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IDENTIFICATION OF INTELLECTUAL DISABILITY GENES SHOWINGCIRCADIAN CLOCK-DEPENDENT EXPRESSION IN THEMOUSE HIPPOCAMPUS

J. RENAUD, a,b,c F. DUMONT, a,c M. KHELFAOUI, e

S. R. FOISSET, e F. LETOURNEUR, a,c T. BIENVENU, a,b,c

O. KHWAJA, d O. DORSEUIL a,b,cy AND P. BILLUART a,b,c*†

a Institut National de la Santé et de la Recherche Mé dicale

(INSERM), U1016, Institut Cochin, Paris, France

b Université Paris Descartes Paris V, Institut Cochin, Paris, France

c Centre National de la Recherche Scientifique (CNRS),

UMR8104, Institut Cochin, Paris, France

d Roche Pharma Research and Early Development,

Roche Innovation Center Basel, Basel, Switzerland e Centre National de la Recherche Scientifique (CNRS), UMR,

INCI, Strasbourg, France

Abstract—Sleep is strongly implicated in learning,

especially in the reprocessing of recently acquired memory.

Children with intellectual disability (ID) tend to have sleep-

wake disturbances, which may contribute to the pathophys-

iology of the disease. Given that sleep is partly controlled by

the circadian clock, we decided to study the rhythmic

expression of genes in the hippocampus, a brain structure

which plays a key role in memory in humans and

rodents. By investigating the hippocampal transcriptome

of adult mice, we identified 663 circadian clock controlled

(CCC) genes, which we divided into four categories basedon their temporal pattern of expression. In addition to the

standard core clock genes, enrichment analysis identified

several transcription factors among these hippocampal

CCC genes, and our findings suggest that genes from one

cluster regulate the expression of those in another. Interest-

ingly, these hippocampal CCC genes were highly enriched

in sleep/wakefulness-related genes. We show here that

several genes in the glucocorticoid signaling pathway,

which is involved in memory, show a CCC pattern of expres-

sion. However, ID genes were not enriched among these

CCC genes, suggesting that sleep or learning and memory

disturbances observed in patients with ID are probably not

related to the circadian clock in the hippocampus.

2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Key words: circadian clock, hippocampus, sleep, memory,

intellectual disability.

INTRODUCTION

Intellectual disability (ID) is defined by an overall

intelligence quotient (IQ) lower than 70 and deficits in

cognitive abilities with an onset before 18 years of

age. This disorder affects 1–3% of the population.

The underlying causes of ID are heterogeneous andinclude genetic and/or environmental factors that

influence the development and function of the central

nervous system. Genetic factors comprise mutations in

genes encoding proteins involved in neurogenesis,

neuronal migration, synaptic function, transcription or

translation. Many children with ID have problems with

memory and learning. Indeed, several memorization

processes and associated structures may be impaired in

ID, which limits the ability to learn broader cognitive

skills. For example, patients with Down’s or Williams

syndrome show working memory deficits (Jarrold et al.,

1999). Numerous studies report that many children with

ID show delayed settling or frequent night waking(Didde and Sigafoos, 2001; Robinson and Richdale,

2004; Sajith and Clarke, 2007).

Sleep is implicated in long-term memory, especially in

the reprocessing of recently acquired memory (Gais and

Born, 2004; Ji and Wilson, 2007). Indeed, sleep plays a

fundamental role in the neurocognitive performance of

infants and children (Born and Wilhelm, 2012). More

precisely, slow-wave sleep activity helps to consolidate

hippocampal-dependent episodic memory. Sleep is con-

trolled by two main regulatory processes: a homeostatic

and a circadian one. The homeostatic process tracks

the need for sleep whereas the distribution of sleep

throughout the day is strongly regulated by circadian

rhythm (CR). Circadian clock genes may also be directlyinvolved in sleep homeostasis (Naylor et al., 2000).

Sleep/wake-related genes have been reported to be

expressed in the cortex, cerebellum, forebrain and

hypothalamus in mice and rats (Terao et al., 2003,

2006; Cirelli et al., 2004).

The circadian clock is a fundamental system

regulating many aspects of behavior and physiology,

including sleep–wake cycles but also blood pressure,

body temperature, and metabolism. The

suprachiasmatic nucleus (SCN) of the hypothalamus

contains the master circadian clock, which is reset daily

http://dx.doi.org/10.1016/j.neuroscience.2015.08.066

0306-4522/ 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

*Correspondence to: P. Billuart, Institut National de la Sante ´ et de laRecherche Me ´ dicale (INSERM), U1016, Institut Cochin, Paris,France

E-mail address: [email protected] (P. Billuart).y These authors contributed equally to this work.

Abbreviations: AA, ascorbic acid; ASD, Autism Spectrum Disorders;CCC, circadian clock controlled; CR, circadian rhythm; CT, CircadianTime; HSPs, heat shock proteins; ID, intellectual disability; IPA,Ingenuity Pathway Analysis; IQ, intelligence quotient; REM, rapid eyemovement; SCN, suprachiasmatic nucleus; SWG, sleep-wakefulness-related genes; SWS, slow wave sleep; TF, transcription factor; TFBSs,transcription factor binding sites.

Neuroscience 308 (2015) 11–50

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http://dx.doi.org/10.1016/j.neuroscience.2015.08.066mailto:[email protected]://dx.doi.org/10.1016/j.neuroscience.2015.08.066http://dx.doi.org/10.1016/j.neuroscience.2015.08.066http://dx.doi.org/10.1016/j.neuroscience.2015.08.066mailto:[email protected]://dx.doi.org/10.1016/j.neuroscience.2015.08.066http://-/?-http://-/?-http://-/?-http://-/?-

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by light that is detected by retinal ganglion cells and

relayed via signaling through the retinohypothalamic

tract. The circadian clock consists of a group of highly

conserved core clock proteins (e.g. Rev-erba, Clock,

Bmal1, Per1, Per2, Per3), the expression of which is

regulated through transcriptional, translational and post-

translational feedback loops. These regulatory mechanismsgenerate and maintain the cyclic expression of clock

genes as well as those they regulate (called output

genes) over a 24-h period (Bell-Pedersen et al., 2005;

Ko and Takahashi, 2006; Dardente and Cermakian,

2007). The SCN coordinates peripheral oscillators like

the liver, kidney and cortex, which can be self-

sustained, although their correct functioning depends on

the other oscillators. Clock mechanisms in the SCN and

peripheral oscillators are identical, and involve the 24-h

cyclic expression of core clock genes (Reppert and

Weaver, 2002; Yoo et al., 2004).

CR has been studied in many non-neuronal tissues,

especially in the liver because it regulates metabolism.Several studies report that about 10% of transcripts

show circadian clock controlled (CCC) expression in the

liver, most of which are involved in metabolic functions

(Panda et al., 2002; Storch et al., 2002; Cho et al.,

2012). Microarrays and RNA-seq have been used to

examine the transcriptome at different Circadian Time

(CT) points in several regions of the mouse brain, includ-

ing the SCN (Panda et al., 2002), prefrontal cortex (Yang

et al., 2007), cerebellum, hypothalamus and brain stem

(Zhang et al., 2014). The core clock genes are expressed

in various brain structures, such as the hippocampus and

cortex (Namihira et al., 1999; Reick et al., 2001;

Wakamatsu et al., 2001; Shieh, 2003; Yang et al., 2007;

Jilg et al., 2010) and studies on mutant mice suggest thatthe disruption of clock genes alters hippocampal-

dependent memory formation (Garcia, 2000; Wang

et al., 2009; Jilg et al., 2010). The hippocampus controls

memory and learning, processes spatial information and

episodic memory, and consolidates information from

short-term to long-term memory. These processes are

regulated by CR (Chaudhury and Colwell, 2002;

Chaudhury et al., 2005; Ruby et al., 2008; Gerstner

et al., 2009). For instance, circadian oscillations in hip-

pocampal MAPK activity are implicated in the formation

and persistence of memory (Eckel-Mahan et al., 2008;

Phan et al., 2011). In addition, adult neurogenesis in the

dentate gyrus is also controlled by the circadian molecular

clock (Bouchard-Cannon et al., 2013; Schnell et al.,

2014).

Although the link between cognitive impairment,

memory and sleep as well as that between CR and

sleep are well established, little is known about the

relationship between the circadian clock and ID. The

enrichment of CCC genes among the known ID genes

would favor the hypothesis that the circadian clock-

mediated deregulation of memory processes is involved

in the pathogenesis of ID.

In this context, we analyzed genome-wide rhythmic

gene expression in the hippocampus, focusing on

circadian regulation. Using the JTK cycle algorithm, we

demonstrate here that about 3.5% of transcripts show

circadian-regulated expression in the mouse hippo-

campus. We divided these genes into four categories

based on their temporal expression, and show that each

category is characterized by specific molecular and

cellular signatures. The category containing genes

that were strongly expressed at the transition phase

between light and dark was highly enriched in sleep/wake-related genes.

Furthermore, from a list of 673 ID-associated genes,

we identified 30 genes (4.4%) showing circadian-

dependent expression in the hippocampus. This

proportion was not significantly higher than that expected

by chance (3.5%), showing that the pathogenesis of ID

is probably not related to disturbances in the circadian

clock in the hippocampus.

EXPERIMENTAL PROCEDURES

Animals

Animal experiments were performed in accordance withthe European Communities Council Directive (86/809/

EEC) on the care and use of animals and were

approved by the local ethics committee. All animals

were housed singly in cages in a room maintained at

2 3 ± 1 C under a 12/12 light–dark cycle (bright light,

200 lx). Nine-week-old male C57/BL6 mice were killed in

the dark and hippocampi were quickly excised under a

red-light lamp and snap-frozen in liquid nitrogen. The

dark/dark period started at 7 p.m. Four CT points were

sampled. The CT18 samples were taken after 30 h of

continuous darkness, after which the other CT samples

were taken at 6-h intervals: CT0, CT6 and CT12 after

36, 42 and 48 h of continuous darkness, respectively.

Microarray analysis

The SurePrint G3 Mouse GE 8 60 K microarray (Agilent

Technologies, Santa Clara, CA, USA) was used for

expression profiling experiments.

Total RNA was extracted using RNA-PLUS (MP

Biomedicals, Santa Ana, CA, USA), treated with rDNase

(Macherey-Nagel EURL, Hoerdt, France) and cleaned

with an RNA clean-up kit (Macherey-Nagel EURL,

Hoerdt, France). RNA transcripts were quantified using

a Nanodrop spectrophotometer (Thermo Fischer

Scientific, Waltham, MA, USA) and quality was

monitored with the Agilent 2100 Bioanalyzer (AgilentTechnologies, Santa Clara, CA, USA). Each biological

replicate, i.e. total RNA from the hippocampus of one

mouse, was hybridized to one chip.

Real-time RT-PCR

Microarray results were validated by performing RT-PCR

on the same samples used for hybridization. DNase

I–treated RNA was reverse transcribed using the

QuantiTect reverse transcription kit (Qiagen, Hilden,

Germany) according to the manufacturer’s instructions.

The expression of two clock genes was evaluated

(Rev-erba and Arntl ). Reactions were performed in

triplicate in an LC480 LightCycler (Roche Applied

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Science, Indianapolis, IN, USA). The Gapdh and

Cyclophilin A housekeeping genes were used as internal

controls. Average normalized delta-Ct values were used

to calculate the fold difference in expression between

samples. The following primers were used: mouse

Rev-erba (ID #Mm00520708_m1), mouse Arntl (ID

#Mm00500226_m1) and Gapdh (ID #Mm99999915_g1)from Applied Biosystems, and cyclophilin A forward:

50-GTG ACT TTA CAC GCC ATA ATG-30 and reverse:

50-ACA AGA TGC CAG GAC CTG TAT-30, gene ID for

cyclophilin A (or Ppia) (uc007hyn.1). A one-way ANOVA

was performed to assess the significance of expression

differences between samples.

Western blotting

Total protein extracted from the hippocampus was

separated by SDS–PAGE, followed by Western blotting

with a rabbit antibody against Clock (1:1000, Merck

Millipore, Fontenay sous Bois, France, AB2203) and a

mouse antibody against Gapdh (1:4000, AmbionThermo Fischer Scientific, Waltham, MA, USA,

AM4300). Data were expressed as the mean ± SEM.

The significance of differences in protein levels between

samples was tested by an unpaired Student t test.

PCA and JTK analysis

Raw data from the Agilent extraction file were first

normalized using g-processed signal values and

quantile normalization. Expression data were then

summarized to obtain the gene-level expression value.

First, variations in gene expression were analyzed

using Principal Component Analysis to eliminate

technical bias and outliers. The JTK cycle algorithm(Hughes et al., 2010) was then applied to identify rhythmic

genes. An adjusted p-value < 0.05 was considered to be

significant (JTK-positive). These two analyses were

performed using R statistical software.

Identification and clustering of genes

To identify clusters of genes among JTK-positive genes,

we applied the k -means clustering algorithm (Pearson

dissimilarity and centroid linkage) as implemented in the

open source clustering software Cluster 3.0 (Eisen

et al., 1998). To view the clustering results generated by

Cluster 3.0, we used Alok Saldanha’s Java Treeview,

which displays hierarchical as well as k -means clusteringresults.

Functional analysis of genes controlled by the

circadian clock

The 663 JTK-positive genes and the genes of each

cluster were uploaded to Ingenuity Pathway Analysis

(IPA, Ingenuity Systems Inc., Redwood City, CA, USA)

to analyze molecular and cellular functions and

canonical pathways associated with the dataset.

Analysis of transcription factor (TF) binding sites

Promoter sequences were analyzed using the‘‘Overrepresented transcription factor binding sites” and

Matinspector tools in Genomatix software (GmbH,

Munich, Germany). The software calculates z -scores

that correspond to the distance from the population

mean expressed in units of the standard deviation of the

population. The z -scores are calculated with a continuity

correction using the formula z = (x E 0.5)/S , where x

is the number of found matches in the input data, E isthe expected value and S is the standard deviation.

To determine the degree of inter-regulation among the

genes in each cluster, for each TF, we calculated the

mean of the z -scores of all the clusters. For each

cluster and each TF, the z -score was normalized by the

mean value of the z -scores. The sum of the normalized

z -scores was then averaged.

RESULTS

CCC genes in the hippocampus

To identify CCC genes in the mouse hippocampus, we

conducted our study in dark–dark conditions to

overcome synchronizing effect of the SCN in response

to light. We previously reported that the expression of

some clock genes (Rev-erba and Arntl ) oscillates in the

hippocampus in such conditions (Valnegri et al., 2011).

To identify systematically genes with a CCC pattern of

expression, we used Agilent microarray technology to

examine the transcriptome of the hippocampus of three

mice at four CT points. We used the JTK cycle algorithm

(Hughes et al., 2010) to identify and characterize genes

with rhythmic expression. This algorithm accurately mea-

sures the period, phase, acrophase and amplitude of

cycling transcripts. Among the 948 JTK-positive genes

(adjusted p-value < 0.05), 663 showed cyclic expressionof a 24-h period and could thus be defined as CCC genes

(Table 1).

To assess the circadian rhythmicity of the

hippocampus, we first examined oscillations in the

expression of core clock genes (Fig. 1 and Table 1). We

found that Clock (1a) and Rev-erba mRNAs (1b)

oscillate with a period of 24 h. Furthermore, we

previously reported that levels of Rev-erba protein show

a rhythmic pattern (Valnegri et al., 2011). We thus

assessed the abundance of Clock protein in the hip-

pocampus over 24 h in dark/dark conditions (1c). Protein

levels peaked at CT0 (the ratio of Clock to Gapdh was

1.539 ± 0.084 at CT0 vs. 0.4174 ± 0.007 at CT18,

0.5473 ± 0.082 at CT12 and 0.4745 ± 0.06515 at CT6, p-values = 0.0056, 0.004 and 0.002, respectively).

These results suggest that the expression of clock genes

in the hippocampus shows circadian oscillations at both

the mRNA and protein level. The analysis of microarray

data revealed that Arntl , also known as Bmal1 (1d),

Per1 (1e), and Per2 (1f) also showed CCC expression.

Real-time PCR analysis of two major clock genes, Rev-

erba and Arntl , the expression of which are temporally

opposed, confirmed the findings of our microarray exper-

iments (Fig. 1c, d, g , h). Thus, these results show that the

abundance of clock genes mRNA oscillates in a typical

cyclic manner in the hippocampus.

We next performed an Ingenuity Pathway Analysis(IPA) on our dataset of 663 genes to identify the

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Table 1. JTK analysis of the 948 genes with an adjusted p-value < 0.05 (JTK-positive). The 663 JTK-positive genes with a period of 24 h are

highlighted in red


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Table 1 (continued )

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pathways and molecular/cellular functions associatedwith these CCC genes. The core-clock genes (Per1,Per2 , Arntl , Rev-erba and Clock ) were significantlyenriched among the top canonical pathways ( p-value =2.34 103) (Fig. 2). We also identified other criticalcomponents of the clock, Cry1 and Cry2, in a list of

upstream regulators ( p-value of overlap = 8.26 105and 8.26 105, respectively). Cry proteins interact with

Per proteins and associate with Clock and Arntl torepress transcription.

Co-regulation of CCC genes

We next defined clusters of genes based on their

expression levels at the four CT points analyzed to

identify co-regulated CCC genes using cluster and java

tree view software. The 663 JTK-positive genes were

grouped into four clusters showing a distinct temporal

expression pattern and containing between 48 and 308

genes (Fig. 3a). Clock genes mapped to different

clusters depending on their peak of expression and weassumed that genes in the same cluster were

co-regulated during circadian activity (Fig. 3b–e).

Given that sleep is a circadian process and implicated

in learning and memory, we wondered whether our 663

CCC genes were enriched in sleep-wakefulness-related

genes (SWG). Approximately 5% of the transcripts

expressed in the rat cortex and cerebellum are

regulated by sleep/wakefulness (Cirelli et al., 2004). By

comparing our list of 663 hippocampal CCC genes with

the mouse orthologs of the genes reported by Cirelli

et al., we found 52 genes potentially regulated by SWG

(i.e. 7.85% of our entire list) (Cirelli et al., 2004). A

Chi-square test with Yates continuity correction, which is

used for large sample sizes, showed that CCC geneswere significantly enriched in SWG ( p < 0.0001). We also

examined for the presence of SWG in each cluster.

Clusters 1 and 4 were significantly enriched in SWG

(11% and 8.3%, respectively) whereas cluster 2 and

cluster 3 did not contain a significant proportion of these

genes (1.06% and 4.25%, respectively). Genes in cluster

4 showed a peak of expression at CT12, which is relevant

because CT12 is a transition phase between light and

dark. Per2 was classified as a cluster 1 gene, the expres-

sion of which peaked at CT18. Individuals presenting a

mutation in the phosphorylation site of PER2 suffer from

familial advanced sleep phase syndrome (FAPS), which

is characterized by early sleep onset and offset (Toh,2001). Our findings suggest that sleep/wakefulness and

circadian activity are two inter-connected processes in

the hippocampus.

Genes involved in post-translational modifications and

protein ubiquitination pathways were highly enriched incluster 1 ( p-value = 3.17 107 and 3.91 104

respectively). These two pathways are two well-known

circadian processes (Mehra et al., 2009). Indeed, post-translational modifications such as phosphorylation,acetylation and ubiquitination of clock proteins contributeto the entrainment of the clock. Ubiquitination, in particu-lar, regulates the half-life of proteins and is clearly essen-

tial to control daily fluctuations in protein abundance. Inour dataset, heat shock proteins (HSPs) were over-represented among the proteins involved in post-translational modifications. Many HSPs show circadianregulation and variations in their expression levels during

sleep and have been suggested to control the clock inperipheral organs (Terao et al., 2003; Reinke et al.,2008; Yan et al., 2008). Furthermore, HSPs interact with

the glucocorticoid receptor in a manner dependent on

the circadian oscillations (Furay et al., 2006). Interest-ingly, glucocorticoid signaling was also in the topcanonical pathway ( p-value = 1.07 103) and wasover-represented in cluster 2 ( p-value = 2.11 104).

Glucocorticoids are strongly implicated in the hippocampal-dependent consolidation of memory (de Quervain et al.,2009), and Arntl-deficient mice present deficits in gluco-corticoid production and a poor behavioral response tostress (Leliavski et al., 2014). Moreover, circadian oscilla-tion of glucocorticoid levels promotes the formation of dendritic spines in the cortex (Liston et al., 2013). Another CCC gene identified in this analysis was the glucocorti-

coid receptor, NR3C1 (nuclear receptor subfamily 3,group C, member 1) identified by IPA as an upstream reg-

ulator of transcription ( p-value of overlap = 1.4 103).This receptor functions both as a transcriptional activator that binds to glucocorticoid response elements in thepromoters of glucocorticoid responsive genes and as a

regulator of other TFs. This result suggests that thecircadian clock influences the hippocampus and memorythrough the regulation of glucocorticoid signaling.

In addition, the antioxidant action of vitamin C was an

enriched pathway ( p-value = 3.68 104) that was over-

represented in cluster 3 ( p-value = 1.09 102).

Ascorbic acid (AA), also known as vitamin C,

accumulates in the brain, especially in the hippocampus

and its antioxidant function is neuroprotective

(MacGregor et al., 1996). Furthermore, the amount of AA oscillates with CR in the SCN (Wang et al., 2012).



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Regarding metabolism, cluster 1, which contains genes

highly expressed during an active phase (dark phase),

was enriched in genes involved in lipid metabolism. Lipids

are important sources of energy and are provided in large

quantities in the diet, with mice feeding preferentially dur-

ing the arousal phase (Pan and Hussain, 2009). Overall,

metabolism is a circadian-regulated process and is clo-

sely related to sleep–wake and feeding–fasting cycles.

Moreover, metabolic processes influence the functionand timing of the clock (Peek et al., 2012). Indeed, cellular

redox state influences DNA binding of the Clock-Arntl het-

erodimer (Rutter et al., 2001). Therefore, it is not surpris-

ing to find metabolism pathways highly represented in our

list of hippocampal CCC genes.

Transcriptional regulation of CCC genes

Gene expression is regulated by the binding of TFs to

promoter regions, which is temporally and spatially

controlled. We thus wondered whether the coordinated

expression of genes in each cluster may be explained

by the presence of specific transcription factor bindingsites (TFBSs) in the promoters of the hippocampal CR

genes. We screened the genes in each cluster for

enrichment in TFBSs using the search tool ‘‘Over-

represented TFBSs” in Genomatix software. The

software analyzes the promoter regions of genes

(500 bp upstream and 100 bp downstream from the

transcription start site) and assigns a statistical

enrichment value, named a z -score, to each TFBS.

First, we noticed that the matrix family v$HIFF (E box-

element target sequence: CACGTG) was highlyrepresented (z -score = 19.3) in cluster 3 (peak of

expression at CT6) (data not shown). This matrix family

is strongly associated with CR and with the expression

of genes regulated by Clock and Arntl. Moreover, this

analysis showed substantial overlap in the TFBSs

identified in each cluster (Fig. 4a). We categorized

TFBSs into four groups based on their enrichment in

each cluster, with group 1, 2, 3 and 4 containing

TFBSs enriched in one, two, three or four clusters,

respectively. Group 4 included 74.3% of the TFBSs

whereas groups 1, 2 and 3 contained only 12.4%,

3.8% and 9.5% of the TFBSs, respectively (Fig. 4b).

We conclude that the transcriptional regulation of hippocampal CCC genes appears to be similar among

Fig. 2. Network of circadian core clock genes. Network of the core clock genes identified as circadian oscillating genes in our microarray analysis

(highlighted in pink). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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Fig. 3. Clustering of the 663 JTK-positive genes according to their temporal expression. (a) Heat map showing the expression profiles of the 663

JTK-positive genes. Four clusters are identified. (b–e) Plots showing the distinct temporal expression patterns of JTK-positive genes in the four

clusters. Clock genes associated with each cluster are shown in red. Cluster 1 (b) contains genes showing an expression peak at CT18, including

Per2 and Clock. Cluster 2 (c) contains genes showing an expression peak at CT0, including Arnt1. Cluster 3 (d) gene contains genes showing an

expression peak at CT6, including Rev-erba. Cluster 4 (e) contains genes showing an expression peak at CT12, including Cry1 and Per1. In panel

(b,c,d,e), expression peak is shown by a blue star. (For interpretation of the references to color in this figure legend, the reader is referred to the

web version of this article.)

Fig. 4. Identification of TFBSs in the 663 CCC genes. (a) Table showing the number of TFBSs shared between clusters. (b) Clustering of TFBSs

based on their enrichment in the various clusters. Four groups can be identified (G1–G4). G4 contains TFBSs present in all the clusters whereas G1

contains TFBSs present specifically in one cluster.


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clusters. Therefore, circadian oscillations in the

expression of these genes may be generated by a

restricted number of specific TFs.

Given that the clock is regulated transcriptionally and

that clock genes are TFs, we determined the proportion

of TFs among the CCC genes in the hippocampus.

Among our four clusters, cluster 2 was the most

enriched in TFs (24.21%) whereas cluster 3 included

very few TFs (7.55%). We then identified the TFs in

each cluster that may regulate the expression of genes

in the other clusters. We quantified the potential inter-

regulation of gene expression between clusters bycalculating a normalized z -score value (Fig. 5). We

found that TFs from cluster 1 may strongly regulate the

expression of genes from clusters 3 and 4. Cluster 2

contained TFs that may control the temporal expression

of genes from clusters 1, 3 and 4. This analysis thus

highlights transcriptional regulation between our clusters

that may underlie their different temporal patterns of

expression, thereby illustrating the typical functioning of

a clock.

Identification of CCC ID genes in the hippocampus

Next, we wanted to explore the link between ID and the

circadian clock. From the literature and from publicdatabases, we identified a list of 673 genes involved in

ID and Autism Spectrum Disorders (ASD) (Table 2), 30

(4.45%) of which showed CCC expression in the

hippocampus according to our analysis (Table 3). This

proportion is not significantly different from that

expected by chance alone ( p-value = 0.1), suggesting

that clock output genes in the hippocampus are not

associated with ID. However, Fgfr3 and Aff2 , both of

which are included in this list of 30 genes, have already

been implicated in sleep-related respiratory disturbances

and sleep enhanced multifocal spiking, respectively

(Musumeci et al., 2000; Schlu ¨ ter et al., 2011).

Furthermore, Per1 was also included in this list of 30CCC regulated ID/ASD genes (Nicholas et al., 2007).

Polymorphisms in Per1, which encodes a critical compo-

nent of the circadian clock, are associated with extreme

diurnal preference (Carpen et al., 2006).

IPA also showed that lipid metabolism was an over-

represented biological function ( p-value = 5.05 105

to 3.89 102). ID and lipid metabolism disorders have

already been described as two potentially related

diseases (Thiel et al., 2003; Voronov et al., 2008;

Matsumata et al., 2014). Lipid metabolism is known to

be a clock-controlled process (Sancar and Brunner,

2014).

Finally, we divided the 30 CCC ID genes in eachcluster according to their temporal pattern of expression

(Fig. 6). These results are of significant interest for

clinicians. Indeed, children with ID may have different

clinical profiles of sleep/wake defects depending on the

expression pattern of the ID gene involved.

DISCUSSION

Circadian clocks drive the CR of biological processes in

various tissues. This system is organized into a master

circadian clock, the SCN that synchronizes the

peripheral oscillators. Instead of a single cerebral clock,

the brain contains a system of multiple local oscillators

exhibiting CR in different structures (Guilding andPiggins, 2007). Indeed, the expression of Per1 and Per2

clock genes only partially overlaps in various brain

regions and their promoters are activated in a cell type-

specific manner (neurons for Per1 vs. glial cells for

Per2 ) (Feillet et al., 2008; Cheng et al., 2009).

The hippocampus is entrained by the SCN suggesting

that these regions are connected physically or in an

indirect manner. Pathways connecting the two

structures have been identified in rats (Moga et al.,

1995). In addition, the release of melatonin from the

pineal gland (Perreau-Lenz et al., 2003) or hypocretin-

expressing neurons of the lateral hypothalamus (Yang

et al., 2013) may constitute an indirect link between themaster clock and the hippocampus.

Fig. 5. Quantification of potential inter-regulation of gene expression between clusters. (a) A normalized mean z -score value was assigned to each

cluster as an enrichment factor. (b) Model of inter-regulation between clusters.


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Table 2. List of the 673 ID-ASD genes. ID genes have been compiled from literature and from public databases ( http://www.genes2cognition.org; http://

www.g2conline.org; http://www.ggc.org/research/molecular-studies/xlid.html; http://www.gencodys.eu; https://gene.sfari.org/autdb/Welcome.do)

Gene symbol Gene name

A2bp1 ataxin 2-binding protein 1

Aass aminoadipate-semialdehyde synthase

Abat 4-aminobutyrate aminotransferaseAbcc8 ATP-binding cassette, sub-family C (CFTR/MRP), member 8

Abcd1 ATP-binding cassette, sub-family D (ALD), member 1

Abhd5 abhydrolase domain containing 5

Acbd6 acyl-Coenzyme A binding domain containing 6

Acox1 acyl-Coenzyme A oxidase 1, palmitoyl

Acsl4 acyl-CoA synthetase long-chain family member 4

Acy1 aminoacylase 1

Ada adenosine deaminase

Adk adenosine kinase

Adora2a adenosine A2a receptor

Adra2b adrenergic receptor, alpha 2b

Adrb2 adrenergic receptor, beta 2

Adsl adenylosuccinate lyase

Aff2 AF4/FMR2 family, member 2

Aga aspartylglucosaminidaseAgtr2 angiotensin II receptor, type 2

Ahi1 Abelson helper integration site 1

Ak1 adenylate kinase 1

Aldh18a1 aldehyde dehydrogenase 18 family, member A1

Aldh3a2 aldehyde dehydrogenase family 3, subfamily A2

Aldh4a1 aldehyde dehydrogenase 4 family, member A1

Aldh5a1 aldehyde dehydrogenase family 5, subfamily A1

Aldoa aldolase A, fructose-bisphosphate

Alg12 asparagine-linked glycosylation 12 (alpha-1,6-mannosyltransferase)



Alg6 asparagine-linked glycosylation 6 (alpha-1,3,-glucosyltransferase)

Alox5ap arachidonate 5-lipoxygenase activating protein

Alx1 ALX homeobox 1

Alx4 aristaless-like homeobox 4Amt aminomethyltransferase

Ankrd11 ankyrin repeat domain 11

Ap1s2 adaptor-related protein complex 1, sigma 2 subunit

Ap4e1 adaptor-related protein complex AP-4, epsilon 1

Ap4m1 adaptor-related protein complex AP-4, mu 1

Apc adenomatosis polyposis coli

Ar androgen receptor

Arg1 arginase, liver

Arhgef6 Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6

Arhgef9 CDC42 guanine nucleotide exchange factor (GEF) 9

Arnt2 aryl hydrocarbon receptor nuclear translocator 2

Arsa arylsulfatase A

Arx aristaless-related homeobox

Asah1 N-acylsphingosine amidohydrolase 1

Ascc3 activating signal cointegrator 1 complex subunit 3Ascl1 achaete-scute complex homolog 1 (Drosophila)

Asl argininosuccinate lyase

Asmt acetylserotonin O-methyltransferase

Aspa aspartoacylase

Aspm asp (abnormal spindle)-like, microcephaly associated (Drosophila)

Ass1 argininosuccinate synthetase 1

Astn2 astrotactin 2

Atic 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase

Atn1 atrophin 1

Atp10a ATPase, class V, type 10A

Atp2a2 ATPase, Ca++ transporting, cardiac muscle, slow twitch 2

Atp5e ATP synthase, H+ transporting, mitochondrial F1 complex, epsilon subunit

Atp6ap2 ATPase, H+ transporting, lysosomal accessory protein 2

Atp7a ATPase, Cu++ transporting, alpha polypeptide


http://www.genes2cognition.org/http://www.g2conline.org/http://www.g2conline.org/http://www.ggc.org/research/molecular-studies/xlid.htmlhttp://www.gencodys.eu/https://gene.sfari.org/autdb/Welcome.dohttps://gene.sfari.org/autdb/Welcome.dohttp://www.gencodys.eu/http://www.ggc.org/research/molecular-studies/xlid.htmlhttp://www.g2conline.org/http://www.g2conline.org/http://www.genes2cognition.org/

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Ehmt1 euchromatic histone methyltransferase 1

Ehmt2 euchromatic histone lysine N-methyltransferase 2

Eif2ak3 eukaryotic translation initiation factor 2 alpha kinase 3

Eif2s3x eukaryotic translation initiation factor 2, subunit 3, structural gene X-linkedEif4e eukaryotic translation initiation factor 4E

Elp2 elongator acetyltransferase complex subunit 2

Emx2 empty spiracles homeobox 2

En2 engrailed 2

Entpd1 ectonucleoside triphosphate diphosphohydrolase 1

Ercc2 excision repair cross-complementing rodent repair deficiency, complementation group 2




Ercc8 excision repaiross-complementing rodent repair deficiency, complementation group 8

Erlin2 ER lipid raft-associated 2

Esr1 estrogen receptor 1 (alpha)

Esr2 estrogen receptor 2 (beta)

Esrrb estrogen-related receptor, beta

F13a1 coagulation factor XIII, A1 subunitFabp5 fatty acid binding protein 5

Fabp7 fatty acid binding protein 7, brain

Fam126a family with sequence similarity 126, member A

Fanca Fanconi anemia, complementation group A

Fancb Fanconi anemia, complementation group B

Fancc Fanconi anemia, complementation group C

Fasn fatty acid synthase

Fbn1 fibrillin 1

Fbxo33 F-box protein 33

Fbxo40 F-box protein 40

Fezf2 Fez family zinc finger 2

Fgd1 FYVE, RhoGEF and PH domain containing 1

Fgf14 fibroblast growth factor 14

Fgfr1 fibroblast growth factor receptor 1

Fgfr2 fibroblast growth factor receptor 2Fgfr3 fibroblast growth factor receptor 3

Fh1 fumarate hydratase 1

Fhit fragile histidine triad gene

Fkrp fukutin-related protein

Fktn fukutin

Flna filamin, alpha

Flt1 FMS-like tyrosine kinase 1

Fmr1 fragile X mental retardation syndrome 1

Folr1 folate receptor 1 (adult)

Foxe1 forkhead box E1

Foxg1 forkhead box G1

Foxp1 forkhead box P1

Foxp2 forkhead box P2

Fras1 Fraser syndrome 1 homolog (human)

Frk fyn-related kinaseFrmpd4 FERM and PDZ domain containing 4

Fry furry homolog (Drosophila)

Ftsj1 FtsJ homolog 1 (E. coli)

Fuca1 fucosidase, alpha-L- 1, tissue

G6pdx glucose-6-phosphate dehydrogenase X-linked

Gabra4 gamma-aminobutyric acid (GABA) A receptor, subunit alpha 4

Gabrb1 gamma-aminobutyric acid (GABA) A receptor, subunit beta 1

Gabrb3 gamma-aminobutyric acid (GABA) A receptor, subunit beta 3

Gale UDP-galactose-4-epimerase

Galnt13 UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 13

Galt galactose-1-phosphate uridyl transferase

Gamt guanidinoacetate methyltransferase

Gatm glycine amidinotransferase (L-arginine:glycine amidinotransferase)

Gcdh glutaryl-Coenzyme A dehydrogenase



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Gch1 GTP cyclohydrolase 1

Gck glucokinase

Gcsh glycine cleavage system protein H (aminomethyl carrier)

Gdi1 guanosine diphosphate (GDP) dissociation inhibitor 1Gfap glial fibril lary acidic protein

Glb1 galactosidase, beta 1

Gldc glycine decarboxylase

Gli3 GLI-Kruppel family member GLI3

Glo1 glyoxalase 1

Glra2 glycine receptor, alpha 2 subunit

Gm2a GM2 ganglioside activator protein

Gnas GNAS (guanine nucleotide binding protein, alpha stimulating) complex locus

Gnpat glyceronephosphate O-acyltransferase

Gns glucosamine (N-acetyl)-6-sulfatase

Gon4l gon-4-like (C.elegans)

Gpc3 glypican 3

Gphn gephyrin

Gpi1 glucose phosphate isomerase 1

Gpx1 glutathione peroxidase 1Grcc10 gene rich cluster, C10 gene

Gria3 glutamate receptor, ionotropic, AMPA3 (alpha 3)

Grik2 glutamate receptor, ionotropic, kainate 2 (beta 2)

Grin2a glutamate receptor, ionotropic, NMDA2A (epsilon 1)

Grin2b glutamate receptor, ionotropic, NMDA2B (epsilon 2)

Grm8 glutamate receptor, metabotropic 8

Grpr gastrin releasing peptide receptor

Gss glutathione synthetase

Gstm1 glutathione S-transferase, mu 1

Gucy2f guanylate cyclase 2f

Gusb glucuronidase, beta

Gyk glycerol kinase

Hadh hydroxyacyl-Coenzyme A dehydrogenase

Hccs holocytochrome c synthetase

Hdac4 histone deacetylase 4Hesx1 homeobox gene expressed in ES cells

Hexa hexosaminidase A

Hexb hexosaminidase B

Hla-a major histocompatibility complex, class I, A

Hla-drb1 major histocompatibility complex, class II, DR beta 1

Hlcs holocarboxylase synthetase (biotin- [propriony-Coenzyme A carboxylase (ATP-hydrolyzing)] ligase)

Hmgcl 3-hydroxy-3-methylglutaryl-Coenzyme A lyase

Hnrnph2 heterogeneous nuclear ribonucleoprotein H2

Hoxa1 homeobox A1

Hoxb1 homeobox B1

Hpd 4-hydroxyphenylpyruvic acid dioxygenase

Hprt hypoxanthine guanine phosphoribosyl transferase

Hras Harvey rat sarcoma virus oncogene

Hs3st5 heparan sulfate (glucosamine) 3-O-sulfotransferase 5

Hsd11b1 hydroxysteroid 11-beta dehydrogenase 1Hsd17b10 hydroxysteroid (17-beta) dehydrogenase 10

Hspd1 heat shock protein 1 (chaperonin)

Htr1b 5-hydroxytryptamine (serotonin) receptor 1B

Htr2a 5-hydroxytryptamine (serotonin) receptor 2A

Htr3a 5-hydroxytryptamine (serotonin) receptor 3A

Htr3c 5-hydroxytryptamine (serotonin) receptor 3, family member C

Htr7 5-hydroxytryptamine (serotonin) receptor 7

Huwe1 HECT, UBA and WWE domain containing 1

Ids iduronate 2-sulfatase

Idua iduronidase, alpha-L-

Igbp1 immunoglobulin (CD79A) binding protein 1

Igbp1b immunoglobulin (CD79A) binding protein 1b

Igf1 insulin-like growth factor 1

Ikbkg inhibitor of kappaB kinase gamma


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Il1rapl1 interleukin 1 receptor accessory protein-like 1

Immp2l IMP2 inner mitochondrial membrane peptidase-like (S. cerevisiae)

Inpp1 inositol polyphosphate-1-phosphatase

Inpp4a inositol polyphosphate-4-phosphatase, type IInpp5e inositol polyphosphate-5-phosphatase E

Iqsec2 IQ motif and Sec7 domain 2

Itga4 integrin alpha 4

Itga7 integrin alpha 7

Itgb3 integrin beta 3

Itgb7 integrin beta 7

Jag1 jagged 1

Jmjd1c jumonji domain containing 1C

Kank1 KN motif and ankyrin repeat domains 1

Kcnj1 potassium inwardly-rectifying channel, subfamily J, member 1

Kcnj10 potassium inwardly rectifying channel, subfamily J, member 10

Kcnj11 potassium inwardly rectifying channel, subfamily J, member 11

Kcnk9 potassium channel, subfamily K, member 9

Kcnma1 potassium large conductance calcium-activated channel, subfamily M, alpha member 1

Kctd7 potassium channel tetramerization domain containing 7Kdm5a lysine (K)-specific demethylase 5A

Kdm5c lysine (K)-specific demethylase 5C

Kdm6b KDM1 lysine (K)-specific demethylase 6B

Kiaa1586 Kiaa1586

Kif7 kinesin family member 7

Kirrel3 kin of IRRE like 3 (Drosophila)

Klf8 Kruppel-like factor 8

Klhl15 kelch-like 15

Kmt2c lysine (K)-specific methyltransferase 2C

Kras v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog

L1cam L1 cell adhesion molecule

L2hgdh L-2-hydroxyglutarate dehydrogenase

Lama1 laminin, alpha 1

Lama2 laminin, alpha 2

Lamb1 laminin B1Lamp2 lysosomal-associated membrane protein 2

Large l ike-glycosyltransferase

Larp7 La ribonucleoprotein domain family, member 7

Las1l LAS1-like (S. cerevisiae)

Lrfn5 leucine rich repeat and fibronectin type III domain containing 5

Lrpprc leucine-rich PPR-motif containing

Lrrc1 leucine rich repeat containing 1

Lzts2 leucine zipper, putative tumor suppressor 2

Macrod2 MACRO domain containing 2

Magt1 magnesium transporter 1

Man1b1 mannosidase, alpha, class 1B, member 1

Man2b1 mannosidase 2, alpha B1

Manba mannosidase, beta A, lysosomal

Maoa monoamine oxidase A

Map2 microtubule-associated protein 2Map2k1 mitogen-activated protein kinase kinase 1

Map2k2 mitogen-activated protein kinase kinase 2

Mark1 MAP/microtubule affinity-regulating kinase 1

Mat1a methionine adenosyltransferase I, alpha

Mbd1 methyl-CpG binding domain protein 1




Mbtps2 membrane-bound transcription factor peptidase, site 2

Mccc1 methylcrotonoyl-Coenzyme A carboxylase 1 (alpha)

Mccc2 methylcrotonoyl-Coenzyme A carboxylase 2 (beta)

Mcoln1 mucolipin 1

Mcph1 microcephaly, primary autosomal recessive 1

Mdga2 MAM domain containing glycosylphosphatidylinositol anchor 2



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Mecp2 methyl CpG binding protein 2

Med12 mediator complex subunit 12

Med13l mediator complex subunit 13-like

Med17 mediator complex subunit 17Med23 mediator complex subunit 23

Med25 mediator complex subunit 25

Mef2c myocyte enhancer factor 2C

Met met proto-oncogene

Mgat2 mannoside acetylglucosaminyltransferase 2

Mid1 midline 1

Mkks McKusick–Kaufman syndrome

Mll2 myeloid/lymphoid or mixed-lineage leukemia 2

Mlycd malonyl-CoA decarboxylase

Mmachc methylmalonic aciduria cblC type, with homocystinuria

Mocs1 molybdenum cofactor synthesis 1

Mocs2 molybdenum cofactor synthesis 2

Mogs mannosyl-oligosaccharide glucosidase

Mpdu1 mannose-P-dolichol utilization defect 1

Mtf1 metal response element binding transcription factor 1Mthfr 5,10-methylenetetrahydrofolate reductase

Mtm1 X-linked myotubular myopathy gene 1

Mtr 5-methyltetrahydrofolate-homocysteine methyltransferase

Mtrr 5-methyltetrahydrofolate-homocysteine methyltransferase reductase

Myo16 myosin XVI

Myo5a myosin VA

Naga N-acetyl galactosaminidase, alpha

Naglu alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB)

Nbea neurobeachin

Nbn nibrin

Nd5 NADH dehydrogenase subunit 5

Ndnl2 necdin-like 2

Ndp Norrie disease (pseudoglioma) (human)

Ndst1 N-deacetylase/N-sulfotransferase (heparan glucosaminyl) 1

Ndufa1 NADH dehydrogenase (ubiquinone) 1 alpha subcomplex, 1Ndufs1 NADH dehydrogenase (ubiquinone) Fe-S protein 1

Ndufs2 NADH dehydrogenase (ubiquinone) Fe-S protein 2




Ndufv1 NADH dehydrogenase (ubiquinone) flavoprotein 1

Neu1 neuraminidase 1

Nf1 neurofibromatosis 1

Nhs Nance-Horan syndrome (human)

Nkx2-1 NK2 homeobox 1

Nlgn1 neuroligin 1

Nlgn3 neuroligin 3

Nlgn4-x neuroligin 4, X-linked

Nlgn4-y neuroligin 4, Y-linked

Nlrp3 NLR family, pyrin domain containing 3Nos1ap nitric oxide synthase 1 (neuronal) adaptor protein

Nos2a nitric oxide synthase 2A, inducible

Npas2 neuronal PAS domain protein 2

Npc1 Niemann-Pick type C1

Npc2 Niemann-Pick type C2

Nras neuroblastoma ras oncogene

Nrcam neuronal cell adhesion molecule

Nrp2 neuropilin 2

Nrxn1 neurexin I

Nsd1 nuclear receptor-binding SET-domain protein 1

Nsdhl NAD(P)-dependent steroid dehydrogenase-like

Ntng1 netrin G1

Ntrk1 neurotrophic tyrosine kinase, receptor, type 1

Ntrk3 neurotrophic tyrosine kinase, receptor, type 3


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Oat ornithine aminotransferase

Ocrl oculocerebrorenal syndrome of Lowe

Odf3l2 outer dense fiber of sperm tails 3-like 2

Ofd1 oral-facial-digital syndrome 1 gene homolog (human)Ophn1 oligophrenin 1

Oprm1 opioid receptor, mu 1

Or1c1 olfactory receptor, family 1, subfamily C, member 1

Otc ornithine transcarbamylase

Oxtr oxytocin receptor

Pafah1b1 platelet-activating factor acetylhydrolase, isoform 1b, subunit 1

Pah phenylalanine hydroxylase

Pak3 p21 protein (Cdc42/Rac)-activated kinase 3

Pank2 pantothenate kinase 2

Park2 Parkinson disease (autosomal recessive, juvenile) 2, parkin

Parp1 poly (ADP-ribose) polymerase family, member 1

Pax8 paired box 8

Pcca propionyl-Coenzyme A carboxylase, alpha polypeptide

Pccb propionyl Coenzyme A carboxylase, beta polypeptide

Pcdh10 protocadherin 10Pcdh19 protocadherin 19

Pcdh9 protocadherin 9

Pcp4 Purkinje cell protein 4

Pcx pyruvate carboxylase

Pde9a phosphodiesterase 9A

Pdha1 pyruvate dehydrogenase E1 alpha 1

Pdhx pyruvate dehydrogenase complex, component X

Pdp1 pyruvate dehydrogenase phosphatase catalytic subunit 1

Pdzd4 PDZ domain containing 4

Pecr peroxisomal trans-2-enoyl-CoA reductase

Pepd peptidase D

Per1 period circadian clock 1

Pex1 peroxisomal biogenesis factor 1


Pex12 peroxisomal biogenesis factor 12Pex13 peroxisomal biogenesis factor 13





Pex5l peroxisomal biogenesis factor 5-like



Pgk1 phosphoglycerate kinase 1

Phf6 PHD finger protein 6

Phf8 PHD finger protein 8

Pik3cg phosphoinositide-3-kinase, catalytic, gamma polypeptide

Pitx1 paired-like homeodomain transcription factor 1

Pla2g6 phospholipase A2, group VI

Pln phospholambanPlp1 proteolipid protein (myelin) 1

Pmm2 phosphomannomutase 2

Pnp purine-nucleoside phosphorylase

Polr3b polymerase (RNA) III (DNA directed) polypeptide B

Pomgnt1 protein O-linked mannose beta 1,2-N-acetylglucosaminyltransferase

Pomt1 protein-O-mannosyltransferase 1

Pon1 paraoxonase 1

Porcn porcupine homolog (Drosophila)

Pou1f1 POU domain, class 1, transcription factor 1

Ppox protoporphyrinogen oxidase

Ppp1r3f protein phosphatase 1, regulatory (inhibitor) subunit 3F

Ppt1 palmitoyl-protein thioesterase 1

Pqbp1 polyglutamine binding protein 1

Prkcb protein kinase C, beta



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Prkcg protein kinase C, gamma

Prkra protein kinase, interferon inducible double stranded RNA-dependent activator

Prmt10 protein arginine methyltransferase 10 (putative)

Prps1 phosphoribosyl pyrophosphate synthetase 1Prrt2 proline-rich transmembrane protein 2

Prss12 protease, serine 12 neurotrypsin (motopsin)

Psap prosaposin

Psmd10 proteasome (prosome, macropain) 26S subunit, non-ATPase, 10

Ptch1 patched homolog 1

Ptch2 patched homolog 2

Ptchd1 patched domain containing 1

Pten phosphatase and tensin homolog

Ptgs2 prostaglandin-endoperoxide synthase 2

Ptpn11 protein tyrosine phosphatase, non-receptor type 11

Pts 6-pyruvoyl-tetrahydropterin synthase

Pvrl1 poliovirus receptor-related 1

Pycr1 pyrroline-5-carboxylate reductase 1

Qdpr quinoid dihydropteridine reductase

Rab39b RAB39B, member RAS oncogene familyRaf1 v-raf-leukemia viral oncogene 1

Rai1 retinoic acid induced 1

Ralgds ral guanine nucleotide dissociation stimulator

Rapgef4 Rap guanine nucleotide exchange factor (GEF) 4

Rb1cc1 RB1-inducible coiled-coil 1

Rbm10 RNA binding motif protein 10

Rbm28 RNA binding motif protein 28

Reep3 receptor accessory protein 3

Reln reelin

Rfwd2 ring finger and WD repeat domain 2

Rgs7 regulator of G protein signaling 7

Rhoxf1 Rhox homeobox family, member 1

Rims3 regulating synaptic membrane exocytosis 3

Rlim ring finger protein, LIM domain interacting

Robo1 roundabout homolog 1 (Drosophila)Rpgrip1l Rpgrip1-like

Rpl10 ribosomal protein L10

Rps6ka2 ribosomal protein S6 kinase, polypeptide 2

Rps6ka3 ribosomal protein S6 kinase polypeptide 3

Sar1b SAR1 gene homolog B (S. cerevisiae)

Satb2 special AT-rich sequence binding protein 2

Sc5d sterol-C5-desaturase (fungal ERG3, delta-5-desaturase) homolog (S. cerevisiae)

Scaper S phase cyclin A-associated protein in the ER

Scn1a sodium channel, voltage-gated, type I, alpha

Scn2a1 sodium channel, voltage-gated, type II, alpha 1

Sdc2 syndecan 2

Sdha succinate dehydrogenase complex, subunit A, flavoprotein (Fp)

Sema5a sema domain, seven thrombospondin repeats (type 1 and type 1-like), transmembrane domain (TM) and short cytoplasmic

domain, (semaphorin) 5A

Setbp1 SET binding protein 1Sez6l2 seizure-related 6 homolog like 2

Sgsh N-sulfoglucosamine sulfohydrolase (sulfamidase)

Sh3kbp1 SH3-domain kinase binding protein 1

Shank2 SH3/ankyrin domain gene 2

Shank3 SH3/ankyrin domain gene 3

Shh sonic hedgehog

Shoc2 soc-2 (suppressor of clear) homolog (C. elegans)

Shroom4 shroom family member 4

Six3 sine oculis-related homeobox 3

Slc12a1 solute carrier family 12, member 1

Slc12a6 solute carrier family 12, member 6

Slc16a2 solute carrier family 16 (monocarboxylic acid transporters), member 2

Slc17a5 solute carrier family 17 (anion/sugar transporter), member 5

Slc1a1 solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter, system Xag), member 1


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Slc25a12 solute carrier family 25 (mitochondrial carrier, Aralar), member 12

Slc25a15 solute carrier family 25 (mitochondrial carrier ornithine transporter), member 15

Slc2a1 solute carrier family 2 (facilitated glucose transporter), member 1

Slc31a1 solute carrier family 31, member 1Slc35c1 solute carrier family 35, member C1

Slc4a10 solute carrier family 4, sodium bicarbonate cotransporter-like, member 10

Slc4a4 solute carrier family 4 (anion exchanger), member 4

Slc5a5 solute carrier family 5 (sodium iodide symporter), member 5

Slc6a4 solute carrier family 6 (neurotransmitter transporter, serotonin), member 4

Slc6a8 solute carrier family 6 (neurotransmitter transporter, creatine), member 8

Slc7a7 solute carrier family 7 (cationic amino acid transporter, y+ system), member 7

Slc9a6 solute carrier family 9 (sodium/hydrogen exchanger), member 6

Slc9a9 solute carrier family 9 (sodium/hydrogen exchanger), member 9

Smc1a structural maintenance of chromosomes 1A

Smc3 structural maintenance of chromosomes 3

Smpd1 sphingomyelin phosphodiesterase 1, acid lysosomal

Sms spermine synthase

Snap29 synaptosomal-associated protein 29

Sos1 son of sevenless homolog 1 (Drosophila)Sox10 SRY (sex determining region Y)-box 10

Sox3 SRY (sex determining region Y)-box 3

Spg11 spastic paraplegia 11

Srd5a3 steroid 5 alpha-reductase 3

Srpx2 sushi-repeat-containing protein, X-linked 2

St7 suppression of tumorigenicity 7

Stag2 stromal antigen 2

Stard8 START domain containing 8

Stk39 serine/threonine kinase 39

Stxbp1 syntaxin binding protein 1

Sucla2 succinate-Coenzyme A ligase, ADP-forming, beta subunit

Suclg2 succinate-Coenzyme A ligase, GDP-forming, beta subunit

Suox sulfite oxidase

Surf1 surfeit gene 1

Syn1 synapsin ISyngap1 synaptic Ras GTPase activating protein 1 homolog (rat)

Syp synaptophysin

Syt17 synaptotagmin XVII

Taf1 TAF1 RNA polymerase II, TATA box binding protein (TBP)-associated factor



Taf7l TAF7-like RNA polymerase II, TATA box binding protein (TBP)-associated factor

Taf9b TAF9B RNA polymerase II, TATA box binding protein (TBP)-associated factor

Tat tyrosine aminotransferase

Tbc1d24 TBC1 domain family, member 24

Tbce tubulin-specific chaperone E

Tcf4 transcription factor 4

Tcn2 transcobalamin 2

Tdgf1 teratocarcinoma-derived growth factor 1

Tdo2 tryptophan 2,3-dioxygenaseTg thyroglobulin

Tgfbr1 transforming growth factor, beta receptor I

Tgfbr2 transforming growth factor, beta receptor II

Tgif1 TGFB-induced factor homeobox 1

Th tyrosine hydroxylase

Thoc2 THO complex 2

Thrb thyroid hormone receptor beta

Timm8a1 translocase of inner mitochondrial membrane 8A1

Tmem135 transmembrane protein 135

Agmo alkylglycerol monooxygenase

Tmem67 transmembrane protein 67

Tph2 tryptophan hydroxylase 2

Tpi1 triosephosphate isomerase 1

Tpo thyroid peroxidase



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We present here data showing clock genes in thehippocampus are expressed in a circadian-dependent

manner in dark/dark conditions. Consistently, the

abundance of Rev-erba protein and mRNA was

inversely correlated, which was expected given that

Rev-erba represses its own transcription (Valnegri et al.,

2011). The abundance of Clock protein peaked at CT0,

about 3 h after the peak in clock transcription. This analy-

sis of mRNA and protein abundance suggests that the

hippocampus presents some characteristics of an

endogenous clock. However, previous experiments

examining the expression of clock genes at the mRNA

and protein level in light/dark conditions showed that the

expression of most clock genes peaks simultaneously,

suggesting the absence of an endogenous oscillator in

the hippocampus (Jilg et al., 2010). Therefore, it is impor-tant to conduct parallel studies in both dark/dark and

light/dark conditions to determine how the SCN synchro-

nization by light influences gene expression in the

hippocampus.

The expression of circadian oscillating genes is very

tissue specific. Indeed, Panda et al. found little overlap

in CCC genes expressed in the SCN and those

expressed in the liver (about 8%) (Panda et al., 2002).

Similarly, among the 663 hippocampal circadian genes

identified in this study, less than 3% correspond to

circadian-regulated genes in the SCN. This tissue speci-

ficity suggests that most of these genes have specific

functions in the hippocampus. Indeed, we identified

pathways related to memory in our list of hippocampal



Tpp1 tripeptidyl peptidase I

Trappc9 trafficking protein particle complex 9

Trmt1 tRNA methyltransferase 1

Tsc1 tuberous sclerosis 1Tsc2 tuberous sclerosis 2

Tshb thyroid stimulating hormone, beta subunit

Tshr thyroid stimulating hormone receptor

Tsn translin

Tspan7 tetraspanin 7

Tuba1a tubulin, alpha 1A

Tuba8 tubulin, alpha 8

Tubb2b tubulin, beta 2B class IIB

Tubb3 tubulin, beta 3 class III

Tusc3 tumor suppressor candidate 3

Ube2a ubiquitin-conjugating enzyme E2A

Ube2h ubiquitin-conjugating enzyme E2H

Ube3a ubiquitin protein ligase E3A

Ubr7 ubiquitin protein ligase E3 component n-recognin 7 (putative)

Upb1 ureidopropionase, betaUpf3b UPF3 regulator of nonsense transcripts homolog B (yeast)

Usp27x ubiquitin specific peptidase 27, X chromosome

Vash1 vasohibin 1

Vldlr very low density lipoprotein receptor

Vps13b vacuolar protein sorting 13B (yeast)

Vrk1 vaccinia-related kinase 1

Wdr45b WD repeat domain 45B

Wdr62 WD repeat domain 62

Wnk3 WNK lysine deficient protein kinase 3

Wnt2 wingless-type MMTV integration site family, member 2

Xpa xeroderma pigmentosum, complementation group A

Xpc xeroderma pigmentosum, complementation group C

Xpnpep3 X-prolyl aminopeptidase (aminopeptidase P) 3, putative

Ywhae tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon polypeptide

Yy1 YY1 transcription factor Zbtb16 zinc finger and BTB domain containing 16

Zbtb40 zinc finger and BTB domain containing 40

Zcchc8 zinc finger, CCHC domain containing 8

Zdhhc15 zinc finger, DHHC domain containing 15

Zdhhc9 zinc finger, DHHC domain containing 9

Zeb2 zinc finger E-box binding homeobox 2

Zfp526 zinc finger protein 526



Zic2 zinc finger protein of the cerebellum 2

Zic3 zinc finger protein of the cerebellum 3


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Table 3. Circadian clock controlled genes involved in ID. List of the 30 ID genes showing a CCC pattern of expression in the mouse hippocampus

among the 673 ID genes

Gene

symbol

Gene name Chromosome

(H/M)

Function Disease(s) also

associated to ID

GO annotations

Human Mus

musculus

AFF2 Aff2 AF4/FMR2 family,

member 2

X RNA-binding protein Fragile XE syndrome G-quadruplex RNA

binding

ALG2 Alg2 asparagine-linked

glycosylation 2

(alpha-1,3-

mannosyltransferase)

9/4 alpha 1,3

mannosyltransferase

congenital disorder of

glycosylation and

congenital disorder of

glycosylation type ii

calcium-dependent

protein binding and

protein

heterodimerization

activity.

ASS1 Ass1 argininosuccinate

synthase 1

9/2 control of blood pressure citrullinemia and

citrullinemia type i

protein binding and

ATP binding

BBS7 Bbs7 Bardet-Biedl

syndrome 7

4/3 ciliogenesis bbs7-related Bardet-

Biedl syndrome and

Bardet-Biedl

syndrome 7

protein binding

BRAF Braf Braf transforming

gene

7/6 transduction of mitogenic

signals from the cell

membrane to the nucleus

colorectal cancer and

hairy cell leukemia

protein kinase

activity and

calcium ion binding

CDH22 Cdh22 cadherin 22 20/2 calcium-dependent cell

adhesion proteins

colorectal cancer calcium ion binding

CLCNKB Clcnkb chloride channel,

voltage-sensitive Kb

1/4 voltage-gated chloride

channel

bartter syndrome type

3 and bartter

syndrome type 4

voltage-gated

chloride channel

activity

CLN6 Cln6 ceroid-lipofuscinosis,

neuronal 6

15/9 degradation of post-

translationally modified

proteins in lysosomes

ceroid-lipofuscinosis,

neuronal 6, variant

late infantile and

ceroid lipofuscinosis

neuronal 6.

protein binding and

protein

homodimerization

activity

DAPK1 Dapk1 death-associated

protein kinase 1

9/13 calcium/calmodulin-

dependent serine/threonine

kinase

pediatric lymphoma

and central nervous

system lymphoma

protein kinase

activity and

identical protein

binding

DBT Dbt dihydrolipoamide

branched chain

transacylase E2

1/3 the branched-chain alpha-

keto dehydrogenase complex

catalyzes the overall

conversion of alpha-keto

acids to acyl-CoA and CO(2)

maple syrup urine

disease and maple

syrup urine disease

type 2

dihydrolipolylysine-

residue (2-

methylpropanoyl)

transferase activity

and cofactor

binding

FABP5 Fabp5 fatty acid binding

protein 5

8/3 high specificity for fatty acids bladder squamous

cell carcinoma and

psoriasis

transporter activity

and fatty acid

binding

FGFR3 Fgfr 3 fibroblast growth

factor receptor 3

4/5 tyrosine-protein kinase acting

as cell-surface receptor for

fibroblast growth factors and

playing an essential role in the

regulation of cell proliferation,

differentiation and apoptosis

achondroplasia and

hypochondroplasia

protein tyrosine

kinase activity and

fibroblast growth

factor-activated

receptor activity

FKRP Fkrp fukutin-related protein 19/7 post-translational modification

of dystroglycan

limb-girdle muscular

dystrophy type 2i and

muscular dystrophy-

dystroglycanopathy

transferase activity

GALE Gale UDP-galactose-4-

epimerase

1/4 epimerization of UDP-glucose

to UDP-galactose and the

epimerization of UDP-N-

acetylglucosamine to UDP-N-

acetylgalactosamine

epimerase deficiency

galactosemia and

erythrocyte galactose

epimerase deficiency

coenzyme binding

and protein

homodimerization

activity

GLDC Gldc glycine

decarboxylase

9/19 degradation of glycine gldc-related glycine

encephalopathy and

glycine

encephalopathy

electron carrier

activity and lyase

activity



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Gene

symbol

Gene name Chromosome

(H/M)

Function Disease(s) also

associated to ID

GO annotations

Human Mus

musculus

GUCY2F Gucy2f guanylate cyclase 2F X resynthesis of cGMP after

light activation of the visual

signal transduction cascade

retinitis and retinal

disease

protein

heterodimerization

activity and protein

homodimerization

activity

HEXB Hexb hexosaminidase B 5/13 degradation of GM2

gangliosides and of other

molecules containing terminal

N-acetyl hexosamines

sandhoff disease and

cervical cancer,

cation binding and

protein

homodimerization

activity

KMT2C Kmt 2c lysine (K)-specific

methyltransferase 2C

7/5 histone methyltransferase transcriptional

coactivation

MAGT1 Magt1 magnesium

transporter 1

X N-glycosylation and Mg(2+)

transport

x-linked magnesium

deficiency with

epstein-barr virus

infection and

neoplasia and t

lymphocyte

deficiency

magnesium ion

transmembrane


MKKS Mkks McKusick–Kaufman

syndrome

20/2 folding of proteins upon ATP

hydrolysis

Bardet-Biedl

syndrome and

McKusick–Kaufman

syndrome

unfolded protein

binding and ATP

binding

PARK2 Park2 Parkinson disease

(autosomal

recessive, juvenile) 2,

parkin

6/17 part of a multiprotein E3

ubiquitin ligase complex,

catalyzing the covalent

attachment of ubiquitin

moieties onto substrate

proteins

Parkinson’s disease

and parkin type of

juvenile Parkinson

disease

protein kinase

binding and

ubiquitin protein

ligase binding.

PCDH10 Pcdh10 protocadherin 10 4/3 potential calcium-dependent

cell-adhesion protein

dravet syndrome and

nasopharyngitis

calcium ion binding

PECR Pecr peroxisomal trans-2-

enoyl-CoA reductase

2/1 chain elongation of fatty acids alcoholism and

hirschsprung’s

disease

2,4-dienoyl-CoA

reductase

(NADPH) activity

and nucleotide

binding

PER1 Per1 period circadian clock

1

17/11 transcription factor acting as a

repressor in the circadian

transcriptional loop

advanced sleep

phase syndrome and

delayed sleep phase

syndrome

E-box binding and

signal transducer

activity

PEX3 Pex3 peroxisomal

biogenesis factor 3

6/10 peroxisome biosynthesis and

integrity

zellweger syndrome,

complementation

group g and

peroxisome

biogenesis disorder

10a

lipid binding and

protein

dimerization

activity

PSMD10 PSMD10 proteasome

(prosome,

macropain) 26S

subunit, non-ATPase,

10

X chaperone during the

assembly of the 26S

proteasome

hepatocellular

carcinoma and

autism spectrum

disorder

protein binding and

transcription factor

binding

RPL10 RPL10 ribosomal protein L10 X protein synthesis, component

of the 60S subunit of the

ribosome

autism spectrum

disorder and wilms

tumor

structural

constituent of

ribosome

SLC7A7 SLC7A7 solute carrier family 7

(cationic amino acid

transporter, y+

system), member 7

14 sodium-independent uptake

of dibasic amino acids and

sodium-dependent uptake of

some neutral amino acids

lysinuric protein

intolerance and

cystinuria

amino acid

transmembrane


ZBTB40 ZBTB40 zinc finger and BTB

domain containing 40

1/4 transcriptional regulation DNA binding


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context. Learning entrains hippocampal memory-specific

neuronal networks, through the cAMP and MAPK path-

ways. The amplification or modulation of oscillations in

these signaling pathways may encode memory before

it is transferred from the hippocampus to the cortex

for long-term storage (Eckel-Mahan and Storm, 2009).

It also appears that this transfer of information betweenthese two structures depends on sleep (Ji and Wilson,

2007).

The role of core clock genes in memory formation,

synaptic plasticity and learning is further strengthened

by the recent derivation of a Per1 KO mouse. These

mice present daytime deficits in working memory

performance together with the loss of CR-regulated

CREB phosphorylation in the hippocampus (Rawashdeh

et al., 2014). Although synaptic plasticity is closely related

to memory and learning (Whitlock et al., 2006), genes

encoding excitatory synaptic proteins were not enriched

in our dataset of CCC genes. The multicellular environ-

ment of the hippocampus may explain this lack of enrichment. Alternatively, although we focused on tran-

scriptional regulation in our study, post-transcriptional

and post-translational modifications may also drive the

circadian expression of synaptic proteins. Indeed, pro-

cesses such as RNA methylation (Fustin et al., 2013)

and protein phosphorylation (Gallego and Virshup,

2007) are key regulators of the circadian clock.

Sleep is partly controlled by CR. Specific sequences

of neuronal activity are replayed during sleep, and sleep

and consolidation of memory are related processes

(Shank and Margoliash, 2009; Cairney et al., 2014).

Furthermore, sleep promotes the formation of dendritic

spines in the cortex after learning (Yang et al., 2014).

We show here that hippocampal CCC genes are enrichedin SWG. This overlap supports the idea that sleep, the

circadian clock and memory are related processes.

Slow wave sleep (SWS), occurring during non REM

sleep (non-rapid eye movement sleep), is the deepest

form of sleep. It is related to the consolidation and

strengthening of memory and reflects communication

between the hippocampus and cortex (Huber and Born,

2014). In humans, slow EEG frequencies are implicated

in long-term memory performance and consolidation

(Marshall et al., 2006; Moroni et al., 2014). The highest

levels of SWS occur during childhood, which supports

the cognitive role of this type of sleep (Fogel et al.,

2012). Children with ID, in particular those with Fragile

X or Down syndrome, show lower than normal levels of

A1 EEG slow oscillations, a subtype that reflects the

deepening of sleep and is associated with higher cogni-

tive function (Miano et al., 2007, 2008). Aff2, also known

as Fmr2 , has also been implicated in the sleep/wake

processes in patients with FRAXE-linked ID (Musumeci

et al., 2000). Interestingly, we also identified Aff2 as a

CCC ID gene, suggesting that its rhythmic expression is

important for memory consolidation.

Overall, our list of CCC ID genes will be very useful for

retrospective and prospective clinical studies. Indeed, it

would be interesting to search for sleep defects in

children with ID and mutations in one of these 30 CCC

genes. In addition these genes are good candidates for

mutation screening in patients with ID and sleep

abnormalities. Our findings may also help clinicians to

decide at what time of day patients should receive

education. Indeed, the benefit of dedicated care may

depend on the rhythmicity of the expression of CCC ID

genes. Further studies examining this link may lead to

new therapeutic strategies.

CONCLUSIONS

Our analysis demonstrates that about 3.5% of genes

expressed in the mouse hippocampus show a CCC

pattern of expression and that ID genes are not

enriched among these CCC genes. In addition, we

identify a list of 30 ID genes showing CCC expression.

Mutations in these genes may lead to defects in the

circadian regulation of hippocampal-dependent memory

consolidation and/or in sleep/wake disturbances

observed in patients with ID.

Acknowledgments—We thank Cochin Institute genomic facility

for technical assistance. We thank Dr. Etienne Challet (from

INCI, Strasbourg, France) for his expertise on CR. This work

was supported by grants from Inserm, Agence Nationale de la

Recherche (ANR-2010-BLANC-1434-03), Europe (Euro FP7,

Gencodys, 241995) and Roche (Agreement, N 121531A10).

J.R. received a postdoctoral fellowship from Roche.

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