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The Transcriptional RepressorDEC2 Regulates Sleep Lengthin Mammals
Christopher R. Jones,
Jimmy L. Holder Jr.,
Moritz J. Rossner,
Sleep deprivation can impair human health and performance. Habitual total sleep time andhomeostatic sleep response to sleep deprivation are quantitative traits in humans. Genetic locifor these traits have been identified in model organisms, but none of these potential animalmodels have a corresponding human genotype and phenotype. We have identified a mutation in atranscriptional repressor (hDEC2-P385R) that is associated with a human short sleep phenotype.Activity profiles and sleep recordings of transgenic mice carrying this mutation showedincreased vigilance time and less sleep time than control mice in a zeitgeber time
dependent manner. These mice represent a model of human sleep homeostasis thatprovides an opportunity to probe the effect of sleep on human physical and mental health.
lthough sleep is an essential process for life, the brain circuits regulating sleep andthe cellular and/or molecular mechanismsinvolved in this complex process are still enigmatic(
). Sleep or a
behavior is present invirtually every animal species where it has beenstudied. Total sleep deprivation can be fatal, and partial deprivation of sleep has serious con-sequences on cognition, mood, and health (
).It is obvious that situational increases in behavioraldrive can transiently delay sleep, but very little isknown about chronic partial sleep curtailment as a possible consequence of a persistent elevation inwaking behavioral drive. The latter trait, sometimesreferred to as a
), isa theoretical third influence on sleep habits.Murine Dec2 (mDec2) is a negative compo-nent of the circadian clock (
). It belongs to a basic helix-loop-helix (bHLH) protein family inwhich members can dimerize with each other andcan affect gene transcription by binding to specificDNA sequences (
). While performing candidategene resequencing in DNAs from human fami-lies, segregating alleles for extremely early wakeup times, we identified an h
point mutationin a small family with two affected individuals(Fig. 1A) (
). Subjects carrying this mutationhad lifelong shorter daily sleep times than normalindividuals (Table 1). The self-reported nonwork-day habitual sleep-offset times of the mutationcarriers were much earlier than those of the non-carriers (including noncarrier family membersand general controls). However, these two indi-viduals have sleep-onset times that are similar tothat of conventional sleepers. The habitual self-reported total sleep time per 24-hour day wasmuch shorter in mutation carriers (average 6.25hours) compared with the noncarriers (aver-age 8.06 hours) in this family. Thus, they rep-resent
natural short sleepers
who routinelysleep less than individuals with familial advancedsleep-phase syndrome (FASPS) or general con-trols (Table 1). The average total sleep time for American adults on nonworkdays is ~7.4 hours(www.sleepfoundation.org). The mutation changesa C to G in the DNA sequence of
, whichis predicted to cause a proline-to-arginine altera-tion at amino acid position 385 of DEC2 (Fig.1B). This change was not found in over 250control DNA samples. The proline at position385 of DEC2 (P385) is conserved in mammals but not invertebrates. P385 is located in a highlyconserved region within a proline-rich domain of unknown function and is close to the C-terminalhistone deacetylase (HDAC)
interacting region of DEC2 (Fig. 1B). Activity-rest recording in onemutation carrier using 10-day sleep logs with co-incident wrist actigraphy demonstrated an ex-tended active period each day (Fig. 1C).To examine the effect of the P385R mutationon Dec2 repressor activity, a wild-type (WT) or a P385R mDec2 construct was used in a luciferaseassay, and the results showed that P385R atten-uated Dec2 repressive activity of Clk/Bmal1-mediated transactivation (fig. S1A). The reductionin Dec2 repressive activity was moderate com- pared with that of the R57A/K mutations (inwhich arginine 57 was replaced by alanine or lysine) reported before (
). Dec2 was previous-ly shown to preferentially bind to class B E-boxelements (CACGTG) as a homodimer and to re- pressthetranscriptionoftargetgenesinanHDAC-dependent manner (
). The effect of HDAC onthemutantDec2repressionwasthenanalyzedbymonitoring m
promoter-driven luciferase ac-tivity with or without a general HDAC inhibitor trichostatin A (TSA) (fig. S1B). HDAC inhibitionresulted in similar increases in luciferase activityfor both WT and mutant Dec2. Coimmunopre-cipitation was then performed for mDec2 (WTor P385R) and human sirtuin-1 (hSIRT1). HEK293cellsweretransientlycotransfectedwithgreenflu-orescent protein (GFP)
tagged (WT or mutated)m
and FLAG-tagged h
, followed byFLAG-peptide pull-down and detection of GFPwith antibodies on Western blots. The resultsshowed similar physical interactions between WTor P385R mDec2 and hSIRT1 (fig. S1C). Takentogether, these results suggest that the P385R mutation affects Dec2 transcriptional repressionactivity independently from its interaction withHDAC/SIRT.Because there are only two human mutationcarriersinthisstudy,thequestionremainedwhether the natural short sleep phenotype was caused bythe
mutation. Thus, we generated WTandP385R
transgenic (Tg) mice using a hu-manbacterialartificialchromosome(BAC)clone(RP11-288E19) carrying the entire h
geneto test this hypothesis. As
has been estab-lished as a component of circadian clock (
),we first set out to determine the circadian period(
deleted[knockout (KO) mice] (
) and WT littermateswere tested in parallel as controls. No significant differences in
were detected among mice of dif-ferent genotypes (table S1).Because the mutation was identified in hu-man short sleepers who, presumably, have cor-
Department of Neurology, University of California at SanFrancisco, Mission Bay, 1550 Fourth Street, San Francisco,CA 94158, USA.
Department of Neurology, University ofUtah, Salt Lake City, UT 84132, USA.
Sleep and CircadianNeurobiology Laboratory, Stanford University, 1201 WelchRoad, P213, Palo Alto, CA 94304, USA.
Mechanical Engi-neering, University of California, Berkeley, Hesse Hall, Room245, Berkeley, CA 94720, USA.
Max Planck Institute of Ex-perimental Medicine, 37075 Göttingen, Germany.*Present address: Model Animal Resource Center, NanjingUniversity, China 210061.
Present address: Department of Pediatrics, Baylor College ofMedicine, Texas Children
s Hospital, Houston, TX 77030, USA.
To whom correspondence should be addressed. E-mail:firstname.lastname@example.org
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