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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 )
(continued on next page)
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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Table 1 (continued )
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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).
Table 1 (continued )
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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.
32 J. Renaud et al. /Neuroscience 308 (2015) 11–50
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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.
J. Renaud et al. / Neuroscience 308 (2015) 11–50 33
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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)
Alg2 asparagine-linked glycosylation 2 (alpha-1,3-mannosyltransferase)
Alg3 asparagine-linked glycosylation 3 (alpha-1,3-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
34 J. Renaud et al. /Neuroscience 308 (2015) 11–50
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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Table 2 (continued )
Gene symbol Gene name
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
Ercc3 excision repair cross-complementing rodent repair deficiency, complementation group 3
Ercc5 excision repair cross-complementing rodent repair deficiency, complementation group 5
Ercc6 excision repair cross-complementing rodent repair deficiency, complementation group 6
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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Table 2 (continued )
Gene symbol Gene name
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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Table 2 (continued )
Gene symbol Gene name
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
Mbd3 methyl-CpG binding domain protein 3
Mbd4 methyl-CpG binding domain protein 4
Mbd5 methyl-CpG binding domain protein 5
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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Table 2 (continued )
Gene symbol Gene name
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
Ndufs4 NADH dehydrogenase (ubiquinone) Fe-S protein 4
Ndufs7 NADH dehydrogenase (ubiquinone) Fe-S protein 7
Ndufs8 NADH dehydrogenase (ubiquinone) Fe-S protein 8
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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Table 2 (continued )
Gene symbol Gene name
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
Pex10 peroxisomal biogenesis factor 10
Pex12 peroxisomal biogenesis factor 12Pex13 peroxisomal biogenesis factor 13
Pex16 peroxisomal biogenesis factor 16
Pex19 peroxisomal biogenesis factor 19
Pex3 peroxisomal biogenesis factor 3
Pex5 peroxisomal biogenesis factor 5
Pex5l peroxisomal biogenesis factor 5-like
Pex6 peroxisomal biogenesis factor 6
Pex7 peroxisomal biogenesis factor 7
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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Table 2 (continued )
Gene symbol Gene name
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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Table 2 (continued )
Gene symbol Gene name
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
Taf2 TAF2 RNA polymerase II, TATA box binding protein (TBP)-associated factor
Taf7 TAF7 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
Table 2 (continued )
Gene symbol Gene name
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
Zfp592 zinc finger protein 592
Zfp711 zinc finger protein 711
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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Table 3 (continued )
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
transporter activity
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
transporter activity
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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