA 94720,美国。 ^(5){ }^{5} 马克斯普朗克实验医学研究所,37075哥廷根,德国。
*Present address: Model Animal Resource Center, Nanjing University, China 210061. * 现地址:南京大学模式动物资源中心,邮编:210061。
†Present address: Department of Pediatrics, Baylor College of Medicine, Texas Children’s Hospital, Houston, TX 77030, USA. ‡\ddagger To whom correspondence should be addressed. E-mail: ying-hui.fu@ucsf.edu †现住址:美国德克萨斯州休斯顿贝勒医学院儿童医院儿科,邮编:77030。#000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000电子邮件:ying-hui. ucsf.edu