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Timing of expression of the core clock gene Bmal1influences its effects on aging and survivalGuangrui Yang,1* Lihong Chen,1* Gregory R. Grant,1,2 Georgios Paschos,1 Wen-Liang Song,1

Erik S. Musiek,3 Vivian Lee,4 Sarah C. McLoughlin,1 Tilo Grosser,1 George Cotsarelis,5

Garret A. FitzGerald1†

The absence of Bmal1, a core clock gene, results in a loss of circadian rhythms, an acceleration of aging, and ashortened life span in mice. To address the importance of circadian rhythms in the aging process, we generatedconditional Bmal1 knockout mice that lacked the BMAL1 protein during adult life and found that wild-type circadianvariations in wheel-running activity, heart rate, and blood pressure were abolished. Ocular abnormalities and brainastrogliosis were conserved irrespective of the timing of Bmal1 deletion. However, life span, fertility, body weight,blood glucose levels, and age-dependent arthropathy, which are altered in standard Bmal1 knockout mice, remainedunaltered, whereas atherosclerosis and hair growth improved, in the conditional adult-life Bmal1 knockout mice, de-spite abolition of clock function. Hepatic RNA-Seq revealed that expression of oscillatory genes was dampened in theadult-life Bmal1 knockout mice, whereas overall gene expression was largely unchanged. Thus, many phenotypes inconventional Bmal1 knockout mice, hitherto attributed to disruption of circadian rhythms, reflect the loss of proper-ties of BMAL1 that are independent of its role in the clock. These findings prompt reevaluation of the systemicconsequences of disruption of the molecular clock.

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INTRODUCTION

Circadian rhythms are biological processes that display endogenousand entrainable oscillations of about 24 hours. They are driven by agroup of clock genes that are widely conserved across plants, animals,and bacteria (1). In mammals, the core clock genes, including Bmal1,Clock, Cry, and Per, are rhythmically expressed in both the suprachias-matic nucleus (SCN)—the master clock that resides in the hypothalamus—and almost all peripheral tissues, in which they control the expressionof numerous target genes in a circadian manner, influencing manyphysiological and biochemical processes (2). Chronic disruption of cir-cadian rhythms has been associated with cardiometabolic morbidity inhumans (3). In mice, deletion of Bmal1 prenatally disrupts clock-dependent oscillatory gene expression and behavioral rhythmicitycoincident with reduced body weight, impaired hair growth, abnormalbone calcification, eye pathologies, neurodegeneration, and a shortenedlife span (4–7). However, although Bmal1 is the sole nonredundant genein the core molecular clock, the degree to which phenotypes expressedin conventional knockout (cKO) mice reflect disruption of clock func-tion is unknown.

Here, we describe the characterization of inducible Bmal1 KO(iKO) mice that express the gene during embryogenesis but not afterbirth. Despite ablation of clock function in both iKO and cKO mice,we observed striking physiological differences between the two modelsystems, prompting reevaluation of the systemic consequences of dis-ruption of the molecular clock.

1The Institute for Translational Medicine and Therapeutics, Perelman School of Med-icine, University of Pennsylvania, Philadelphia, PA 19104, USA. 2Department of Genet-ics, University of Pennsylvania, Philadelphia, PA 19104, USA. 3Hope Center forNeurological Disorders, Washington University School of Medicine, St. Louis, MO63110, USA. 4Department of Ophthalmology, University of Pennsylvania Scheie EyeInstitute, Philadelphia, PA 19104, USA. 5Department of Dermatology, Perelman Schoolof Medicine, University of Pennsylvania, PA 19104, USA.*These authors contributed equally to this work.†Corresponding author. E-mail: garret@upenn.edu

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RESULTS

Loss of circadian rhythmsBmal1 deletion in various tissues of the iKO mice (Bmal1f/f-EsrCre),including brain, heart, lung, liver, kidney, spleen, skeletal muscle, andepididymal fat, was confirmed by quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR). The iKO mice showed an80% (liver) to 99% (brain and muscle) reduction of Bmal1 mRNAlevels at Zeitgeber time 0 (ZT0) when Bmal1 expression is high (fig.S1A). Disruption of circadian behavior in iKOs was confirmed usingrunning wheels. Before tamoxifen treatment, the Bmal1f/f-EsrCre miceshowed normal rhythmic locomotor activities under both 12-hour/12-hour light/dark (LD) and constant darkness (DD), which is in-distinguishable from their Cre− Bmal1f/f control littermates (Fig. 1A).Whenmice were administered tamoxifen, Bmal1f/f mice (Ctrl) maintainedtheir circadian behavior under DD, whereas all Bmal1f/f-EsrCre mice lostrhythmicity immediately after the treatment (Fig. 1A), which is similar tocKO mice (fig. S2A). Loss of circadian behavior in iKOs was still evident15 months after Bmal1 deletion (Fig. 1C), suggesting the permanent dis-ruption of circadian rhythms. The reduction in overall locomotor ac-tivity in cKOs (fig. S2B) (8) was not recapitulated in iKOs (Fig. 1, B andD), indicating that adult-life deletion of Bmal1 does not predisposemice to the usual age-related decline of wheel-running activity (9).

Consistent with disruption of core clock function, diurnal variation inheart rate (HR) and blood pressure (BP) was lost in iKOs (Fig. 1E), andcircadian expression of hepatic clock genes was dampened (Fig. 1F). Thevariation of clock gene expression between ZT0 and ZT12 was alsodampened in other tissues in iKOs (fig. S1B). Thus, behavioral, physiolog-ical, and molecular evidence for molecular clock disruption was present inthe iKOs, consistentwithwhat haspreviously been reported in cKOs (8,10).

Conserved life span, weight, fertility, and blood glucoseDespite permanent loss of circadian rhythmicity in iKOs, the micehad a normal average life span of more than 2 years (Fig. 2A and

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fig. S3A). By contrast, the average life span of cKOs was just 9 months(fig. S3B) (6). Except for ocular abnormalities, the iKO mice generallyexhibit no gross morphological defects, and body weight was con-served in both genders (Fig. 2B and fig. S2C). Similarly, the weightof organs examined in the iKOs did not differ from controls, exceptfor the liver at 2 months after Bmal1 deletion (Fig. 2C). Although iKOmice are less fertile than normal mice (tamoxifen untreated), the fer-tility percentage was comparable to their tamoxifen-treated littermatecontrols (36% versus 30% in male, and 22% versus 27% in female; Fig.2D), suggesting that the defect in fertility resulted from the tamoxifentreatment, not the consequent gene deletion or disruption of circadianrhythms. In contrast, the cKOs were completely sterile (fig. S2D). Glu-cose tolerance tests (GTTs) and insulin tolerance tests (ITTs) did notdiffer between Ctrls and iKOs (Fig. 2E).

Hair growth and arthropathyLoss, graying, and growth inactivity of hair (telogen) are hallmarks ofaging (11, 12). Indeed, conventional Bmal1 and Clockmutant mice dem-onstrate an increase in telogen follicles compared to controls (6, 13).Here, in an assay to assess hair follicle cycling in which hair is shaved

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and new hair regrowth is assessed (Fig. 3A), cKOs were shown not toenter the hair growth phase (anagen) as frequently as did wild-type mice(Fig. 3C), as previously reported. Unexpectedly, however, hair follicles iniKO mice entered anagen more frequently than did controls (Fig. 3B)even in aged (6 months and 1.5 years) mice (Fig. 3D). In young cKOmice, anagen follicles were absent 7 weeks after shaving (Fig. 3B), indi-cating that the follicles stayed in telogen. Anagen was also rare in wild-type and Ctrl mice; however, two-thirds of the iKO mice entered anagenat 7 weeks after shaving (Fig. 3B). Similarly, 12 weeks after shaving, allwild-type, Ctrl, and iKOmice had entered anagen, but more than half ofthe cKOs still had not done so (Fig. 3C). Expression of genes associatedwith anagen (Ccnd1 and Mki67) was up-regulated in iKOs comparedwith controls (fig. S4).

Accelerated age-related arthropathy has been reported in cKOsand was studied here using Alizarin Red–stained ribcages and hind-limbs, as previously described (4, 5). As expected, we observed ab-normal calcification in the costosternal junctions and calcanealtendons of all cKOs (n = 5) at 24 weeks of age (Fig. 3E). By contrast,abnormal calcification was not evident in any 30-week-old iKOs (n =5, 24 weeks after Bmal1 deletion). Calcification of the calcaneal tendon

Fig. 1. Loss of circadian rhythms in iKO mice. (A) Representative double-plotted actograms of wheel-running activity of 3-month-old Bmal1f/f and

(D) Counts of wheel revolutions from 18-month-old Ctrls and iKOs under DD(n = 6 to 7; Student’s t test). (E) Representative radiotelemetry results of loco-

Bmal1f/f-EsrCremice (red dots, tamoxifen treatment). Similar results were ob-tained in n = 8 to 9mice per group. (B) Counts of wheel revolutions per hourfrom control mice (Ctrls) and iKO mice under conditions of DD (n = 8 to 9,Student’s t test; ns, no significant difference). (C) Representative double-plotted actograms of wheel-running activity from 18-month-old Ctrl andiKOmice under DD. Similar results were obtained in n= 6 to 7mice per group.

motor activity, systolic BP (SBP), and HR in Bmal1f/f and Bmal1f/f-EsrCre mice(red inverted triangles, tamoxifen treatment). Similar results were obtainedin n = 3 mice per group. (F) Hepatic mRNA levels of canonical clock genesand clock-controlled geneDbpwere determined by qRT-PCR [n= 4 per geno-typeper timepoint; x axis, circadian time (CT); y axis, relativemRNA levels; *P<0.05; **P < 0.01; ***P < 0.001, two-way analysis of variance (ANOVA)].

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was observed in some 48-week-old iKO mice (3 of 8, 24 weeks afterBmal1 deletion) but was indistinguishable from that in littermate con-trols (4 of 10).

Differential impact on atherogenesisThe molecular clock has been implicated in various aspects ofcardiovascular function, including BP homeostasis and thrombogen-esis (14). Atherogenesis is accelerated in ClockD19/D19 mutant mice(15). Here, we compared conventional and adult-life Bmal1 deletionin mice on a hyperlipidemic low-density lipoprotein receptor–deficient(LDLR−/−) background that were fed a high-fat diet (HFD). Similar toClockD19/D19 mutant mice, en face aortic lesion analysis revealedmarked acceleration of atherogenesis in both male and female cKOs

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(Fig. 4A), whereas, unlike ClockD19/D19 mutant mice, cKOs show plas-ma total cholesterol and triglyceride levels comparable to those ofwild-type mice (fig. S5A). By contrast, atherogenesis relative to Ctrlmice was retarded when Bmal1 was deleted in adulthood (Fig. 4B).Adult-life deletion of Bmal1 did not affect BP or HR, at least at ZT8to ZT9 (fig. S5B), whereas the increase in body weight in HFD-fed micewas reduced in male iKOs. Female iKOs showed a trend toward a re-duction in body weight (fig. S5C). Consistently, aortae from iKOs showedreduced expression of proinflammatory genes, including CD68 (clusterof differentiation 68),MCP-1 (monocyte chemotactic protein 1), and iNOS(inducible nitric oxide synthase), compared to controls (fig. S5D). Amongthese genes,MCP-1 is a direct target gene of Bmal1, as revealed by ChIP-Seq (chromatin immunoprecipitation sequencing) in mouse liver (16).

Fig. 2. General status of iKO mice. (A) Life span of Ctrls and iKOs withmedians of 745 and 765 days, respectively (P = 0.9852, log-rank test).

tion). (D) Fertility analysis in both male and female Ctrl, iKO, and untreatedCre+ mice (**P < 0.01; ***P < 0.001, c2 test). (E) Blood glucose concen-

TAM, tamoxifen. (B) Body weight of Ctrls and iKOs for both genders(Student’s t test). (C) Ratio of organ to body weight in 5- and 11-month-old Ctrls and iKOs (**P < 0.01, multiple t tests with Holm-Sidak correc-

trations of Ctrls and iKOs that underwent GTT and ITT (n = 6 to 7; two-way ANOVA; no significant difference between Ctrls and iKOs at anytime point).

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Ocular abnormalities and astrogliosisDespite the differential effects on hair regrowth, arthropathy, and ath-erogenesis, ocular abnormalities similar to those observed with prenatalBmal1 depletion were evident in the iKOs (Fig. 5, A to D). By 8 monthsof age (5 months after Bmal1 deletion), about 80% of iKOs developedcloudy changes in one or both eyes, whereas none of their littermatecontrols displayed any visible abnormalities. Histological examinationrevealed various pathological changes, including corneal neovascularization,keratinization, and progressive inflammation in the iKOs.

We have reported that cKOs and neural-specific Bmal1 KO mice(Bmal1f/f-NestinCre; nKO) exhibit severe astrogliosis (7). Here, we ex-amined the brains of 11- to 12-month-old iKO mice and found them tobe nearly indistinguishable from those of cKO mice (aged 6 months) ornKOmice (aged 11 to 12 months), with undetectable BMAL1 immuno-reactivity in neurons and severe astrogliosis, whichwas reflected byGFAP(glial fibrillary acidic protein) staining throughout the cerebral cortex (Fig.5E). Transcriptional analysis revealed similar changes inGfapmRNA ex-pression (fig. S6).However, suchneurodegenerative changesdidnot affect

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the life spans of iKOs (Fig. 2A) or nKOs (fig. S3C). It is not surprising thatthis pathological brain phenotype did not lead to premature death, be-cause similar levels of inflammation and synapse loss are observed insome mouse models of amyloid b–mediated cerebral amyloidosis,and these mice have normal life spans (17).

Loss of the circadian transcriptomeTo characterize gene expression profiles, we performed RNA-Seq usingsamples of liver, where most circadian genes are expressed (18). Asexpected, all clock genes oscillated in control mice, but were expressedat relatively constant levels in iKOs (fig. S7). This expression patternwashighly consistent with the qRT-PCR results (Fig. 1F). A circadian analysiswas performed using JTK (Jonckheere-Terpstra-Kendall)_CYCLEalgorithm (19) to explore 24-hour oscillations in transcript abundance.We used a 5% false discovery rate (FDR) threshold to detect circadiangenes in control mice and found that 14.5% of genes (5457 among37681; data file S1) were rhythmically expressed (Fig. 6A). By contrast,even those genes that top the list in control mice displayed no circadian

Fig. 3. Hair cycling and arthropathy. (A) Six-week-old Bmal1f/f andBmal1f/f-EsrCre mice were treated with tamoxifen. Meanwhile, dorsal hair

strains. The hair regrowthwas observed 7weeks (B) or 12weeks (C) after hairshaving.WT,wild type. (D) Hair regrowth assay in 6-month-old and1.5-year-old

was shaved. Pictures were taken 7 weeks after hair shaving. Mice with obvi-ous hair regrowth are highlightedwith orange boxes. (B and C) Contingencybar graphs show frequencydistribution of hair regrowth inboth cKOand iKO

mice. Thehair regrowthwasobserved7weeksafterhair shaving. (E) Represent-ative photographs of Alizarin Red–stained ribcages andhindlimbs frombothcKO and iKO strains. Blue circles indicate calcification.

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expressionpattern in iKOs (table S1).Only one gene,Erh, showed circadianvariability at the mRNA level in iKOs (Fig. 6B), illustrating how few genesare rhythmically expressed independent of the classic circadian clock.

Phenotype enrichment analysis ofdifferentially expressed genesTo understand the potential reasons why there is a striking phenotypicdifference between iKOs and cKOs despite a shared ablation of circadianrhythms, we compared hepatic gene expression profiles between wild-type versus cKOs and Ctrl versus iKOs, irrespective of time (data fileS2). An FDR threshold of 5% was used for detection of differentiallyexpressed genes for both KO strains, although the specific value of thiscutoff did not alter the fact that the iKOs had many fewer differentiallyexpressed genes than did the cKOs (table S2); from these analyses, wefound 462 differentially expressed genes in cKOs and 3 in iKOs (Fig.6C). Enrichment analysis of these 462 genes revealed multiple potentialphenotypes that are consistent with what might be expected, includingabnormal cardiovascular function, induced morbidity and mortality, ab-normal skin morphology, abnormal metabolism, abnormal survival, ab-normalmuscle physiology, and abnormal immune function (Fig. 6D anddata file S3). However, only three genes in the iKOs (without overlapwiththe cKOs) were identified as differentially expressed (Fig. 6, C and E). En-richment analysis showed that no category reached the cutoff (P < 0.05),perhaps because of the small number of genes involved (data file S3).

DISCUSSION

Among canonical clock genes, Bmal1 is the only one whose deletionresults in complete loss of circadian rhythms (8); therefore, Bmal1 KO

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mice have become a valuable resource for study of the effects of circa-dian disruption. Other than loss of circadian rhythms, however, thesemice also exhibit a greatly reduced life span and develop various featuresconsistent with premature aging (4–7). Because of the absence of Bmal1expression during development in cKO mice, it is not clear whetherthese phenotypes relate to its absence during embryogenesis or postnataldevelopment or, indeed, whether these phenotypes reflect disruption ofthe molecular clock or loss of other properties of BMAL1. Here, wecompared iKOs with cKOs and found that both approaches to genedeletion similarly disrupt behavioral and transcriptional indicators ofclock-dependent circadian function, and both types of KO mice exhibitmarkers consistent with accelerated aging [such as ocular abnormalitiesand brain astrogliosis (table S3)]. In contrast, other biomarkers of agingand premature death, so evident in cKOs, including a markedlyshortened life span, infertility, reduced body weight, organ shrinkage(6), impaired glucose homeostasis (20, 21), and abnormal bone calcifi-cation (4), are not replicated in the iKOs. Further, two aging-relatedphenotypes, hair growth inactivity and a predisposition to athero-sclerosis, were reversed in iKOs. To characterize Bmal1 KO mice atthe level of gene expression as well as to determine potential mecha-nisms underlying the general difference between cKO and iKO strains,we performed RNA-Seq using liver samples. Although the various phe-notypes may not be explained simply by changes of the transcriptome inany given tissue, liver affords an appropriate initial opportunity to ad-dress this question, because it expresses most circadian genes (18). Alltime-effect genes were dampened in iKO mice without obvious changesin time-independent genes. These striking differential effects betweenthe two strains of mice, despite a shared ablative effect on clock-dependentcircadian biology, question the attribution of many phenotypes in thewidely used cKOs to disruption of the molecular clock.

Fig. 4. HFD-induced atherosclerosis. (A and B) Both cKO (A) and iKO(B) mice and their littermate controls were in an LDLR−/− background

dissected and stained with oil red O. Plaque areas (shown in red) werequantified with Image-Pro. The percentage of plaque area relative to

and fed an HFD (42% kcal from fat) from 12 weeks of age. Sixteen weeksafter HFD treatment, mice were euthanized and whole aortas were

the entire area was calculated (*P < 0.05; **P < 0.01; ***P < 0.001, Student’st test).

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Circadian recruitment of clock proteins including BMAL1 to DNAbinding sites is believed to contribute to the oscillation of target genes(16). However, Bmal1 is expressed in mice throughout embryogenesis(22) but does not oscillate, either in the SCN (23, 24) or in peripheral tissues(25), suggesting that clock regulatory loopsmaynot bewell establisheduntillate in development. Hence, Bmal1 expressionmight exert important non-clock functions during early development, and its deletion during this pe-riod might account for premature aging and various related phenotypesobserved in the adult mice. Alternatively, the physiologic effects of Bmal1may vary across tissues and defects in one tissuemay compensate for thosein another when analyzing multi-tissue mutants. This possibility is not ad-dressed in the present study. However, although shortened life span has yetto be reported in any prenatal tissue-specific Bmal1KOmice, some do ex-hibit phenotypes that are not evident in adult-life global KOs. For example,impaired glucose tolerance was reported in prenatal hepatocyte–specific(19, 25) and pancreatic b cell–specific (26) Bmal1 KO mice. This is con-sistent with cKOs, but is not seen in adult-life global KOs.

To our surprise, our cKO and iKO mice showed opposite pheno-types in hair growth and atherogenesis. We found increased growing

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follicles in all young, middle-aged, and old iKO mice as well as con-sistent expression of hair growth–promoting genes Ccnd1 and Mik67in the skin. ChIP-Seq has revealed that theCcnd1 gene contains bindingsites for all core clock proteins including BMAL1 (16), which impliesthatCcnd1 is directly clock-controlled. However, its expression in cKOswas not changed. It is possible that this regulation is masked and hairgrowth is retarded by the overall premature aging phenotype. Pan et al.found impaired cholesterol metabolism and enhanced atherosclerosisin HFD-fed Clock mutant mice generated in an LDLR−/− background,implicating the circadian clock in atherogenesis (15). Consistent withtheir finding, we observed increased atherogenesis in Bmal1 cKOs.However, unlike the case for Clock mutant mice, Bmal1 deletion doesnot alter cholesterol metabolism, which suggests that disruption ofthe two clock-directed genes influences atherogenesis by distinct themechanisms. By contrast, adult-life Bmal1 depletion restrains athero-genesis coincident with dampened expression of proinflammatorygenes in the vasculature and a reduction in the weight gain inducedby the HFD. The relative contribution of local and systemic effects tothis phenotype remains to be determined. Ocular abnormalities and

Fig. 5. Ocular abnormalities and astrogliosis in iKO mice. (A toD) Rep-resentative gross images (left) and hematoxylin and eosin (H&E)–stained

ened. The retina is adherent to the lens. The corneal epithelium is attenuated(right, arrow) with chronic inflammation and neovascularization in the stroma.

sections of eyes (middle and right) from Ctrl (A) and iKOs (B to D). (A) Un-remarkable globe from a Ctrl mouse. AC, anterior chamber; Co, cornea; Ir, iris;Le, lens; Re, retina. (B) Pathologic changes in amalemouseeye. Grossly, there isa leukoplakic plaque on the cornea (left, arrow). Histologically, the corneaappears thickened with keratinization of the epithelium (right, arrow) andchronic inflammation and neovascularization of the stroma (right, arrowhead).(C) In the contralateral eye of the same mouse, the corneal surface appearsirregular with a flattened chamber. Histologically, the cornea appears thick-

A subcapsular anterior cataract is present (right, arrowhead). (D) A femalemouse eye shows an irregular corneal surface with leukoplakia (left, arrow)and corneal neovascularization (right, arrow). (E) Immunofluorescence stainingof 4′,6-diamidino-2-phenylindole (DAPI) (all nuclei), NeuN (neuronal nuclei),BMAL1, and GFAP (activated astrocytes) in the motor cortex from cKO, iKO,and nKO mouse strains. Representative images show loss of Bmal1 immuno-reactivity in all KOs,which is accompaniedby severe astrogliosis (GFAPpanels).Similar results were obtained in n = 5 mice per group. Scale bar, 150 mm.

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brain astrogliosis were preserved in iKO mice, consistent with Bmal1exerting a direct role in the eye and neural system irrespective of devel-opmental issues. However, it remains unknown if these phenotypes arecaused by loss of circadian rhythms or loss of nonclock functions ofBmal1, because deletion of core clock genes not only disrupts rhythmic-ity of genes relevant to expression of these phenotypes but also mayhave striking consequences on their overall expression levels (26, 27).

The apparent restraining influence of Bmal1 expression duringembryogenesis on aging-related phenotypes and, indeed, life span inthe adult mice is reminiscent of the Barker hypothesis, which postu-lated that factors impinging on fetal development would influence theexpression of aging and life span in adult humans (28). Insight intothe impact of environmental factors and maternal behavior, such assmoking and alcohol consumption, on postnatal health has suggestedan epigenetic basis for this hypothesis (29). Here, in the case of Bmal1,

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we raise the additional possibility of a direct genetic effect on suchphenomena. A provocative consideration is that the anticipatory roleof the clock might condition the impact of environmental insults, at-tenuating them at certain times of day.

In summary, despite a shared impact on circadian function, manyphenotypes observed in conventional KOs are not observed whenBmal1 is deleted in adultmice. This prompts reevaluation of the system-ic role of the molecular clock and its importance in the biology of aging.

MATERIALS AND METHODS

Study designThe primary objective of this study was to investigate the role of cir-cadian rhythms in aging in adult mice. We used Bmal1-inducible KO

Fig. 6. Hepatic transcriptome. RNA samples from iKOs and their litter-mates under DD condition were used for RNA-Seq. (A) Of the 37,681 tran-

shown [x axis, circadian time; y axis, fragments per kilobase of transcript permillion mapped reads (FPKM)]. (C) Venn diagram (sizes not to scale) depict-

scripts, 5457 exhibited circadian variability in Ctrl mice. By contrast, only onegene in iKOs exhibited a circadian pattern. Heat map rendering of the tem-poral gene expression pattern of these 5457 circadian genes is shown withthe average gene expression level measured for each time point. (B) Erh isthe only hepatic gene that shows a circadian expression pattern in iKOs, andthe q value (JTK_CYCLE analysis) and fold change (ratio of peak/trough) are

ing the number of differentially expressed genes in cKO and iKO strains.(D) Mouse Genome Informatics Mammalian Phenotype Level 3 enrichmentanalysis for cKOdifferentially expressedgenes. The top 17 (adjusted P<0.05)phenotypes related to these genes are given. Overlap, number of appearinggene/number of background genes. (E)Mup3, Serpina3k, and Apoa1 are theonly differentially expressed genes in the iKO strain.

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mice to bypass possible issues about developmental defects in con-ventional KOs. For all experiments, the number of independent repli-cates is outlined in each figure or legend unless individually plotted.Sample sizes were based on the desired magnitude of the difference tobe detected, and the variance of the estimates was based on previousdata where available and setting a = 0.05 and 1 − b = 0.9. Mice in allexperiments were randomized into various groups. The studies werenot performed under double-blind conditions. The experimenterswere not blinded to the identities of individual groups. Animals werenot withdrawn from the studies according to predetermined criteria inthe Institutional Animal Care and Use Committee (IACUC) pro-tocols, nor were they excluded from any of the analyses.

MiceBoth cKO and Bmal1f/f mice (30) were obtained from C. Bradfield (Uni-versity ofWisconsin,Madison,WI). The floxedmice were crossed with atamoxifen-inducible universal Cre (EsrCre) mice orNestinCremice (TheJackson Laboratory) to yield Bmal1f/f-EsrCremice and Bmal1f/f-NestinCre(nKO) mice, respectively. To generate iKO mice, 3-month-old (unlessspecified) Bmal1f/f-EsrCre mice were treated with tamoxifen as previ-ously described (31). Cre− Bmal1f/f littermates treated with tamoxifenserved as controls (Ctrl). For atherogenesis, all mice were crossed intoan LDLR−/− background. All mice were housed under 12-hour/12-hourLD conditions with free access to food and water unless specified. Allprocedures were approved by the University of Pennsylvania IACUC.

Wheel-running activityYoung (3months, both cKO and iKO strains) and aged (18months, iKOstrainonly)micewere individually housed in cages equippedwith runningwheels (Coulbourn Instruments). Cages were kept within a ventilated andlight-tight isolation chamber with a computer-controlled lighting system.Locomotor activity was continuously recorded and analyzed using Clock-Lab software (Actimetrics). For the young group, mice from both cKO(n = 4 per genotype) and iKO (n = 8 to 9 per genotype) strains werehoused in 12-hour/12-hourLDand then released intoDD.After 1week,Bmal1f/f and Bmal1f/f-EsrCremice were treated with 5 mg of tamoxifenby gavage for five consecutive days followed by recording for 10 days.

Radiotelemetry readingThe implantation of radiotelemetry (model no. TA11PA-C20, DataSciences International) was performed as previously described (31).After 1 week of recovery, BP, HR, and locomotor activity weremonitored continuously in 3-month-old Bmal1f/f (n = 3) andBmal1f/f-EsrCre (n = 3) mice using the Dataquest LabPro AcquisitionSystem. Mice were housed under LD and then released into DD. After1 week, mice were treated with 5 mg of tamoxifen by gavage for fiveconsecutive days followed by recording for 15 days.

Fertility analysisBoth genders were used for fertility analysis. Eight-week-old Bmal1f/f

and Bmal1f/f-EsrCre mice were treated with tamoxifen. At 10 weeksold, each iKO, control, or untreated Cre+ littermate was mated with awild-type C57Bl/6 mouse. All breeding cages were monitored for birthdaily for 9 months.

Glucose tolerance testTwelve-month-old mice (10 months after tamoxifen treatment) werefasted for 16 hours before the GTT. Blood was obtained from a tail cut

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and was assessed for fasting glucose levels using a OneTouch Ultra 2(LifeScan, Johnson & Johnson) glucometer. The mice then received aglucose solution (0.2 g/ml; 2 g/kg body weight) delivered by oral ga-vage. At 15, 30, 60, 90, and 120 min after the administration, dried bloodwas quickly removed from the tail wound and fresh blood was collectedagain to measure the glucose concentration.

Insulin tolerance testTwenty-month-old mice (18 months after tamoxifen treatment) werefasted for 4 hours before ITT. After baseline blood glucose measure-ment, insulin (1 U/kg) (Eli Lilly) was injected intraperitoneally andblood glucose was measured again at time points of 30, 60, 90, and120 min after injection.

Hair regrowth assayTo characterize hair growth, the backs ofmice were shaved and animalsweremonitored for hair regrowth for 3months. For RNA analysis, dor-sal skins from 8-week-old mice (1 week after tamoxifen treatment foriKO strain) were collected for RNA extraction and quantitative RT-PCR analysis for Ccnd1 andMki67.

Bone stainingSkeletons were stained using Alizarin Red as previously described (5)with modifications. Briefly, skinned and eviscerated animal carcasseswere submerged in 1% KOH for 4 hours followed by incubation in 2%KOH overnight. Carcasses were then stained in 2% KOH containing0.004% Alizarin Red (Sigma) for 2 days. After clearing in 2:1:2 glyc-erin/benzyl alcohol/70% ETOH, stained skeletons were photographed.

AtherosclerosisAll mice were on an LDLR−/− background and fed an HFD (42% kcalfrom fat, Harlan Laboratories) for 16 weeks from 12weeks of age. Mouseaortic trees were prepared and stained, and atherosclerotic lesions werequantified as described (32). Briefly, the entire aorta from the aortic rootto the iliac bifurcation was collected and fixed in formalin-free fixativePrefer (AnatechLtd.). Adventitial fatwas removed. The aortawas openedlongitudinally, stainedwith Sudan IV (Sigma), andpinneddownonblackwax to expose the intima. The extent of atherosclerosis was determinedby the en face lesion area percentage to the entire intimal area.

Lipid analysis in plasmaBlood was collected from the vena cava of CO2-euthanizedmice. Plasmatotal cholesterol and triglyceridesweremeasuredby commercial kits fromWako Chemicals.

Tail-cuff BP and HR measurementSBP and HR were measured in conscious mice at ZT8–9 using a tailcuff system (Visitech Systems Inc.) as previously described (33).

Ocular pathologyAnimals were euthanized, and both eyes were harvested and fixed in10% paraformaldehyde solution. After gross examination, globes wereroutinely processed and embedded in paraffin. Sections were obtained,stained with H&E, and reviewed by an ocular pathologist.

Immunostaining of brain sectionsCryosections from the prefrontal cortex to caudal hippocampus wereprepared as previously described (7). Sections were washed in tris-buffered

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saline (TBS), blocked for 30 min in TBS containing 3% donkey serum and0.25% Triton X-100, and then incubated overnight in TBS plus 0.25%Triton X-100 with 1% donkey serum containing rabbit GFAP (Dako),BMAL1 (Novus), ormouse NeuN primary antibodies at 4°C. Sections werewashed, then incubated in TBS containing Alexa Fluor 488–conjugatedanti-rabbit or Alexa Fluor 568–conjugated anti-mouse secondary anti-bodies for 1 hour at room temperature, and then washed again in TBS.Sections were mounted and imaged using an epifluorescent microscope.

Quantitative RT-PCRTotal RNA from various tissues was isolated using the Qiagen RNeasyKit. Reverse transcription was performed using an RNA-cDNA kit (Ap-plied Biosystems). Real-time PCR was performed using ABI TaqManprimers and reagents on an ABI Prism 7500 thermocycler accordingto the manufacturer’s instructions. All mRNA measurements were nor-malized to b-actin mRNA levels.

Sample preparation for RNA-SeqTwenty-four male iKO mice aged 4 to 6 months (2 weeks after tamox-ifen treatment) and their littermates were maintained under 12-hour/12-hour LD conditions and then released into DD condition. Startingat 36 hours after release, four mice per genotype were euthanized indarkness every 4 hours for 20 hours (six time points). Liver sampleswere quickly excised and snap-frozen in liquid nitrogen. Total RNAwas isolated using Qiagen RNeasy Kit, and the RNA integrity waschecked using an Agilent Technologies 2100 Bioanalyzer. RNAsamples were subjected to two rounds of hybridization to oligo(dT)beads. The resulting mRNA was then used as template for double-stranded complementary DNA (cDNA) synthesis. After purification,cDNA samples were fragmented with nebulization technique to yieldfragment sizes of 100 to 300 base pairs and subjected to end-repair,adenylation, ligation of Illumina sequencing adapters, and PCR amplifica-tion. The PCR product was purified and used directly for cluster gen-eration and sequencing (HiSeq 2000).

When comparing iKOs with cKOs irrespective of time points, werandomly picked one mouse per genotype per time point from iKOsand littermates. To match these samples, six male cKO mice and sixlittermate wild-type controls (one mouse per genotype per time point)were euthanized for liver collection. Samples for RNA-Seq wereprepared as described above.

RNA-Seq analysisRNA-Seq data were aligned to the mouse genome build mm9 bySTAR version 2.4.2a (34). Data were normalized using a resamplingstrategy as described previously (35); the code is available at https://github.com/itmat/Normalization. The normalized counts thus pro-duced were then used for the subsequent statistical analyses. JTK_CYCLEwas used to explore 24-hour oscillations in transcript abundance (19).Differential expression analysis was performed by standard permuta-tion methods to control the FDR (36). The list of resulting differen-tially expressed genes was used for enrichment analysis using Enrichr(http://amp.pharm.mssm.edu/Enrichr/). All data have been submittedto the Gene Expression Omnibus (GEO) database (accession nos.GSE70497, GSE70499, and GSE70500).

Statistical analysisAll statistical tests were two-sided. A log-rank test was used for sur-vival curve analysis, and c2 test was used for contingency data. For

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other comparisons except RNA-Seq results, Student’s t test or one-way ANOVA was used when a single variable was compared betweentwo or more genotypes, and two-way ANOVA with Bonferroni’sposttest was used when multiple variables were compared betweengenotypes. The cutoff for significance was P < 0.05 (*). #P < 0.1, **P <0.01, ***P < 0.001. In all figures with error bars, the graphs depictmeans ± SEM.

SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/8/324/324ra16/DC1Fig. S1. Validation of Bmal1 deletion and dampening effect of other clock genes.Fig. S2. Wheel-running activity, body weight, and fertility in cKO mice.Fig. S3. Life span of iKO, cKO, and nKO mice.Fig. S4. mRNA levels of Ccnd1 and Mki67 in skin.Fig. S5. Other parameters in HFD-fed mice.Fig. S6. iKO mice display similar Gfap induction in the brain as cKO.Fig. S7. RNA-Seq results revealed dampening effect in core clock genes in iKO mice.Table S1. Top 20 oscillating hepatic genes (JTK_CYCLE q value) in Ctrl mice show no circadianpattern in iKO mice.Table S2. The ratio of differentially expressed gene numbers in cKO strain to the numbers iniKO strain.Table S3. Summary of phenotypes of cKO and iKO mice.Data file S1. Circadian transcriptome.Data file S2. Differentially expressed genes irrespective of time points.Data file S3. Phenotype enrichment analysis of differentially expressed genes.

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Acknowledgments: We thank C. A. Bradfield (University of Wisconsin, Madison, WI) forconventional Bmal1 KO mice and Bmal1f/f mice. We thank E. J. Kim, N. Lahens, K. Hayer, F. Colden,A. Pizarro, and A. Srinivasan (University of Pennsylvania, Philadelphia, PA) for their support in RNA-Seq analysis. We thank Y. Zheng (University of Pennsylvania, Philadelphia, PA) for discussionsabout hair regrowth. We thank W. Yan (University of Pennsylvania, Philadelphia, PA) for technicalassistance. We thank A. Sehgal for reading and commenting on this manuscript. Funding: Thiswork was supported by a grant from the NIH (HL097800) and the University of PennsylvaniaGenomics Frontiers Institute’s Translational and Personalized Genomics Centers Initiative. G.A.F.is the McNeil Professor of Translational Medicine and Therapeutics. Author contributions: G.Y.and G.A.F. conceived the project. G.Y. and L.C. performed and analyzed most of the experiments inthis study. G.P. prepared cKO samples for RNA-Seq. E.S.M. and V.L. conducted staining of brain andeye, respectively. W.-L.S. performed atherosclerosis experiment for cKO mice. S.C.M. collected iKOorgans for weight measurement. T.G. designed and conducted the RNA-Seq experiments, andG.R.G. did the data analysis. G.C. discussed the data of hair growth assay. G.R.G., E.S.M., V.L.,and G.C. provided critical intellectual input in manuscript preparation of their pertinent parts.G.Y., L.C., and G.A.F. wrote the paper. Competing interests: The authors declare that theyhave no competing interests. Data and materials availability: RNA-Seq data can be retrievedfrom the GEO database under accession nos. GSE70497, GSE70499, and GSE70500.

Submitted 28 August 2015Accepted 29 December 2015Published 3 February 201610.1126/scitranslmed.aad3305

Citation: G. Yang, L. Chen, G. R. Grant, G. Paschos, W.-L. Song, E. S. Musiek, V. Lee,S. C. McLoughlin, T. Grosser, G. Cotsarelis, G. A. FitzGerald, Timing of expression of the coreclock gene Bmal1 influences its effects on aging and survival. Sci. Transl. Med. 8, 324ra16(2016).

us

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survival influences its effects on aging andBmal1Timing of expression of the core clock gene

Sarah C. McLoughlin, Tilo Grosser, George Cotsarelis and Garret A. FitzGeraldGuangrui Yang, Lihong Chen, Gregory R. Grant, Georgios Paschos, Wen-Liang Song, Erik S. Musiek, Vivian Lee,

DOI: 10.1126/scitranslmed.aad3305, 324ra16324ra16.8Sci Transl Med

increase the likelihood of translation for preclinical studies of the aging process.functions. Thus, the systemic role of the molecular clock in the biology of aging requires reinvestigation in order to

knockout mice apparently manifest as a result of clock-independent BMAL1Bmal1disruption in conventional Taken together, these findings reveal that many phenotypes thought to be caused by circadian rhythm

postnatal knockout mice displayed similar ocular abnormalities and brain astrogliosis. knockout mice, even though there's a quelling of expression of oscillating genes. Both prenatal andBmal1

function. Another surprising observation was little changes in overall gene expression in the livers of adult-life age-dependent arthropathy; in fact, atherosclerosis and hair growth actually improved, despite obliteration of clockheart rate, and blood pressure, but exhibited normal life spans, fertility, body weight, blood glucose levels, and

knockout mice displayed loss of circadian rhythm in wheel-running activity,Bmal1expression, the new conditional Bmal1mice that are missing the BMAL1 protein only during adult life. Unlike knockout mice that perpetually lack

knockoutBmal1To assess the role of circadian rhythms in the aging process, the authors made conditional the circadian clock's effects on aging and survival.

expression influencesBmal1. show that the developmental timing of et alrhythms in the aging process. Now Yang knockout mice often serve as a model system in studies of the role of circadianBmal1span in mice. As a result,

accelerates aging and shortens the life−−, a core clock geneBmal1for example, by knocking out −−circadian clock Ironically, antiaging product advertisements often promise to ''slow down the clock.'' But abolishing the

For clock ticking, timing matters

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