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The Nuclear Receptor REV-ERBa Regulates Fabp7 andModulates Adult Hippocampal NeurogenesisAnna Schnell1, Sylvie Chappuis1¤a, Isabelle Schmutz1¤b, Emanuele Brai2, Jurgen A. Ripperger1,

Olivier Schaad3¤c, Hans Welzl4, Patrick Descombes3¤d, Lavinia Alberi2, Urs Albrecht1*

1 Dept. of Biology, Unit of Biochemistry, University of Fribourg, Fribourg, Switzerland, 2 Dept. of Medicine, Unit of Anatomy, University of Fribourg, Fribourg, Switzerland,

3 NCCR frontiers in Genetics, University of Geneva, Geneva, Switzerland, 4 Dept. of Anatomy, University of Zurich, Zurich, Switzerland

Abstract

The function of the nuclear receptor Rev-erba (Nr1d1) in the brain is, apart from its role in the circadian clock mechanism,unknown. Therefore, we compared gene expression profiles in the brain between wild-type and Rev-erba knock-out (KO)animals. We identified fatty acid binding protein 7 (Fabp7, Blbp) as a direct target of repression by REV-ERBa. Loss of Rev-erba manifested in memory and mood related behavioral phenotypes and led to overexpression of Fabp7 in various brainareas including the subgranular zone (SGZ) of the hippocampus, where neuronal progenitor cells (NPCs) can initiate adultneurogenesis. We found increased proliferation of hippocampal neurons and loss of its diurnal pattern in Rev-erba KO mice.In vitro, proliferation and migration of glioblastoma cells were affected by manipulating either Fabp7 expression or REV-ERBa activity. These results suggest an important role of Rev-erba and Fabp7 in adult neurogenesis, which may open newavenues for treatment of gliomas as well as neurological diseases such as depression and Alzheimer.

Citation: Schnell A, Chappuis S, Schmutz I, Brai E, Ripperger JA, et al. (2014) The Nuclear Receptor REV-ERBa Regulates Fabp7 and Modulates Adult HippocampalNeurogenesis. PLoS ONE 9(6): e99883. doi:10.1371/journal.pone.0099883

Editor: Henrik Oster, University of Lubeck, Germany

Received February 20, 2014; Accepted May 19, 2014; Published June 16, 2014

Copyright: � 2014 Schnell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: Funding by the Swiss National Science Foundation, the Velux Foundation and the State of Fribourg is gratefully acknowledged. The funders had norole in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

¤a Current address: Vifor Pharma, Fribourg, Switzerland¤b Current address: The Rockefeller University, New York, New York, United States of America¤c Current address: Dept. of Biochemistry, Sciences II, University of Geneva, Geneva, Switzerland¤d Current address: Nestle Institute of Health Sciences, EPFL Innovation Park, Lausanne, Switzerland

Introduction

In mammals the circadian clock system regulates many aspects

of systemic biology such as biochemistry, physiology and behavior

with the suprachiasmatic nuclei (SCN) as the main coordinating

entity to synchronize all cellular clocks in the body. At the cellular

level, the circadian clockwork consists of interwoven positive and

negative feedback loops, or ‘limbs’. The positive limb involves

BMAL1/CLOCK heterodimers that bind to E-boxes located in

the regulatory region of the period (Per) and cryptochrome (Cry) genes.

CRY and PER proteins form oligomers that are transported from

the cytoplasm to the nucleus, where they repress their own

transcription by inhibiting BMAL1/CLOCK (negative limb). The

positive and negative limbs are further interlaced as BMAL1/

CLOCK also induces the expression of the nuclear receptor REV-

ERBa (NR1D1), which represses the transcription of Bmal1 via

direct binding to a REV-ERBa response element (RORE) in the

Bmal1 promoter [1]. In addition to its action in the circadian clock

mechanism, REV-ERBa also has strong regulatory functions in

liver metabolism [2,3] and drugs targeting it may have potential

applications for treatment of metabolic syndrome [4]. However,

the roles of REV-ERBa in the central nervous system remain

unclear.

Components of the clock mechanism modulate neurogenesis.

For example Per2 regulates neural stem/progenitor cell prolifer-

ation in the adult hippocampus [5] while Bmal1/Clock seems to

regulate neurogenic transcription factors such as Neuro D1 and

differentiation of neuronal stem/progenitor cells in the subven-

tricular zone (SVZ) of the lateral ventricle [6]. Furthermore, gene

expression profiling revealed an increased expression of Rev-erba in

neural progenitor cells (NPCs) compared to immature neurons [7].

Outside of the central nervous system, in the skin, the clock

appears to play a role in the regulation of stem cell differentiation

[8,9].

Adult neurogenesis is an important process, because it may

replace lost or dysfunctional cells by generating new neurons via

neural stem cells (NSCs) [10]. A dysfunction of this process may

lead to neuropsychiatric diseases such as age-related cognitive

decline [11] and depression (reviewed in [12]). Substantial

generation of new neurons occurs mainly in two brain areas: the

subventricular zone (SVZ) lining the lateral ventricles [13] and the

subgranular zone (SGZ) of the hippocampal dentate gyrus (DG)

[14]. Adult hippocampal neurogenesis in mammals is a sensitive

process, which is affected by environmental stimuli, such as stress

[15,16], physical activity [17], sleep deprivation [18], enriched

living conditions [19], and jet-lag [20,21]. Such environmental

changes directly affect the circadian clock [22], suggesting that the

clock may be a mediator between environmental change and

neurogenesis. This hypothesis is supported by the observation that

neurogenesis fluctuates over the day [23–26] indicating that the

circadian clock or components of it may influence neurogenesis.

PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e99883

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Since REV-ERBa is strongly expressed in the brain [27] and in

NPCs [7] we performed genome wide expression profiling in the

SCN of wild-type and Rev-erba KO mice. We found fatty acid

binding protein 7 (FABP7), also termed brain lipid binding protein

(BLBP), to be strongly up-regulated in Rev-erba KO animals.

FABP7 is a family member of the fatty acid binding protein family,

which facilitates the solubility of hydrophobic long chain fatty

acids. They function primarily in fatty acid uptake/transport [28],

and have been widely implicated in cell growth and differentiation

[29]. FABP7 is a well-known marker for NPCs [30] in neurogenic

niches of the hippocampal SGZ [31] and in the forebrain SVZ

[32]. It is expressed in type 2 and 3 NSCs and early transitory

amplifying cells (TAPs) but not in late TAPs and neuroblasts [32].

Interestingly, Fabp7 mRNA is expressed in a time of day

dependent manner in hippocampal granule precursors in adult

mice [33] and its localization and grade of polyadenylation are

diurnal [34]. These observations implicate an involvement of

circadian clock components in the regulation of Fabp7 and adult

neurogenesis.

In this study we show that Fabp7 is a direct target gene of Rev-

erba and that both genes are involved in aspects of adult

neurogenesis in mice.

Methods

Animal experimentsAnimal handling and care was performed in accordance with

the guidelines of the Schweizer Tierschutzgesetz (TSchG, SR455)

and the declaration of Helsinki. The protocol was approved by the

state veterinarian of the Canton of Fribourg. Suffering of animals

was minimized by anesthesia that was induced at 4.5 to 5%

isoflurane and lowered to 2–1.5% isoflurane mixed with oxygen

(0.8l/min). Rev-erba2/2 knockout mice [27] were obtained from

heterozygous Rev-erba breeding pairs originally provided by Prof.

U. Schibler, Geneva. Two to four month old animals were used

for experiments and wild-type mice served as controls. Animals

were kept under 12 h light and 12 h dark (LD 12:12) with food

and water ad libitum.

Cell cultureNG 108-15, mouse neuroblastoma x rat glioblastoma cells [35]

and U-251 MG, human malignant glioblastoma tumour [36] were

used for in vitro experiments. Cells were maintained in Dulbecco’s

Modified Eagle Medium (DMEM), high glucose [4.5 g/l](Sigma

6429) containing 10% fetal calf serum (FCS) and 100 U/ml

penicillin/streptomycin at 37uC in a humidified atmosphere

containing 5% CO2. Sub-confluent cultures were split 1:3 to 1:6.

Affymetrix oligonucleotide microarray hybridizationTo obtain sufficient amounts of SCN, tissue of 18 male mice (3

months) were used per genotype. Dissection was performed at time

point ZT 14. SCN of 6 animals were pooled to yield 3 samples of

wild-type and Rev-erba2/2 mice each, and homogenized in RNA-

Bee (AMS Biotechnology) using syringe and needle (Ø 0.19 mm).

RNA extraction was performed with chloroform, followed by

isorpropanol precipitation and wash with EtOH. For further

purification RNA was precipitated again with 0.4 M NaOAc/

0.2% SDS and extracted using phenol:chloroform:isoamylalcohol.

RNA quality and integrity was checked by absorbance ratio A260/

A280, on denaturing agarose gels and by using the Agilent 2100

Bioanalyzer. 5 mg of total RNA were employed for the synthesis of

biotinylated cRNA and 17.5 mg of this cRNA were hybridized to

Affymetrix Mouse Genome 430 2.0 array (according to the

Affymetrix protocol). The signal intensities were analyzed using

Partek Genomics suites (Partek, St. Louis, MI, USA) and Matlab

(The MathWorks Inc., Natick, Massachusetts, USA) The data

were normalized using RMA [37]. The selections were based on

the fold-change intensities and p values (p,0.05). Genes for which

the concordance in the pairwise comparisons exceeded the

imposed threshold of 77% (seven out of nine comparisons) were

considered as statistically significant and only transcripts whose

accumulation had an average change of at least 1.5-fold were

extracted (Tables S1 and S2).

In situ hybridizationThe in situ hybridization probe for mFabp7 was cloned from

cDNA corresponding to nucleotides 34–588 (accession number:

NM_021272). Primer used for pCR II TOPO (Invitrogen) cloning

are displayed in Table S1. Specimen preparation, 35S-rUTP

labeled riboprobe synthesis and hybridization steps were per-

formed as described earlier [38]. Quantification was performed by

densitometric analysis of autoradiograph films (Amersham Hy-

perfilm MP) using the Quantity One 1-D analysis software

(Biorad). Data from the region of interest was normalized by

comparison with the signal intensities in an equal area of the

lateral hypothalamus. Relative mRNA abundance was calculated

by defining the maximal value of each experiment as 100%. Slides

were further analyzed by dipping in NTB-2 emulsion and

microscopy (Zeiss Axioplan 2). Silver grains were visualized with

dark field illumination and tissue was visualized by counterstaining

of nuclei with Hoechst-dye.

Luciferase reporter assays and transfectionA 1.4 kb fragment of the mouse Fabp7 promoter region

(nucleotides -19348 to the transcriptional start site, containing

RORE at 2934 and 2257) was cloned into the pGL3 basic vector

(Promega) using the primers indicated in Table S1. Deletion of the

proximal RORE (nucleotide 2257) was achieved by site directed

mutagenesis using primers displayed in Table S1, which led to

replacement of the proximal RORE (TGACCT) by nucleotides

GATATC. Expression vectors for Rev-erba (NM_145434) and Rora(NM_013646) have been described [39] and an expression vector

for b-galactosidase was used as control. Transfection and luciferase

reporter assays were performed with NG108-15 cells (Neuroblas-

toma) according to [40]. Empty pGL3-vector and Bmal1 promoter

region cloned into pGL3 [39], were used as negative and positive

controls, respectively. Real-time bioluminescence was monitored

as described in [39] using a LumiCycle apparatus (Actimetrics).

Chromatin immunoprecipitation (ChIP)Hippocampal tissue was dissected using a mouse brain slicer

(Zivic instruments). Freshly isolated tissue of two animals was

combined for homogenization in 1% formaldehyde/1xPBS

pH 7.4 and cross-linked for 5 min at RT. Nuclei and soluble

chromatin fragments were obtained by ultracentrifugation through

1.8 M sucrose cushions and sonication according to [41].

Chromatin was precipitated with antibodies raised against REV-

ERBa [42] and co-immunoprecipitated DNA was quantified with

TaqMan real-time PCR using the primers and probes described in

Table S1. ChIP data was normalized against corresponding input

data and results were presented as percent of input.

Quantitative Real-time PCR (qRT-PCR)Total RNA was extracted and purified from snap frozen brain

tissue using RNeasy kit (Qiagen) with on column DNAse digestion.

RNA from cultured cells was extracted using RNA-Bee (AMS

Biotechnology), purified by phenol:chloroform extraction and

REV-ERBa and Adult Hippocampal Neurogenesis


ethanol precipitation. cDNA was synthesized with SuperScript II

(Invitrogen) and random priming. SYBR green fluorescence-based

real-time PCR was performed for RNA quantification (KAPA

SYBR FAST Universal, KAPA Biosystems, RotorGene 6000,

Corbett Research). All RNA samples were normalized to Gapdh.

Primers are listed in Table S3.

Western blot analysisUsing RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl,

1 mM EDTA, 0.1% SDS, 1% Triton X-100, 0.5% sodium

deoxycholate containing protease and phosphatase inhibitors),

protein of cultured cells and brain tissue was extracted. Proteins

were separated on 12.5% SDS-PAGE and transferred to

nitrocellulose (Protran BA 83, 0.2 mm pores, GE healthcare).

Primary antibodies were incubated over night at 4uC, Anti-rabbit

FABP7 1:250 (Abcam ab27171), Anti-rabbit actin 1:5000 (Sigma,

A5060) and Anti-BMAL1 1:1000 [42]. Detection of the immune

complexes was performed using Western Bright Quantum system

(Advansta) and quantification was done with the Quantity One

analysis software (BioRad). Actin was used for normalization and

relative protein levels were calculated by defining maximal protein

levels as 1.

Behavioral studiesPorsolt Forced swim tests were performed using a cylindrical tank

(35 cm height, 25 cm diameter) filled with water to a height of

20 cm. The water temperature was maintained at 2762uC. An

initial period of 2 min was given for habituation, then immobility

time was recorded during 4 min using a stopwatch. Mice were

considered immobile, when no obvious limb movements were

observed and the floating body did not move actively through the

water. After a total session of 6 min, mice were warmed up on a

heating pad, and then placed back into their home cage. Mice

were tested the same time of day (ZT 6 and ZT 18) at three

subsequent days and mean values were plotted as cumulative

immobility time in seconds.

For Tail suspension tests mice were suspended individually from

the tail, fixed to a cord hanging in a box (36.5 cm high,

30.5630.5 cm2) (according to the EMPRESS standard operating

procedure http://empress.har.mrc.ac.uk). Animals were judged to

be immobile when not agitating and not attempting to escape.

Immobility was recorded during 6 min with a stopwatch. Tests

were repeated for three subsequent days at the same time point

(ZT 6 and ZT 18) and mean values were plotted as cumulative

immobility time in seconds.

Prepulse inhibition (PPI) tests were carried out with acoustic stimuli.

Mice were tested in a startle chamber (SR-lab System, San Diego

instruments) positioned within a sound-proof cabinet in a sound-

attenuating room according to standard methodology [43]. A

constant background white noise of 64 dB was presented

throughout the test. To measure prepulse inhibition, mice were

presented with a 68, 72, 76, 80 and 84 dB prepulse (for 20 ms)

followed by a 120 dB pulse (for 40 ms in length) 100 ms later. The

percentage PPI of the startle response was calculated using the

following formula: 100-[(SRPP/SR)x100]. SR denotes the startle

response to the pulse stimulus, whereas SRPP denotes the startle

response to the pulse with prepulse stimulus.

Elevated O-maze test was used to test anxiety, which affects mood-

related behaviors. The relationship between curiosity/exploration

and fear/hiding in a protected area is investigated. The elevated

O-maze consisted of an elevated (42 cm above the floor) annular

runway (outer diameter was 46 cm with 5.5 cm in with) divided

into 4 sectors. The two 90u closed sectors were protected by 11 cm

high inner and outer walls, while the remaining two open sectors

were unprotected. Animals were released at the interface of the

closed and open area and recorded for 5 min using a video

camera. Number of entries and time spent in the open sector were

counted. In order to avoid habituation to the maze, mice were

tested once for a total session of 5 min.

Y-maze spontaneous alternations test was performed to test the

working memory of mice using a Y-shaped maze with three plastic

arms (height: 12.7 length: 38.3, width: 7.6 cm) at 120u angles.

After introduction in the middle of the maze, mice were allowed to

freely explore the three arms for 5 minutes. Sessions were

videotaped and the sequential entries into each arm (A, B, C) were

noted. An arm entry was scored when all four limbs of the animal

were within an arm. Each set of three consecutive choices where

no repeated entries occurred (counting also overlapping triplets)

was scored as alternation. The Y-maze score was calculated as

follows [number of alternations/(number of total entries-2)*100], a

Y-maze score of 50% indicates random selection of arm entries.

The maze was cleaned with 70% ethanol after each test. Tests

were performed during the resting phase of mice between ZT 4 to

ZT 6.

Spatial object recognition (SOR) tasks were performed in an

arena (30630630 cm) with two objects, a plastic square

(6.562.568.5 cm) and a metal cylinder (h: 9 cm, r: 2.2 cm). The

bottom plate of the arena was decorated on one side with black

and white stripes as a spatial cue. Mice were habituated to the

empty arena for 10 min, subsequently the arena was cleaned with

70% ethanol and two objects were placed in the arena at opposite

corners (upper left and lower right). The mice were introduced in

the center of the arena and allowed to explore the arena for

10 min (object training). Object training was repeated on three

consecutive days (24-h intervals) for 10 min each. Twenty-four

hours after the third training one object was displaced to a new

location (displaced object, DO) while the other object was not

moved (non-displaced object, NOD) and the mice were allowed to

explore the new situation during 10 min. The identity of the DO

(plastic square or metal cylinder) was balanced between groups.

The third training session and test session were videotaped. The

response to spatial change was assessed by calculating the

percentage of time spent exploring the DO vs. NOD. Exploration

was scored when mice were facing and sniffing the objects within

very close proximity and/or touching them.

ImmunohistochemistryAnimals used for immunohistochemistry were sacrificed at ZT

6. Perfused brains were cryoprotected and sectioned (40 mm,

coronal) using a cryostat. Sections chosen for staining were placed

in 24-well plates (up to 4 sections of one sample per well), washed 3

times in 1xTBS and twice in 2xSSC pH 7.0 (0.3 M NaCl/0.03 M

tri-Na-citrate). Antigen retrieval was performed with 50%

formamide/26SSC by heating to 65uC for 50 min. Then, sections

were washed twice in 2xSSC and 3 times in 1xTBS pH 7.5 (0.1 M

Tris/0.15 M NaCl), before blocking them for 1 h in 10% fetal

bovine serum (FBS)/0.1% Triton X-100/1xTBS at room

temperature (RT). Directly after the blocking step, primary

antibodies (DCX [Santa Cruz, SC8066], NeuN [Millipore,

MAB377], FABP7 [abcam ab27171]) diluted in 1% FBS/0.1%

Triton X-100/1xTBS were added to the sections and incubated

overnight at 4uC. The next day sections were washed 3 times in

1xTBS and incubated with the appropriate fluorescent secondary

antibodies diluted 1:500 in 1% FBS/0.1% Triton X-100/16TBS

for 3 h at RT (Dk-Anti-mouse Cy5 [715-605-150], Dk-Anti-rabbit

Cy2 [711-545-152], Dk-Anti-rabbit Cy3 [711-165-152], Dk-Anti-

goat Cy3 [705-165-147], all from Jackson Immuno Research).

After 3 washes with 1xTBS, nuclei were counterstained with DAPI



http://empress.har.mrc.ac.uk

for 10 min. Finally the tissue sections were washed again twice in

1xTBS and mounted on glass microscope slides. Slides were stored

horizontally for at least one day at 4uC to allow the mounting

medium to solidify. Fluorescent images were taken by using a

confocal microscope (Leica TCS SP5), equipped with objectives

10x, 20x, and 40x, and an inverted DMI6000 stand with

motorized stage. Images were taken with a resolution of

102461024, scan speed 400 Hz and Z-stack of 1.5 mm through

the whole section with frame average 3. Images were processed

with LAS AS software from LEICA.

Assessment of cell proliferation and neurogenesisMice aged 6–12 weeks were used for the assessment of

neurogenesis. To assess the total amount of newborn cells in the

adult dentate gyrus, bromodesoxyuridin (BrdU) was administered

by intraperitoneal injection (ZT6) at 100 mg/kg body weight and

the mice (3 per genotype) were sacrificed 4 days later at ZT6. For

the diurnal evaluation of proliferation, mice received a single dose

of BrdU (100 mg/kg body weight) for 10 hour labeling. The

injection schedule was as follows: injection at ZT 1 and perfusion

at ZT 11 for light phase labeling; injection at ZT 13 and perfusion

at ZT 23 for dark phase labeling (4 mice per genotype and time

point). The tissue was fixed by cardiovascular perfusion, cryopre-

served and sections of 40 mm were cut using a cryostat. For

immunohistochemical detection of BrdU streptavidin-biotin de-

tection was chosen. Free-floating sections were incubated in 1 M

HCl on ice for 10 min, then in 2 M HCl at RT for 10 min and

finally in 2 M HCl at 37uC for 20 min. Incubation in 0.1 M boric

acid at pH 8.5 for 12 min was performed for neutralization.

Sections were blocked for 1 h in 10% FBS/0.1% Triton X-100/

16TBS at RT, followed by specific blocking of streptavidin and

biotin binding sites in the tissue (Streptavidin-Biotin blocking kit

Vector labs). Primary antibodies diluted in 1% FBS/0.1% Triton

X-100/16TBS were added to the sections and incubated

overnight at 4uC. Antibodies were Anti-DCX (abcam ab18723),

Anti-BrdU [BU1/75 (ICR1)] (abcam ab6326) and Anti-NeuN

(Millipore MAB377). Secondary antibodies were biotinylated Anti-

rat (Vector Laboratories BA9400), Anti-mouse Cy5 and Anti-

rabbit Cy3 (Jackson Immuno Research 715-605-150 and 711-165-

152) for 3 h at RT and subsequently Streptavidin-FITC conjugate

(Vector Laboratories SA5001) 2 h at RT. Mounted tissue sections

were analyzed with a confocal microscope (Leica TCS SP5).

Fluorescent images covering the DG region were taken with 406magnification and Z-stack of 1.5 mm through the entire coronal

section with frame average 3. Images were processed with LAS AS

software from LEICA. To estimate the number of immunolabelled

BrdU+ cells in the dentate gyrus (DG), systematic random

sampling of every sixth 40-mm coronal section along the rostro-

caudal axis of the DG (21.06 mm to 23.80 mm from bregma)

was chosen and performed according to [5]. Immunopositive cells

were counted and the total amount of cells per DG was calculated

by multiplying the results by six (because every sixth section had

been used).

Knockdown of Fabp7 by siRNASiRNA-mediated gene knockdown was achieved by using

Lipofectamine RNAiMAX transfection kit (Invitrogen). U-251

MG cells plated to 6-well plates and grown to 30-50% confluence

were transfected with 10 nM Stealth siRNA duplexes (Invitrogen):

FABP7HSS103516, FABP7HSS103517, FABP7HSS103518 and

siRNA negative control medium GC. Knockdown efficiency was

assessed 72 h post-transfection by western blotting and real-time

PCR.

SR8278 (REV-ERBa antagonist) treatment25 mM SR8278 (Sigma) stock solution in DMSO was prepared.

Confluent cells were incubated during 24 h in presence of 10 mM

SR8278, if not otherwise stated. Equal volumes of DMSO were

used as control treatment.

Cell migration assayExperiments were carried out with 24-well plates and polycar-

bonate Trans-well membrane inserts containing 8 mm pores

(Corning, 3422). 72 h after siRNA mediated gene knockdown

and 18 h after addition of 10 mM SR8278 (antagonist of REV-

ERBa) in DMSO (equal volumes of DMSO were used as control

treatment), U-251 MG cells were removed from the plate using

0.1% trypsin in 1xPBS and counted. 20’000 cells, in DMEM

without FCS, were plated to trans-well inserts and put in the

receiver-wells. DMEM containing 10% FCS in the receiver-well

was used as attractant. To allow migration, cells were incubated

for 6 h in a CO2 incubator. A Q-tip was used to remove non-

migrated cells from the upper side of the membrane, whereas

migrated cells on the lower side of the membrane were fixed and

stained for 10 min in 0.5% crystal violet/25% methanol. The

number of migrated cells was determined by counting them in

three random big squares of a Neubauer chamber and the results

were displayed as percent of migrated cells of the total amount of

cells plated per trans-well (3 mm2). Experiments were performed

in duplicates and repeated least three times. Representative

pictures of migrated cells were taken with a Zeiss Axioplan 2

microscope.

Proliferation studyThe Luna automated cell counter (Logos Biosystems) was used

to assess proliferation of U-251 MG cells. Experiments were

carried out 72 h post transfection and 18 h after treatment with

10 mM SR8278. Cells were detached using 0.1% trypsin in 1xPBS

(2 min at 37uC), resuspended in growth medium and mixed with

an equal volume of trypan blue stain (0.4% in 1xPBS). 10 ml of

stained cell suspension was used per cell counting chamber,

samples were counted twice and experiments were performed four

times. Results were displayed as total cell number per well, since

the ratio of living and dead cells did not vary between samples.

Statistical analysisStatistical evaluation of all experiments was performed using

GraphPad Prism4 software. Depending on the type of data, either

unpaired t-test, 1- or 2-way ANOVA with Bonferroni post-test was

performed. Values were considered significantly different with p,

0.05 (*), p,0.01 (**), or p,0.001 (***).

Results

Genome wide analysis reveals an increase of Fabp7 in theSCN of Rev-erba KO mice

In order to detect differences in gene expression in brains of

wild- type versus Rev-erba KO mice we performed a microarray

analysis. We focused our analysis on the SCN, because REV-

ERBa is a component of the circadian clock of which the

pacemaker resides in the SCN [44]. In order to identify Rev-erbaregulated genes, we collected tissue 2 hours after the beginning of

the activity phase at zeitgeber time (ZT) 14, which is 2–4 hours

after maximal mRNA expression of Rev-erba [27]. Using Affimetrix

whole genome arrays we identified a number of differentially

expressed genes between the two genotypes (up-regulated genes

Table S1, down-regulated genes Table S2). The strongest



differences in gene expression are summarized in Figure 1. The list

of up-regulated genes includes Bmal1 (Arntl) and Npas2, two clock

components that are directly regulated by REV-ERBa [27,45].

We focused on genes that were up-regulated in Rev-erba KO mice

(red, lower part in Fig. 1A), because REV-ERBa acts as a

repressor binding to RORE elements in the promoter of target

genes. Lack of Rev-erba will therefore lead to up-regulation of direct

target genes. Plotting the RMA (Robust Multi-array Analysis)

signals [37] from Rev-erba KO versus wild-type mice clearly

identified Fabp7 as the most up-regulated (4.5-fold) gene in the

SCN of Rev-erba KO animals (Fig. 1B).

Fabp7 is over-expressed in different brain regions of Rev-erba KO mice

As a next step we verified the increased expression of Fabp7 in

Rev-erba KO mice using in situ hybridization, quantitative RT-

PCR (qRT-PCR) and further extended the validation at the

protein level by Western blotting. In situ hybridization experi-

ments performed on brain slices of wild-type, Per2Brdm1 mutant and

Rev-erba KO animals revealed increased expression of Fabp7

mRNA in Rev-erba KO mice. This expression was increased not

only in the SCN, but also throughout various brain regions,

including the hippocampus (HIP), the habenula (HB) (Figs. 2A and

S1) and the cortex (CX) (Fig. S1), which are known sites of Fabp7

expression [33]. In wild-type and Per2Brdm1 mutant mice, Fabp7

mRNA displayed a shallow diurnal pattern of expression (black

and red lines, respectively) in the SCN and HB, whereas in Rev-

erba KO animals, this expression was elevated at all time points

(green line, Fig. S1). More detailed analysis of the in situ

hybridization experiment revealed that in Rev-erba KO mice,

Fabp7 expression appeared to be elevated in the molecular layer

(Fig. 2B) and the SGZ of the hippocampus (arrows, Fig. 2B). Next

we quantified Fabp7 by qRT-PCR in hippocampus and found it to

be expressed in a phase consistent with the repression of its

expression by REV-ERBa (Fig. 2C). Similar to the increase in

mRNA expression of Fabp7 Western blot analysis on hippocampal

extracts from wild-type and Rev-erba KO mice revealed elevated

levels of FABP7 protein in the brain of Rev-erba KO mice (Fig. 2D),

suggesting that Fabp7 is a target gene of REV-ERBa.

REV-ERBa regulates Fabp7 expression in vitro and in vivoExpression analysis suggested that Fabp7 may be directly

regulated by REV-ERBa. To test this hypothesis we performed

transactivation experiments using a part of the Fabp7 promoter

fused to luciferase (Fabp7::luc) as a reporter and transfected this

construct into the neuroblastoma cell line NG108-15. We found

that REV-ERBarepressed the activity of the Fabp7 promoter in a

dose dependent manner comparable to the known REV-ERBamediated repression of the Bmal1 promoter (Fig. 3A). Deletion of

the proximal REV-ERBa binding element (RORE) on the Fabp7

promoter (2257 nt upstream of the transcription initiation site)

abolished REV-ERBa mediated repression (Fig. 3B). Interestingly,

the positive acting counterpart of REV-ERBa, the retinoic acid

related orphan receptor alpha (RORa), which also binds to

ROREs, activated the Fabp7 promoter in a similar fashion as it

activates Bmal1 (Fig. 3C). These results indicate that Fabp7 is

regulated by the nuclear receptors REV-ERBa and RORa. Since

these two nuclear receptors are components of the circadian clock,

we tested whether Fabp7 is activated in a time dependent fashion.

First, we verified that Fabp7 is regulated by a similar mechanism in

NIH 3T3 fibroblasts (Fig. S2). Thereafter, we monitored cyclic

expression of Fabp7::luc after synchronization of cells with

dexamethasone (Fig. 3D). Our experiments indicated a time

dependent regulation of the Fabp7 promoter in vitro in phase with

Bmal1.

In a next step, we tested whether REV-ERBa binds to the Fabp7

promoter in vivo by performing chromatin immunoprecipitation

(ChIP) using chromatin prepared from the hippocampal area.

Figure 1. Genome wide expression profiling of wild-type and Rev-erba2/2 tissue from suprachiasmatic nuclei (SCN). (A) The strongestup- and down-regulated genes are displayed with red color marking the up-regulated genes and blue the down-regulated genes. The heat map is aselection of genes from the array based on p,0.01 (t-test) and an absolute fold change greater than 1.75. The color code is based on the log2 valueof the fold change of the RMA values. The three columns per genotype represent the individual experiments (n = 3). (B) Plot of the RMA signals fromwild-type versus Rev-erba2/2 SCN reveals Fabp7 as the most up-regulated gene in Rev-erba2/2 mice (dotted lines represent 2 fold changes).doi:10.1371/journal.pone.0099883.g001



Figure 2. Expression profile of Fabp7 mRNA and protein in brain tissue. (A) In situ hybridization on coronal brain sections of wild-type,Per2Brdm1, and Rev-erba2/2 mice at ZT4. The sections in the left column contain the SCN, the sections in right column the hippocampus (HIP) andhabenula (HB). (B) The panel shows dark-field microscopy of the hippocampus (HIP) in the dentate gyrus region. Blue represents Hoechst-dye stainedcell nuclei and the yellow signal represents the hybridization signal detecting Fabp7 mRNA. (C) Quantification of Fabp7 mRNA in the hippocampus ofwild-type (black) and Rev-erba2/2 mice (green) over the period of 24 hours (left panel). The right panel depicts the Rev-erba mRNA in thehippocampus of wild-type (black) and Rev-erba2/2 mice (green). 2-way ANOVA reveals a significant difference between wild-type and Rev-erba2/2

mice (n = 3, p,0.05, mean 6 SEM). (D) The left panel shows a Western blot illustrating FABP7 protein levels in the hippocampus area of wild-type andRev-erba2/2 mice. The right panel illustrates the quantification of FABP7 signal. 2-way ANOVA reveals a significant difference between wild-type andRev-erba2/2 mice (n = 3, p,0.05, mean 6 SEM).doi:10.1371/journal.pone.0099883.g002



Figure 3. Molecular regulation of the Fabp7 promoter. (A) The top panel depicts the murine Fabp7 (mFabp7) promoter with its two REV-ERBaresponse elements (ROREs). Transactivation experiments in NG108-15 neurobalstoma cells show a repression potential of REV-ERBa that is similar forboth the Bmal1::luc and Fabp7::luc reporter constructs (n = 3, *p,0.05, mean 6 SD). (B) Deletion of the RORE element 257 nucleotides upstream ofthe transcription start site of Fabp7 (Fabp7DRORE) abolishes the repression by REV-ERBa (n = 3, *p,0.05, mean 6 SD). (C) RORa activates the



REV-ERBa bound to the Fabp7 promoter in a time dependent

fashion and this binding was absent in Rev-erba KO mice (Fig. 3E).

This suggests that our observations made in cell cultures most

likely also apply in vivo.

Rev-erba KO mice show alterations in mood-relatedbehaviors and hippocampus-dependent cognitiveperformance

Next, we performed behavioral tests comparing wild-type and

Rev-erba KO mice. Fabp7 maps to a quantitative trait locus for a

schizophrenia endophenotype [46] and therefore we employed a

prepulse inhibition (PPI) test, which is used as a measure for

schizophrenia. However, we found no difference between wild-

type and Rev-erba KO mice (Fig. 4A), which overexpress Fabp7, in

contrast to animals lacking Fabp7, which displayed a reduced

response in the PPI test [46]. In order to test anxiety related

behavior we used the elevated O-maze test during the light phase.

Both genotypes spent the same amount of time in the open area

(Fig.4B, left panel) and also the number of entries into the open

area was similar (Fig. 4B, right panel), indicating no significant

differences in anxiety. Next, we performed despair-based behav-

ioral tests that detect differences in mood-related behavior such as

mania and depression. The two genotypes did not differ in their

behavior in the tail suspension test (TST) at ZT6 as well as at

ZT18 (Fig. 4C). The data are shown over 3 consecutive days to

illustrate no changes due to learning or adaptation. A more

sensitive mood-related behavioral test, the forced swim test (FST)

revealed a tendency of wild-type animals towards higher

immobility at ZT6 compared to ZT18 (Fig. 4D). This is consistent

with our previous observations [47]. Interestingly, Rev-erba KO

mice showed significantly reduced immobility compared to wild-

type animals at ZT6 (Fig. 4D), although the total locomotor

activity levels are similar to wild-type animals [27]. In the

literature reduced immobility is often associated with ‘mania-like’

behavior, however, it may also reflect a deficit in learning to adapt

to a hopeless situation [48,49]. Therefore, we tested both

genotypes in memory related tests. Spontaneous alterations in a

Y-maze are considered a test of short term or working memory.

Mice tend to avoid an arm they just have visited and alternate

their entries among the arms, so re-entry into an arm just visited

suggests memory impairment [50]. In the Y-maze task, Rev-erbaKO mice showed reduced spontaneous alterations between the

arms compared to wild-type animals (Fig. 4E), suggesting a deficit

in working and short-term memory. To assess long-term memory

we employed the spatial object recognition test (SOR), which relies

on the innate propensity of mice to explore their environment and

recall where objects are located [51]. After training the mice to

learn the position of objects, one object was moved 24 hours later.

Wild-type mice will recall the position of the nondisplaced object

(NDO) and the exploration of the displaced object (DO) will be

favored. During the training session neither wild-type nor the Rev-

erba KO animals showed a preference for either object, but Rev-

erba KO mice showed less preference for the DO than wild-type

mice when tested 24 hours later (Fig. 4F, left panel). However, it

appeared that Rev-erba KO animals were in general less

explorative than wild-type animals (Fig. 4F, right panel). The

SOR test suggests long-term memory deficits of Rev-erba KO mice

as previously observed [52]. Taken together the results indicate

that Rev-erba KO animals display impaired hippocampal functions

regarding mood-related behavior and memory. We do not know,

however, whether these two behavioral phenotypes are function-

ally related, since Rev-erba may be responsible for the regulation of

several transcriptional events in the brain as evidenced in

figure 1A. In addition to Fabp7 up-regulation many other genes

including the glucocorticoid receptor (Nr3c1) are down-regulated

in Rev-erba KO mice and therefore it is very likely that the

behavioral phenotypes are the result of changes in more than one

transcriptional network.

Neurogenesis and FABP7 protein expression areincreased in the dentate gyrus of Rev-erba KO mice

Neurogenesis-deficient mice exhibit increased immobility in the

FST thus indicating a direct role of adult neurogenesis in

depressive illness [53]. Therefore we hypothesized that Rev-erbaKO animals, which show decreased immobility in the FST

(Fig. 4D), may display increased neurogenesis.

To test this hypothesis we performed immunohistochemistry to

visualize the formation of neurons in the SGZ of the hippocampal

DG. We found that expression of doublecortin (Dcx), a marker for

immature neurons, is increased in the Rev-erba KO hippocampus

(Fig. 5A). Furthermore, an increased number of cells has divided in

Rev-erba KO animals, as evidenced by the cell-cycle dependent

incorporation of bromodeoxyuridine (BrdU) (Fig. 5B). Dcx

staining and BrdU staining partially overlapped (Fig. 5B, magni-

fication), consistent with the accepted model of neurogenesis [32].

In the molecular layer of the hippocampus a partial overlap in

expression was observed between FABP7 and GFAP (Fig. S3).

This is consistent with previous observations describing FABP7 as

a marker for a subpopulation of glial cells [54].

Since we observed increased Fabp7 mRNA expression in the

SGZ of Rev-erba KO mice (Fig. 2B) we investigated its expression

at the protein level. Similar to its mRNA expression, more FABP7

protein containing cells were observed in the SGZ of Rev-erba KO

mice (Fig. 5C). Its expression did not co-localize with Dcx,

indicating that FABP7 may be a marker of a subpopulation of

neuronal stem cells (NSCs, Type-2 and Type-3) and early

transitory amplifying cells (TAPs) before Dcx starts to be expressed

in neuroblasts [32]. Overall these results suggest a correlation

between neurogenesis, FABP7 and REV-ERBa function.

The diurnal pattern of hippocampal neurogenesis is lostand constantly high in Rev-erba KO mice

Because REV-ERBa appears to be responsible for the diurnal

expression of the NPC marker Fabp7 (Fig. 2, 3), we tested whether

the known diurnal pattern of neurogenesis [23–26] is lost in

FABP7 overexpressing Rev-erba KO mice. In wild-type animals we

observed a time of day dependent BrdU incorporation into

newborn cells in the SGZ of the hippocampus with higher

incorporation during the dark phase (ZT13-23) as compared to the

light phase (ZT1-11) (Fig. 6A left panel, 6B). In contrast

incorporation of BrdU into newly formed cells of Rev-erba KO

was constantly high and did not show a diurnal pattern. This

observation correlates with the observed constant overexpression

of Fabp7 in Rev-erba KO animals, suggesting that Rev-erba is

involved in establishing the diurnal pattern of adult neurogenesis.

Fabp7::luc reporter in a similar fashion as the Bmal1::luc reporter (n = 3, *p,0.05, mean 6 SD). (D) Real-time monitoring of NIH 3T3 cells transfectedwith the Bmal1::luc and Fabp7::luc reporters, respectively. (E) Chromatin immunoprecipitation (ChIP) reveals time of day dependent binding of REV-ERBa on the Fabp7 promoter in hippocampal tissue (n = 4, ***p,0.001, mean 6 SEM, one-way ANOVA). denotes background binding of REV-ERBa atthe unrelated Fgf21 promoter.doi:10.1371/journal.pone.0099883.g003



Migration and proliferation of glioblastoma cells aremodulated by Rev-erba and Fabp7 in vitro

Approximately 80% of dividing progenitors in the SGZ are

directed to the neuronal fate and develop into dentate granule

neurons. They migrate radially into the inner third of the granule

layer where they start to display the morphology of mature granule

neurons (reviewed in [55]). Hence, migration is part of adult

neurogenesis. In order to establish a functional link between

increased expression of FABP7 in Rev-erba KO mice and migration

of neuronal cells, we looked at the migration properties of FABP7

expressing U-251 MG glioblastoma cells [36] in a transwell

migration assay.

Migration of the cells through micropores from one compart-

ment to the other was observed if the latter contained 10% fetal

calf serum (FCS). In contrast, no migration was observed if it was

left serum-free (Fig. 7A, B). Addition of the REV-ERBa antagonist

Figure 4. Mood-related behavior and hippocampus-dependent cognitive performance is altered in Rev-erba2/2 mice. (A) Mice weresubjected to prepulse inhibition (PPI) during the light phase. Startle response after a prepulse at 68, 72, 76, 80 and 84 dB, followed by a pulse at120 dB represented as percentage of PPI with 100% as the first absolute startle values. Wild-type and Rev-erba2/2 mice display a comparable amountof PPI, which is a measure related to schizophrenia (mean 6 SEM, n = 6). (B) Mice were tested at ZT0-2 in the anxiety related elevated O-maze test andno significant differences between the two genotypes were observed (mean 6 SEM, n = 12, 2-way ANOVA). (C) Mice were subjected to the tailsuspension test (TST) at ZT6 and ZT18. No differences between the genotypes could be observed (mean 6 SEM, n = 6, 2-way ANOVA). (D) Mice weresubjected to the forced swim test (FST) at ZT6 and ZT18 for 3 consecutive days. Rev-erba2/2 mice were significantly less immobile compared to wild-type animals at ZT6 (mean 6 SEM, n = 10, ***p,0.001, 2-way ANOVA). (E) Short term spatial memory was assessed between ZT4-6 using the Y-mazetest (mean 6 SEM, Student’s t-test, **p,0.01, n = 12). (F) Long term spatial memory was assessed between ZT4-6 using the spatial object recognitiontest (SOR). The left panel shows the preference for the displaced object (DO) 6 SEM (2-way ANOVA, p,0.05, n = 10). The right panel shows the totaltime exploring objects in general (mean 6 SEM, 2-way ANOVA, **p,0.01, ***p,0.001, n = 10).doi:10.1371/journal.pone.0099883.g004



Figure 5. Immunohistochemistry in the dentate gyrus (DG) of wild-type and Rev-erba2/2 mice at ZT6. (A) Cell nuclei stained with DAPIare in blue and antibodies recognizing doublecortin (Dcx) are in red. Dcx expression is increased in the subgranular zone of Rev-erba2/2 miceindicating the presence of more neuroblasts in these animals. Scale bar: 100 mm. (B) Left panel: Visualization of cell division using bromodeoxyuridine



SR8278 [56] increased FABP7 expression (Fig. S3A) and

migration of the cells compared to the solvent control DMSO,

indicating that suppression of REV-ERBa had a positive influence

on the migration properties of U-251 MG glioblastoma cells

(Fig. 7A, B). Introduction of siRNA against Fabp7 into the cells

suppressed both FABP7 expression (Fig. S3B) and migration in the

absence and presence of SR8278, (Fig. 7A, B) further supporting

the notion that Rev-erba modulates migration via Fabp7.

Another hallmark of neurogenesis is cell proliferation. Therefore

we tested in the same glioblastoma cell line whether the REV-

ERBa antagonist SR8278 and siRNA against Fabp7 can affect

proliferation. 72 hours after transfection of control and Fabp7

siRNAs, respectively, we counted the number of cells that have

grown in presence or absence of SR8278. Suppression of REV-

ERBa by its antagonist SR8278 increased proliferation, whereas

down regulation of FABP7 by its siRNA decreased it (Fig. 7C).

These results were in agreement with our in vivo finding that lack

of Rev-erba in mice increased proliferation in the DG (Fig. 5B). This

indicates that our observations in U-251 MG glioblastoma cells are

likely to be applicable to the DG. Overall it appears that Rev-erbaand Fabp7 are involved in the regulation of proliferation and

migration during the process of neurogenesis.

Discussion

In this study we used genome wide profiling to compare gene

expression in the SCN of wild-type and Rev-erba KO mice. We

identified Fabp7 as a direct target gene of REV-ERBa. In situ

hybridization and immunohistochemistry revealed an increase in

FABP7 expression in Rev-erba KO mice in several brain regions

including the SGZ of the hippocampus, suggesting an involvement

of REV-ERBa and FABP7 in adult neurogenesis. In accordance

with this notion Rev-erba KO mice displayed constantly high

proliferation of cells over the day compared to wild-type mice,

which displayed a diurnal pattern of neurogenesis in the SGZ. In

addition, in vitro manipulation of REV-ERBa and FABP7

affected migration and proliferation properties of glioblastoma

cells.

Gene expression profiling of NSCs and their neuronal progeny

in adult hippocampal tissue revealed many genes to be involved in

neurogenesis [7]. Interestingly, this study indicated that in NSCs

Rev-erba (Nr1d1) is about 4 times up-regulated compared to

immature neurons expressing Dcx [7], suggesting an involvement

of Rev-erba in the early steps of adult neurogenesis. Among the

potential target genes of Rev-erba identified in our study (Fig. 1,

Table S1), Fabp7 was found to affect neuronal differentiation [57].

However, because Fabp7 levels are low in NSCs (type 1) and

absent in Dcx expressing immature neurons (Fig. 5C, [32]) the

study by Bracko et al. [7] did not identify Fabp7 to be differentially

expressed between NSCs and immature neurons.

Analysis of Fabp7 over 24 hours revealed a diurnal expression of

its mRNA in brain tissue which is comparable to a previous study

[33] with a trough of expression around ZT16 that is almost anti-

phasic to the expression of Rev-erba (Fig. 2C). Although FABP7

protein levels do not fluctuate over time, lack of Rev-erbasignificantly increased FABP7 protein levels in the brain

(Fig. 2D). Our transactivation and ChIP studies indicate that

REV-ERBa is a regulator of Fabp7 mRNA expression (Fig. 3).

Thus FABP7 appears to be one of the mediators of REV-ERBafunction in the brain.

Lack of Fabp7 in mice leads to altered emotional behavioral

responses [54] and has been associated with a schizophrenia

endophenotype [46]. In particular, Fabp7 KO mice exhibited a

differential response in the PPI test accompanied by reduced

proliferation in the SGZ [46]. In Rev-erba KO mice, which

overexpress Fabp7 (Fig. 2), the opposite phenotype with increased

proliferation in the SGZ (Fig. 5B, 6) and no change in the PPI test

(Fig. 4A) was observed. Similarly, anxiety related behavior is

(BrdU). Antibodies recognizing NeuN in blue mark nuclei of mature neurons, antibodies recognizing Dcx are in red and antibodies against BrdU are ingreen. Scale bar: 50 mm. Right panel: Quantification of the BrdU+ cells after 4 days. Rev-erba2/2 mice display more BrdU positive cells (mean 6 SEM,n = 3, **p,0.005, t-test). (C) FABP7 protein expression (green) does not overlap with Dcx protein expression and both expression levels are higher inRev-erba2/2 mice. The orthogonal sectioning to the right and at the bottom show reconstructions from a confocal z-stack in xz and yz direction,respectively. The dotted white lines mark the granular layer of the DG. Scale bar: 50 mm.doi:10.1371/journal.pone.0099883.g005

Figure 6. Neurogenesis in Rev-erba2/2 mice is constantly high and not diurnal. (A) BrdU was injected at ZT1 (upper panels) or ZT13 (lowerpanels) and incorporation was assessed 10 hours later (ZT11 and ZT23, respectively) in wild-type (left panels) and Rev-erba2/2 KO (right panels).Antibodies recognizing Dcx are in red and antibodies against BrdU are in green. The dotted white lines mark the granular layer of the DG. Scale bar:50 mm. (B) Quantification of the BrdU+ cells after 10 hours. Rev-erba2/2 mice display more BrdU positive cells during the light phase (ZT1-11)compared to wild-type (mean 6 SEM, n = 4, *p,0.05, 2-way ANOVA) whereas in the dark phase (ZT13-23) no difference in the number of BrdUpositive cells was observed between the genotypes.doi:10.1371/journal.pone.0099883.g006



altered in Fabp7 KO mice [54] but in the Fabp7 overexpressing

Rev-erba KO mice this is not the case (Fig. 4B). Furthermore, Rev-

erba KO animals did not differ in the tail suspension test (TST)

compared to wild-type, however, they responded differently to the

FST, spending less time immobile than their wild-type counter-

parts (Fig. 4D). Decreased immobility has been associated with

mania while increased immobility with depression [48] and

reduced neurogenesis [53]. Accordingly, one would expect an

increase of neurogenesis in Rev-erba KO mice. Our results support

this notion as more Dcx positive neuroblasts are observed in the

SGZ of Rev-erba KO animals (Fig. 5A). However, the effect of

REV-ERBa regulation via FABP7 appears to manifest before

neuroblasts are committed, as FABP7 expression did not co-

localize with Dcx positive cells (Fig. 5C). This is consistent with

previous observations describing a transitory expression of FABP7

in type 2 and 3 NSCs and early TAPs but not in late TAPs and

Dcx positive neuroblasts in the SVZ [32].

Adult neurogenesis in the hippocampus has also been associated

with learning and memory (for review see [55,58]). The short-term

memory and long-term memory tests we applied to the Rev-erbaKO mice revealed, that these animals had a deficit in the process

of memory formation (Fig. 4E, F). An increase in adult

neurogenesis in the hippocampus, as observed in Rev-erba KO

animals would, however, predict an improvement of memory

formation. This contradiction may be rooted in the multiple

functions of Rev-erba. Our microarray analysis (Fig. 1, Tables S1

and S2) clearly shows that many genes are altered in their

expression in Rev-erba KO mice. Of special interest in this context

is the down-regulation of the nuclear glucocorticoid receptor

(Nr3c1), because decreased signaling of this receptor in the

hippocampus impaired spatial memory in rats [59,60]. Interest-

ingly, a recent study shows that adult hippocampal neurogenesis

regulates forgetting indicating that too much neurogenesis may

jeopardize memory retention [61]. This notion correlates with our

findings.

The challenge for NSCs as for any other type of stem cells is to

keep the balance between proliferation and quiescence. This

balance is extremely important not only to keep a certain amount

of pluripotent NSCs in their niche, but also to avoid cancer

development due to over-proliferation. Niche signals, such as

notch signaling, can control dormant NSCs and push them

towards proliferative or keep them in a quiescent state [62]. Fabp7

may serve as a potential marker for mitotically activated NSCs in

the SVZ [32]. Rev-erba, as a repressor of Fabp7, may provide an

additional niche stimulus and therefore function as a brake to

avoid excessive proliferation of NSCs. This may explain why we

observe a strong increase in neurogenesis in the SGZ of Rev-erbaKO animals. If proliferation is constantly increased, gliomas may

develop, which are the most common primary malignancy in the

central nervous system of humans. In mice, however, gliomas are

hardly observed and we did not note development of gliomas in

Rev-erba KO mice. This may be due to compensation of increased

neurogenesis by elevation of cell death and/or apoptosis in Rev-

erba KO animal, which has been observed in the developing

cerebellum of these mice [63]. Alterations in cell death and

apoptosis in the adult hippocampus will have to be investigated in

Rev-erba KO mice in the future.

Glioblastoma tumors appear to contain cells with stem cell-like

properties, which contribute to invasion and chemoresistance

[64,65]. The stem cell-like cells grow as neurospheres in culture

and in comparison to adherent glioblastoma cells, they express

elevated levels of Fabp7 accompanied by elevated migration and

proliferation [57]. Manipulation of Rev-erba and Fabp7 in U-251

MG glioblastoma cells shows effects on migration and proliferation

(Fig. 7) that are in line with the observations described above.

Hence, it can be speculated that agonists for REV-ERBamay help

to reduce proliferation and migration in gliomas, which may

Figure 7. Influence of Rev-erba and Fabp7 on migration andproliferation of U-251 MG glioblastoma cells. (A) Cells wereanalyzed under non-migrating (0% FCS) and migrating (10% FCS)conditions 6 hours after treatment. The Rev-erba antagonist SR8278(10 mM) increased migration of the cells compared to DMSO control.siRNA against Fabp7 (Fabp7 16) reduced this migration. Scale bar:200 mm. (B) Quantification of the experiment in A. Shown is the mean6 SD for n = 3 independent experiments (*p,0.05, **p,0.005, t-test).(C) Number of cells 72 hours after transfection with either control orFabp7 siRNA in presence or absence of the Rev-erba antagonist SR 8278.The mean 6 SD for n = 4 experiments (*p,0.05, **p,0.01, t-test) isshown.doi:10.1371/journal.pone.0099883.g007



represent a novel avenue to combat this type of tumors in humans.

Interestingly, it appears that circadian genes are to some extent

related to glioma risk and outcome [66]. In particular elevated

levels of CLOCK contribute to cell proliferation and migration in

glioma [67]. Of note is that Clock is directly regulated by REV-

ERBa [45] and therefore agonists for REV-ERBa [4] may not

only reduce Fabp7 expression but also Clock levels, which may

reduce neurogenesis and lower the potential of glioblastoma

development.

Neurogenesis in the brain continuously declines with age [68].

This might be partially due to an increased quiescence of NSCs

and loss of Fabp7 expressing cells, as it has been observed in the

SVZ of aged mice [32]. Increasing Fabp7 by the application of

REV-ERBa antagonists may awake potentially dormant NSCs in

neurogenic pools and they may replenish dying cells. Hence,

REV-ERBa antagonists may improve the performance of the

ageing brain and help in the treatment of neurodegenerative

diseases such as Alzheimer disease [58].

Furthermore, antagonists for REV-ERBa may serve as anti-

depressants by increasing proliferation and migration (Fig. 7)

leading to a reduction of depressive symptoms (Fig. 4D). However,

future experiments will show to what extent the above predictions

can be met.

Overall our study shows that Rev-erba regulates Fabp7 and that

both genes appear to be involved in the modulation of

neurogenesis. This finding has far reaching implications, as

pharmacological targeting of Rev-erba may lead to improved

treatments for gliomas as well as neurological and depressive

disorders.

Supporting Information

Figure S1 Expression profile of Fabp7 mRNA and protein in

brain tissue. (A) Dark-field microscopy of the SCN and the

habenula (HB) comparing wild-type and Rev-erba2/2 mice at ZT4

and ZT16. The yellow signal represents the hybridization signal

detecting Fabp7 mRNA and blue represents Hoechst-dye stained

cell nuclei. (B) Quantification of the signal in the SCN, the HB and

cortex (CX) over time: black line = wild-type, red line =

Per2Brdm1, green line = Rev-erba2/2. The signal at ZT4 is double

plotted. The values comparing wild-type (or Per2Brdm1) with Rev-

erba2/2 are significantly different (n = 3, p,0.05, 2-way ANOVA,

mean 6 SEM).

(TIF)

Figure S2 Inhibition of Fabp7 transcription by REV-ERBa in

NIH 3T3 fibroblasts. Transactivation experiments show a dose

dependent repression potential of REV-ERBa that is similar for

both the Bmal1::luc and Fabp7::luc reporter constructs (n = 3, *p,

0.05, mean 6 SD).

(TIF)

Figure S3 Immunohistochemistry in the dentate gyrus (DG) of

wild-type and Rev-erba2/2 mice at ZT6. Overlapping signals

(yellow) of FABP7 (green) with GFAP expressing cells (red). The

orthogonal sectioning to the right and on the bottom depict

reconstructions from a confocal z-stack in xz and yz direction to

confirm that the FABP7 signal cell belongs in fact to the GFAP-

positive cell. Scale bar: 50 mm.

(TIF)

Figure S4 Immunobots showing efficiency of inhibition of REV-

ERBa activity and verification of siRNA knockdown of Fabp7 in

U-251 MG glioblastoma cells. (A) Quantification of FABP7 and

BMAL1 protein expression after treatment for 24 h with different

concentrations of REV-ERBa antagonist SR8278. Actin was used

for normalization. (n = 3, *p,0.05, mean 6 SD, t-test). (B) Left

panel: Comparison of Fabp7 knock down efficiency between three

different siRNAs against Fabp7 (16, 17, 18) and negative control

siRNA. Efficiency was tested 72 h post transfection with siRNA

and actin was used for normalization control. The fold change of

FABP7 protein expression was calculated setting the control

siRNA to 1 (n = 4, **p,0.01, mean 6 SD, t-test). Right panel:

Quantification of Fabp7 expression 72 h after siRNA knock down

or 18 h after treatment with 10 mM REV-ERBa antagonist

SR8278 by qRT-PCR. The fold change of Fabp7 mRNA

expression was calculated setting the control siRNA or solvent

control, DMSO, to 1. Experimental conditions were the same as

used for migration and proliferation assays (n = 3, *p,0.05, **p,

0.01, ***p,0.001, mean 6 SD, t-test).

(TIF)

Table S1 List of genes up-regulated in Rev-erba KO SCN.

(PDF)

Table S2 List of genes down-regulated in Rev-erba KO SCN.

(PDF)

Table S3 List of oligonucleotides used in this study.

(PDF)

Acknowledgments

We like to thank James Delorme for comments on the manuscript,

Antoinette Hayoz and Stephanie Baeriswyl-Aebischer for expert technical

assistance, Dr. U. Schibler for Rev-erba KO mice and the members of the

NCCR genomics platform at the University of Geneva for their help with

expression arrays.

Author Contributions

Conceived and designed the experiments: AS IS JAR HW PD LA UA.

Performed the experiments: AS SC IS EB JAR. Analyzed the data: AS SC

IS EB JAR OS HW PD LA UA. Contributed reagents/materials/analysis

tools: JAR UA. Wrote the paper: AS UA.

References

1. Buhr ED, Takahashi JS (2013) Molecular components of the Mammalian

circadian clock. Handb Exp Pharmacol 217: 3–27.

2. Bugge A, Feng D, Everett LJ, Briggs ER, Mullican SE, et al. (2012) Rev-

erbalpha and Rev-erbbeta coordinately protect the circadian clock and normal

metabolic function. Genes Dev 26: 657–667.

3. Cho H, Zhao X, Hatori M, Yu RT, Barish GD, et al. (2012) Regulation of

circadian behaviour and metabolism by REV-ERB-alpha and REV-ERB-beta.

Nature 485: 123–127.

4. Solt LA, Wang Y, Banerjee S, Hughes T, Kojetin DJ, et al. (2012) Regulation of

circadian behaviour and metabolism by synthetic REV-ERB agonists. Nature

485: 62–68.

5. Borgs L, Beukelaers P, Vandenbosch R, Nguyen L, Moonen G, et al. (2009)

Period 2 regulates neural stem/progenitor cell proliferation in the adult

hippocampus. BMC Neurosci 10: 30.

6. Kimiwada T, Sakurai M, Ohashi H, Aoki S, Tominaga T, et al. (2009) Clock

genes regulate neurogenic transcription factors, including NeuroD1, and the

neuronal differentiation of adult neural stem/progenitor cells. Neurochem Int

54: 277–285.

7. Bracko O, Singer T, Aigner S, Knobloch M, Winner B, et al. (2012) Gene

expression profiling of neural stem cells and their neuronal progeny reveals IGF2

as a regulator of adult hippocampal neurogenesis. J Neurosci 32: 3376–3387.

8. Janich P, Pascual G, Merlos-Suarez A, Batlle E, Ripperger J, et al. (2011) The

circadian molecular clock creates epidermal stem cell heterogeneity. Nature 480:

209–214.

9. Janich P, Toufighi K, Solanas G, Luis NM, Minkwitz S, et al. (2013) Human

epidermal stem cell function is regulated by circadian oscillations. Cell Stem Cell

13: 745–753.



10. Lie DC, Song H, Colamarino SA, Ming GL, Gage FH (2004) Neurogenesis in

the adult brain: new strategies for central nervous system diseases. Annu RevPharmacol Toxicol 44: 399–421.

11. Zhao C, Deng W, Gage FH (2008) Mechanisms and functional implications of

adult neurogenesis. Cell 132: 645–660.12. Samuels BA, Hen R (2011) Neurogenesis and affective disorders. Eur J Neurosci

33: 1152–1159.13. Lois C, Alvarez-Buylla A (1994) Long-distance neuronal migration in the adult

mammalian brain. Science 264: 1145–1148.

14. Kuhn HG, Dickinson-Anson H, Gage FH (1996) Neurogenesis in the dentategyrus of the adult rat: age-related decrease of neuronal progenitor proliferation.

J Neurosci 16: 2027–2033.15. Gould E, McEwen BS, Tanapat P, Galea LA, Fuchs E (1997) Neurogenesis in

the dentate gyrus of the adult tree shrew is regulated by psychosocial stress andNMDA receptor activation. J Neurosci 17: 2492–2498.

16. Gould E, Tanapat P, Cameron HA (1997) Adrenal steroids suppress granule cell

death in the developing dentate gyrus through an NMDA receptor-dependentmechanism. Brain Res Dev Brain Res 103: 91–93.

17. van Praag H, Kempermann G, Gage FH (1999) Running increases cellproliferation and neurogenesis in the adult mouse dentate gyrus. Nat Neurosci 2:

266–270.

18. Guzman-Marin R, Suntsova N, Stewart DR, Gong H, Szymusiak R, et al.(2003) Sleep deprivation reduces proliferation of cells in the dentate gyrus of the

hippocampus in rats. J Physiol 549: 563–571.19. Kempermann G, Kuhn HG, Gage FH (1997) More hippocampal neurons in

adult mice living in an enriched environment. Nature 386: 493–495.20. Gibson EM, Wang C, Tjho S, Khattar N, Kriegsfeld LJ (2010) Experimental ‘jet

lag’ inhibits adult neurogenesis and produces long-term cognitive deficits in

female hamsters. PLoS One 5: e15267.21. Kott J, Leach G, Yan L (2012) Direction-dependent effects of chronic ‘‘jet-lag’’

on hippocampal neurogenesis. Neurosci Lett 515: 177–180.22. Golombek DA, Rosenstein RE (2010) Physiology of circadian entrainment.

Physiol Rev 90: 1063–1102.

23. Goergen EM, Bagay LA, Rehm K, Benton JL, Beltz BS (2002) Circadian controlof neurogenesis. J Neurobiol 53: 90–95.

24. Kochman LJ, Weber ET, Fornal CA, Jacobs BL (2006) Circadian variation inmouse hippocampal cell proliferation. Neurosci Lett 406: 256–259.

25. Tamai S, Sanada K, Fukada Y (2008) Time-of-day-dependent enhancement ofadult neurogenesis in the hippocampus. PLoS One 3: e3835.

26. Gilhooley MJ, Pinnock SB, Herbert J (2011) Rhythmic expression of per1 in the

dentate gyrus is suppressed by corticosterone: implications for neurogenesis.Neurosci Lett 489: 177–181.

27. Preitner N, Damiola F, Lopez-Molina L, Zakany J, Duboule D, et al. (2002) Theorphan nuclear receptor REV-ERBalpha controls circadian transcription within

the positive limb of the mammalian circadian oscillator. Cell 110: 251–260.

28. Chmurzynska A (2006) The multigene family of fatty acid-binding proteins(FABPs): function, structure and polymorphism. J Appl Genet 47: 39–48.

29. Feng L, Hatten ME, Heintz N (1994) Brain lipid-binding protein (BLBP): anovel signaling system in the developing mammalian CNS. Neuron 12: 895–

908.30. Yun SW, Leong C, Zhai D, Tan YL, Lim L, et al. (2012) Neural stem cell

specific fluorescent chemical probe binding to FABP7. Proc Natl Acad Sci U S A

109: 10214–10217.31. Young JK, Heinbockel T, Gondre-Lewis MC (2013) Astrocyte fatty acid binding

protein-7 is a marker for neurogenic niches in the rat hippocampus.Hippocampus 23: 1476–1483.

32. Giachino C, Basak O, Lugert S, Knuckles P, Obernier K, et al. (2014) Molecular

diversity subdivides the adult forebrain neural stem cell population. Stem Cells32: 70–84.

33. Gerstner JR, Bremer QZ, Vander Heyden WM, Lavaute TM, Yin JC, et al.(2008) Brain fatty acid binding protein (Fabp7) is diurnally regulated in

astrocytes and hippocampal granule cell precursors in adult rodent brain. PLoS

One 3: e1631.34. Gerstner JR, Vanderheyden WM, LaVaute T, Westmark CJ, Rouhana L, et al.

(2012) Time of day regulates subcellular trafficking, tripartite synapticlocalization, and polyadenylation of the astrocytic Fabp7 mRNA. J Neurosci

32: 1383–1394.35. Hamprecht B (1977) Structural, electrophysiological, biochemical, and phar-

macological properties of neuroblastoma-glioma cell hybrids in cell culture. Int

Rev Cytol 49: 99–170.36. Ponten J, Westermark B (1978) Properties of human malignant glioma cells in

vitro. Med Biol 56: 184–193.37. Bolstad BM, Irizarry RA, Astrand M, Speed TP (2003) A comparison of

normalization methods for high density oligonucleotide array data based on

variance and bias. Bioinformatics 19: 185–193.38. Albrecht U, Sun ZS, Eichele G, Lee CC (1997) A differential response of two

putative mammalian circadian regulators, mper1 and mper2, to light. Cell 91:1055–1064.

39. Schmutz I, Ripperger JA, Baeriswyl-Aebischer S, Albrecht U (2010) Themammalian clock component PERIOD2 coordinates circadian output by

interaction with nuclear receptors. Genes Dev 24: 345–357.

40. Langmesser S, Tallone T, Bordon A, Rusconi S, Albrecht U (2008) Interaction

of circadian clock proteins PER2 and CRY with BMAL1 and CLOCK. BMCMol Biol 9: 41.

41. Ripperger JA, Schibler U (2006) Rhythmic CLOCK-BMAL1 binding to

multiple E-box motifs drives circadian Dbp transcription and chromatintransitions. Nat Genet 38: 369–374.

42. Stratmann M, Stadler F, Tamanini F, van der Horst GT, Ripperger JA (2010)Flexible phase adjustment of circadian albumin D site-binding protein (DBP)

gene expression by CRYPTOCHROME1. Genes Dev 24: 1317–1328.

43. Braff DL, Geyer MA (1990) Sensorimotor gating and schizophrenia. Humanand animal model studies. Arch Gen Psychiatry 47: 181–188.

44. Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals.Nature 418: 935–941.

45. Crumbley C, Burris TP (2011) Direct regulation of CLOCK expression byREV-ERB. PLoS One 6: e17290.

46. Watanabe A, Toyota T, Owada Y, Hayashi T, Iwayama Y, et al. (2007) Fabp7

maps to a quantitative trait locus for a schizophrenia endophenotype. PLoS Biol5: e297.

47. Hampp G, Ripperger JA, Houben T, Schmutz I, Blex C, et al. (2008)Regulation of monoamine oxidase A by circadian-clock components implies

clock influence on mood. Curr Biol 18: 678–683.

48. Petit-Demouliere B, Chenu F, Bourin M (2005) Forced swimming test in mice: areview of antidepressant activity. Psychopharmacology (Berl) 177: 245–255.

49. Renard CE, Dailly E, David DJ, Hascoet M, Bourin M (2003) Monoaminemetabolism changes following the mouse forced swimming test but not the tail

suspension test. Fundam Clin Pharmacol 17: 449–455.50. Lalonde R (2002) The neurobiological basis of spontaneous alternation.

Neurosci Biobehav Rev 26: 91–104.

51. Wimmer ME, Hernandez PJ, Blackwell J, Abel T (2012) Aging impairshippocampus-dependent long-term memory for object location in mice.

Neurobiol Aging 33: 2220–2224.52. Jager J, O’Brien WT, Manlove J, Krizman EN, Fang B, et al. (2014) Behavioral

Changes and Dopaminergic Dysregulation in Mice Lacking the Nuclear

Receptor Rev-erbalpha. Mol Endocrinol 28: 490–498.53. Snyder JS, Soumier A, Brewer M, Pickel J, Cameron HA (2011) Adult

hippocampal neurogenesis buffers stress responses and depressive behaviour.Nature 476: 458–461.

54. Owada Y, Abdelwahab SA, Kitanaka N, Sakagami H, Takano H, et al. (2006)Altered emotional behavioral responses in mice lacking brain-type fatty acid-

binding protein gene. Eur J Neurosci 24: 175–187.

55. Gu Y, Janoschka S, Ge S (2013) Neurogenesis and hippocampal plasticity inadult brain. Curr Top Behav Neurosci 15: 31–48.

56. Kojetin D, Wang Y, Kamenecka TM, Burris TP (2011) Identification ofSR8278, a synthetic antagonist of the nuclear heme receptor REV-ERB. ACS

Chem Biol 6: 131–134.

57. De Rosa A, Pellegatta S, Rossi M, Tunici P, Magnoni L, et al. (2012) A radialglia gene marker, fatty acid binding protein 7 (FABP7), is involved in

proliferation and invasion of glioblastoma cells. PLoS One 7: e52113.58. Mu Y, Gage FH (2011) Adult hippocampal neurogenesis and its role in

Alzheimer’s disease. Mol Neurodegener 6: 85.59. Bizon JL, Helm KA, Han JS, Chun HJ, Pucilowska J, et al. (2001)

Hypothalamic-pituitary-adrenal axis function and corticosterone receptor

expression in behaviourally characterized young and aged Long-Evans rats.Eur J Neurosci 14: 1739–1751.

60. Lee SY, Hwang YK, Yun HS, Han JS (2012) Decreased levels of nuclearglucocorticoid receptor protein in the hippocampus of aged Long-Evans rats

with cognitive impairment. Brain Res 1478: 48–54.

61. Akers KG, Martinez-Canabal A, Restivo L, Yiu AP, De Cristofaro A, et al.(2014) Hippocampal neurogenesis regulates forgetting during adulthood and

infancy. Science 344: 598–602.62. Basak O, Giachino C, Fiorini E, Macdonald HR, Taylor V (2012) Neurogenic

subventricular zone stem/progenitor cells are Notch1-dependent in their active

but not quiescent state. J Neurosci 32: 5654–5666.63. Chomez P, Neveu I, Mansen A, Kiesler E, Larsson L, et al. (2000) Increased cell

death and delayed development in the cerebellum of mice lacking the rev-erbA(alpha) orphan receptor. Development 127: 1489–1498.

64. Singh SK, Clarke ID, Terasaki M, Bonn VE, Hawkins C, et al. (2003)Identification of a cancer stem cell in human brain tumors. Cancer Res 63:

5821–5828.

65. Alcantara Llaguno, Chen J, Parada LF (2009) Signaling in malignantastrocytomas: role of neural stem cells and its therapeutic implications. Clin

Cancer Res 15: 7124–7129.66. Madden MH, Anic GM, Thompson RC, Nabors LB, Olson JJ, et al. (2014)

Circadian pathway genes in relation to glioma risk and outcome. Cancer Causes

Control 25: 25–32.67. Li A, Lin X, Tan X, Yin B, Han W, et al. (2013) Circadian gene Clock

contributes to cell proliferation and migration of glioma and is directly regulatedby tumor-suppressive miR-124. FEBS Lett 587: 2455–2460.

68. Encinas JM, Michurina TV, Peunova N, Park JH, Tordo J, et al. (2011)Division-coupled astrocytic differentiation and age-related depletion of neural

stem cells in the adult hippocampus. Cell Stem Cell 8: 566–579.



Probeset ID Entrez GeneGene Symbol Gene Title

RefSeq Transcript ID p-‐value

stepup(p-‐value) t

Mean(Rev Erb (-‐-‐)) Mean(WT)

MeanDiff(Rev Erb (-‐-‐)-‐WT)

FoldChange(Rev Erb (-‐-‐)/WT)

FoldChange(Rev Erb (-‐-‐)/WT) (Description)

1450779_at 12140 Fabp7 fatty acid binding protein 7, brainNM_021272 1.91E-‐05 0.172295 37.0984 12.1854 10.0876 2.0978 4.28056 Greater than 01415806_at 18791 Plat plasminogen activator, tissueNM_008872 0.00014977 0.35551 33.9192 8.74698 8.04574 0.701239 1.6259 Greater than 01437056_x_at 78892 Crispld2 cysteine-‐rich secretory protein LCCL domain containing 2NM_030209 0.00019083 0.374199 13.3599 8.1312 7.17358 0.957616 1.9421 Greater than 01440773_at 382010 BC088983 cDNA sequence BC088983NM_0010099510.00047366 0.417158 10.5234 6.18471 5.51505 0.669655 1.59069 Greater than 01421907_at 19014 Med1 mediator complex subunit 1NM_001080118 /// NM_013634 /// NM_1340270.0005204 0.417158 19.5181 8.82237 7.82005 1.00232 2.00322 Greater than 01452975_at 71760 Agxt2l1 alanine-‐glyoxylate aminotransferase 2-‐like 1NM_001163587 /// NM_0279070.0005284 0.417158 41.1949 10.9419 10.1908 0.7511 1.68308 Greater than 01418090_at 84094 Plvap plasmalemma vesicle associated proteinNM_032398 0.00072297 0.417158 10.1107 9.39363 8.36452 1.0291 2.04076 Greater than 01436115_at 212539 Gm266 predicted gene 266NM_0010332480.00143176 0.448151 7.8492 8.07414 6.85337 1.22077 2.33072 Greater than 01450725_s_at 23831 Car14 carbonic anhydrase 14NM_011797 0.00174933 0.462159 13.101 8.5954 7.48064 1.11476 2.16559 Greater than 01460458_at 78892 Crispld2 cysteine-‐rich secretory protein LCCL domain containing 2NM_030209 0.00186117 0.463036 17.4074 6.51532 5.85033 0.66499 1.58556 Greater than 01441430_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00215782 0.474935 8.25034 8.18611 7.39473 0.791379 1.73073 Greater than 01421037_at 18143 Npas2 neuronal PAS domain protein 2NM_008719 0.00267762 0.493072 7.30434 6.33878 5.38711 0.951671 1.93411 Greater than 01417130_s_at 57875 Angptl4 angiopoietin-‐like 4NM_020581 0.00276587 0.501202 6.68926 7.20628 6.5425 0.663774 1.58422 Greater than 01450712_at 16524 Kcnj9 potassium inwardly-‐rectifying channel, subfamily J, member 9NM_008429 0.00277093 0.501202 6.76756 7.0472 5.68973 1.35747 2.56235 Greater than 01421679_a_at 12575 Cdkn1a cyclin-‐dependent kinase inhibitor 1A (P21)NM_001111099 /// NM_0076690.00436461 0.511218 8.27631 7.53148 6.88655 0.644929 1.56366 Greater than 01436870_s_at 226250 Afap1l2 actin filament associated protein 1-‐like 2NM_001177796 /// NM_001177797 /// NM_1461020.00454795 0.511218 7.01968 7.53911 6.79534 0.743769 1.67454 Greater than 01454886_x_at 94090 Trim9 tripartite motif-‐containing 9NM_001110202 /// NM_001110203 /// NM_0531670.00547535 0.52282 6.59694 9.26429 8.56613 0.698156 1.62243 Greater than 01434758_at 78892 Crispld2 cysteine-‐rich secretory protein LCCL domain containing 2NM_030209 0.00560114 0.52282 6.46118 6.7046 5.91638 0.788217 1.72694 Greater than 01435176_a_at 15902 Id2 inhibitor of DNA binding 2NM_010496 0.00592162 0.525199 10.2613 10.1823 9.33931 0.842959 1.79373 Greater than 01435189_at 666060 Frmpd1 FERM and PDZ domain containing 1NM_0010811720.00602684 0.526446 8.64103 7.73445 6.85984 0.874608 1.83351 Greater than 01424638_at 12575 Cdkn1a cyclin-‐dependent kinase inhibitor 1A (P21)NM_001111099 /// NM_0076690.00622519 0.533769 6.22729 8.68739 7.75419 0.933201 1.90951 Greater than 01457373_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00631852 0.535608 5.27497 7.47467 6.77008 0.704592 1.62968 Greater than 01423110_at 12843 Col1a2 collagen, type I, alpha 2NM_007743 0.00754798 0.55235 9.18846 5.86131 5.239 0.622311 1.53934 Greater than 01425099_a_at 11865 Arntl aryl hydrocarbon receptor nuclear translocator-‐likeNM_001243048 /// NM_0074890.00766915 0.55235 10.1276 8.43153 7.67722 0.754313 1.68683 Greater than 01447307_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0080578 0.558231 5.09358 4.87734 4.13313 0.744215 1.67506 Greater than 01448383_at 17387 Mmp14 matrix metallopeptidase 14 (membrane-‐inserted)NM_008608 0.0115719 0.586241 8.93396 8.54713 7.49856 1.04857 2.06848 Greater than 01450371_at 22094 Tshb thyroid stimulating hormone, beta subunitNM_001165939 /// NM_001165940 /// NM_0094320.0118457 0.586241 6.7474 7.82556 5.84379 1.98177 3.94977 Greater than 01433670_at 13731 Emp2 epithelial membrane protein 2NM_007929 0.0120236 0.586241 5.23603 8.28111 7.46516 0.815946 1.76045 Greater than 01435998_at 239083 Ccnb1ip1 cyclin B1 interacting protein 1NM_0011111190.0144965 0.601418 6.07221 6.26297 4.56532 1.69765 3.24373 Greater than 01422537_a_at 15902 Id2 inhibitor of DNA binding 2NM_010496 0.0163346 0.618833 5.10731 9.86748 9.21214 0.655346 1.57499 Greater than 01456231_at 237625 Pla2g3 phospholipase A2, group IIINM_172791 0.0167002 0.618833 4.7171 5.43104 4.79071 0.64033 1.55869 Greater than 01416572_at 17387 Mmp14 matrix metallopeptidase 14 (membrane-‐inserted)NM_008608 0.017676 0.622453 4.76771 6.62833 5.85898 0.76935 1.7045 Greater than 0

urs.albrecht

Text Box

Table S1

1450784_at 53614 Reck reversion-‐inducing-‐cysteine-‐rich protein with kazal motifsNM_016678 0.0177013 0.622453 3.94779 6.78119 6.17917 0.60202 1.51784 Greater than 01451901_at 20585 Hltf helicase-‐like transcription factorNM_009210 /// NM_1449590.018316 0.627674 4.03194 4.77893 4.09076 0.688168 1.61124 Greater than 01437671_x_at 76453 Prss23 protease, serine, 23NM_029614 0.0209231 0.634794 3.70598 6.28019 5.61357 0.666627 1.58736 Greater than 01442424_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0222042 0.646038 3.94146 6.0272 5.28157 0.745628 1.6767 Greater than 01458282_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0253181 0.653128 3.6794 6.8863 6.2662 0.620109 1.53699 Greater than 01460604_at 73649 Cybrd1 cytochrome b reductase 1NM_028593 0.0284839 0.668046 3.59038 6.94961 6.18991 0.759701 1.69314 Greater than 01434249_s_at 94090 Trim9 tripartite motif-‐containing 9NM_001110202 /// NM_001110203 /// NM_0531670.0313854 0.680144 4.97624 8.04332 7.1751 0.868221 1.82541 Greater than 01457111_at 103570 AA415038 expressed sequence AA415038-‐-‐-‐ 0.0316847 0.68113 3.23967 7.83945 7.20433 0.635112 1.55306 Greater than 01455050_at 320736 E130203B14RikRIKEN cDNA E130203B14 geneNM_178791 0.0349747 0.692791 4.91004 6.87443 6.01602 0.858414 1.81304 Greater than 01422789_at 19378 Aldh1a2 aldehyde dehydrogenase family 1, subfamily A2NM_009022 0.0365687 0.701318 3.13756 6.34446 5.54955 0.794904 1.73496 Greater than 01428738_a_at100039192 /// 100039257 /// 66039D14Ertd449e /// Gm10395 /// Gm9746DNA segment, Chr 14, ERATO Doi 449, expressed /// predicted gene 10395 /// predicted geNM_025311 /// NM_026679 /// XM_001472492 /// XM_001472510 /// XM_001472657 /// XM_001470.0374852 0.70729 3.93042 8.80065 8.06658 0.734076 1.66333 Greater than 01418937_at 13371 Dio2 deiodinase, iodothyronine, type IINM_010050 0.037696 0.709469 4.79264 7.53049 6.85241 0.678076 1.60001 Greater than 01422230_s_at13086 /// 13087Cyp2a4 /// Cyp2a5cytochrome P450, family 2, subfamily a, polypeptide 4 /// cytochrome P450, family 2, suNM_007812 /// NM_009997 /// XM_0036894050.0425058 0.718655 2.93842 4.55088 3.94786 0.603025 1.5189 Greater than 01439143_at 328399 A930018M24RikRIKEN cDNA A930018M24 geneXR_141243 /// XR_1417390.04317 0.719272 2.92299 5.85523 5.19769 0.657543 1.57739 Greater than 0

Probeset ID Entrez GeneGene Symbol Gene Title

RefSeq Transcript ID p-‐value

stepup(p-‐value) t

Mean(Rev Erb (-‐-‐)) Mean(WT)

MeanDiff(Rev Erb (-‐-‐)-‐WT)

FoldChange(Rev Erb (-‐-‐)/WT)

FoldChange(Rev Erb (-‐-‐)/WT) (Description)

1454675_at 21833 Thra thyroid hormone receptor alphaNM_178060 8.29E-‐06 0.145299 -‐57.5004 10.3989 11.537 -‐1.13814 -‐2.20097 Less than 01428563_at 77591 Ddx10 DEAD (Asp-‐Glu-‐Ala-‐Asp) box polypeptide 10NM_029936 9.66E-‐06 0.145299 -‐40.076 6.26929 6.99888 -‐0.729584 -‐1.65816 Less than 01440282_at 68842 Tulp4 tubby like protein 4NM_001033529 /// NM_001103181 /// NM_0540401.81E-‐05 0.172295 -‐23.9887 4.68877 5.96144 -‐1.27267 -‐2.41608 Less than 01438565_at 231570 A830010M20RikRIKEN cDNA A830010M20 geneNM_001007574 /// NM_0011685572.50E-‐05 0.188073 -‐26.9724 6.54156 7.6672 -‐1.12564 -‐2.18198 Less than 01438207_at 107338 Gbf1 golgi-‐specific brefeldin A-‐resistance factor 1NM_178930 4.64E-‐05 0.298818 -‐19.2931 5.8974 7.52782 -‐1.63042 -‐3.09602 Less than 01450051_at 22589 Atrx alpha thalassemia/mental retardation syndrome X-‐linked homolog (human)NM_009530 5.65E-‐05 0.318414 -‐22.9907 6.72001 7.88493 -‐1.16492 -‐2.24221 Less than 01421064_at 56217 Mpp5 membrane protein, palmitoylated 5 (MAGUK p55 subfamily member 5)NM_019579 9.18E-‐05 0.343755 -‐16.3982 8.53567 9.35092 -‐0.815258 -‐1.75961 Less than 01425600_a_at 18795 Plcb1 phospholipase C, beta 1NM_001145830 /// NM_0196770.00010242 0.343755 -‐15.6636 6.61235 7.70958 -‐1.09723 -‐2.13943 Less than 01429592_at 269629 Lhfpl3 lipoma HMGIC fusion partner-‐like 3NM_001081231 /// NM_0299900.00010583 0.343755 -‐28.8713 6.77337 7.66255 -‐0.889185 -‐1.85213 Less than 01429362_a_at 319322 Sf3b2 splicing factor 3b, subunit 2NM_030109 0.00012957 0.343755 -‐14.6097 9.77683 10.7194 -‐0.942587 -‐1.92197 Less than 01431191_a_at 20979 Syt1 synaptotagmin INM_001252341 /// NM_001252342 /// NM_0093060.00016482 0.371673 -‐15.1937 8.11886 8.98122 -‐0.862362 -‐1.81801 Less than 01426464_at 217166 Nr1d1 nuclear receptor subfamily 1, group D, member 1NM_145434 0.00026119 0.40141 -‐16.6078 5.6937 7.53226 -‐1.83856 -‐3.57653 Less than 01421592_at 17968 Ncam2 neural cell adhesion molecule 2NM_001113208 /// NM_0109540.00026185 0.40141 -‐14.1617 6.9476 7.61969 -‐0.672097 -‐1.59339 Less than 01453860_s_at 14815 Nr3c1 nuclear receptor subfamily 3, group C, member 1NM_008173 0.00028481 0.40141 -‐15.7622 5.30114 6.22606 -‐0.924923 -‐1.89858 Less than 01453735_at 70591 5730455P16RikRIKEN cDNA 5730455P16 geneNM_027472 0.00034631 0.417158 -‐11.5992 7.00214 7.66978 -‐0.667648 -‐1.58848 Less than 01438040_a_at 22027 Hsp90b1 heat shock protein 90, beta (Grp94), member 1NM_011631 0.00037194 0.417158 -‐19.7865 9.08621 9.72152 -‐0.635318 -‐1.55328 Less than 01437657_at 244891 Scaper S phase cyclin A-‐associated protein in the ERNM_001081341 /// NM_1755360.00042978 0.417158 -‐10.7366 7.04936 8.05105 -‐1.00169 -‐2.00234 Less than 01428574_a_at 69993 Chn2 chimerin (chimaerin) 2NM_001163640 /// NM_0235430.00043203 0.417158 -‐17.9418 6.31732 7.14272 -‐0.825401 -‐1.77203 Less than 01435770_at 52837 Tmx4 thioredoxin-‐related transmembrane protein 4NM_029148 0.00051243 0.417158 -‐10.2443 7.37744 8.87368 -‐1.49625 -‐2.82108 Less than 01436850_at 263764 Creg2 cellular repressor of E1A-‐stimulated genes 2NM_170597 0.00054542 0.417158 -‐10.3106 6.73221 7.56694 -‐0.834733 -‐1.78353 Less than 01456112_at 108989 Tpr translocated promoter regionNM_133780 0.00055579 0.417158 -‐11.1511 8.48123 9.32965 -‐0.848423 -‐1.80053 Less than 01424598_at 13209 Ddx6 DEAD (Asp-‐Glu-‐Ala-‐Asp) box polypeptide 6NM_001110826 /// NM_007841 /// NM_1813240.00058353 0.417158 -‐11.0825 8.79573 9.81184 -‐1.01611 -‐2.02246 Less than 01426444_at 215160 Rhbdd2 rhomboid domain containing 2NM_031398 /// NM_1460020.00060305 0.417158 -‐11.6061 8.03791 8.6863 -‐0.648393 -‐1.56742 Less than 01416062_at 66687 Tbc1d15 TBC1 domain family, member 15NM_025706 0.0006314 0.417158 -‐9.73818 5.73914 6.38127 -‐0.642122 -‐1.56062 Less than 01452360_a_at 214899 Kdm5a lysine (K)-‐specific demethylase 5ANM_145997 0.00063605 0.417158 -‐10.0428 8.0063 8.76201 -‐0.75571 -‐1.68846 Less than 01459563_x_at-‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00067641 0.417158 -‐10.7449 4.73154 5.36093 -‐0.62939 -‐1.54691 Less than 01453787_at 52837 Tmx4 thioredoxin-‐related transmembrane protein 4NM_029148 0.00070536 0.417158 -‐13.3261 7.36139 8.72698 -‐1.36559 -‐2.57682 Less than 01429660_s_at 14211 Smc2 structural maintenance of chromosomes 2NM_008017 0.00071179 0.417158 -‐9.4094 3.70653 4.40251 -‐0.695988 -‐1.61999 Less than 01446914_at 96999 C80425 expressed sequence C80425-‐-‐-‐ 0.00071911 0.417158 -‐9.80337 4.28355 5.1542 -‐0.870659 -‐1.8285 Less than 0

urs.albrecht

Text Box

Table S2

1455151_at 100986 Akap9 A kinase (PRKA) anchor protein (yotiao) 9NM_194462 0.00082171 0.417158 -‐16.9228 6.88396 7.56801 -‐0.684054 -‐1.60665 Less than 01416482_at 22129 Ttc3 tetratricopeptide repeat domain 3NM_009441 0.00083769 0.417158 -‐9.07615 9.67229 10.7011 -‐1.02885 -‐2.04039 Less than 01420973_at 71371 Arid5b AT rich interactive domain 5B (MRF1-‐like)NM_023598 0.00085473 0.417158 -‐9.0334 6.78029 7.37206 -‐0.591762 -‐1.50709 Less than 01424207_at 93762 Smarca5 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily aNM_053124 0.00088795 0.417158 -‐14.8229 8.19611 8.97151 -‐0.775399 -‐1.71166 Less than 01450915_at 11774 Ap3b1 adaptor-‐related protein complex 3, beta 1 subunitNM_009680 0.00092129 0.42212 -‐8.79862 7.32067 8.13032 -‐0.809649 -‐1.75278 Less than 01452236_at 224742 Abcf1 ATP-‐binding cassette, sub-‐family F (GCN20), member 1NM_013854 0.00094044 0.42212 -‐12.4387 8.26334 8.85783 -‐0.594496 -‐1.50995 Less than 01439300_at 12212 Chic1 cysteine-‐rich hydrophobic domain 1NM_009767 0.00096272 0.42212 -‐9.26171 8.03765 8.80972 -‐0.772066 -‐1.70771 Less than 01419038_a_at 12995 Csnk2a1 casein kinase 2, alpha 1 polypeptideNM_007788 0.00096402 0.42212 -‐10.832 7.2935 8.02841 -‐0.734907 -‐1.66429 Less than 01430511_at 208898 Unc13c unc-‐13 homolog C (C. elegans)NM_0010811530.00098904 0.424827 -‐8.81433 4.99692 6.35374 -‐1.35682 -‐2.56121 Less than 01436343_at 107932 Chd4 chromodomain helicase DNA binding protein 4NM_145979 0.00102706 0.436995 -‐25.0867 8.57392 9.41203 -‐0.838113 -‐1.78771 Less than 01425019_at 217379 Ubxn2a UBX domain protein 2ANM_145441 0.00104383 0.439979 -‐8.6101 8.67635 9.27266 -‐0.596317 -‐1.51185 Less than 01452788_at 26932 Ppp2r5e protein phosphatase 2, regulatory subunit B (B56), epsilon isoformNM_012024 0.00114816 0.448151 -‐12.1307 8.4145 9.02585 -‐0.611348 -‐1.52769 Less than 01460304_a_at 21429 Ubtf upstream binding transcription factor, RNA polymerase INM_001044383 /// NM_0115510.00116304 0.448151 -‐8.46376 5.66177 6.64695 -‐0.985184 -‐1.97957 Less than 01429432_at 226562 Prrc2c proline-‐rich coiled-‐coil 2CNM_0010812900.00123428 0.448151 -‐8.68462 3.4898 4.41393 -‐0.924135 -‐1.89755 Less than 01422842_at 24128 Xrn2 5'-‐3' exoribonuclease 2NM_011917 0.00124464 0.448151 -‐8.1725 6.85421 7.64615 -‐0.791943 -‐1.7314 Less than 01453263_at 78689 Naa35 N(alpha)-‐acetyltransferase 35, NatC auxiliary subunitNM_030153 0.00124631 0.448151 -‐9.11406 7.49804 8.16073 -‐0.662687 -‐1.58303 Less than 01456489_at 74737 Pcf11 cleavage and polyadenylation factor subunit homolog (S. cerevisiae)NM_029078 0.00132201 0.448151 -‐9.52918 6.26184 7.00035 -‐0.738513 -‐1.66846 Less than 01438975_x_at 224454 Zdhhc14 zinc finger, DHHC domain containing 14NM_146073 0.00133523 0.448151 -‐8.12341 7.91915 8.66103 -‐0.741878 -‐1.67235 Less than 01458385_at 18415 Hspa4l heat shock protein 4 likeNM_011020 0.00135242 0.448151 -‐9.67776 7.70508 8.32114 -‐0.616067 -‐1.53269 Less than 01437614_x_at 224454 Zdhhc14 zinc finger, DHHC domain containing 14NM_146073 0.00137688 0.448151 -‐8.39227 7.8059 8.97614 -‐1.17024 -‐2.25049 Less than 01417831_at 24061 Smc1a structural maintenance of chromosomes 1ANM_019710 0.00137693 0.448151 -‐10.8343 7.56789 8.49408 -‐0.926186 -‐1.90025 Less than 01456088_at 11798 Xiap X-‐linked inhibitor of apoptosisNM_009688 0.00144412 0.448151 -‐11.4223 7.08267 8.08023 -‐0.997559 -‐1.99662 Less than 01424922_a_at 57261 Brd4 bromodomain containing 4NM_020508 /// NM_1980940.00147872 0.448151 -‐9.36023 8.78371 9.46619 -‐0.682479 -‐1.60489 Less than 01430568_at 67302 Zc3h13 zinc finger CCCH type containing 13NM_026083 /// NM_0273770.00150258 0.448151 -‐8.65974 7.0321 7.89956 -‐0.867464 -‐1.82445 Less than 01428046_a_at 22764 Zfx zinc finger protein X-‐linkedNM_001044386 /// NM_0117680.00160795 0.450394 -‐8.59517 4.3965 5.39484 -‐0.998343 -‐1.9977 Less than 01456110_at 77087 Ankrd11 ankyrin repeat domain 11NM_001081379 /// NR_0378650.0016364 0.450394 -‐7.57194 7.90871 8.77301 -‐0.8643 -‐1.82046 Less than 01452885_at 72193 Scaf11 SR-‐related CTD-‐associated factor 11NM_028148 0.00163776 0.450394 -‐9.22071 7.11032 8.14832 -‐1.038 -‐2.05337 Less than 01426485_at 67812 Ubxn4 UBX domain protein 4NM_026390 0.00175156 0.462159 -‐9.55496 9.16767 9.81618 -‐0.64851 -‐1.56755 Less than 01449042_at 13018 Ctcf CCCTC-‐binding factorNM_181322 0.00190988 0.463036 -‐19.9927 8.76159 9.52539 -‐0.763806 -‐1.69796 Less than 01440248_at 319996 Casc4 cancer susceptibility candidate 4NM_001205369 /// NM_001205370 /// NM_001205371 /// NM_177054 /// NM_1990380.00192485 0.463036 -‐13.6699 4.83599 6.36467 -‐1.52868 -‐2.88523 Less than 01421504_at 20688 Sp4 trans-‐acting transcription factor 4NM_001166385 /// NM_0092390.00194249 0.463036 -‐8.31278 7.00713 7.59429 -‐0.58716 -‐1.50229 Less than 01456874_at 399558 Flrt2 fibronectin leucine rich transmembrane protein 2NM_201518 0.00194439 0.463036 -‐7.78557 5.80015 6.51149 -‐0.711338 -‐1.63732 Less than 01459295_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00197836 0.463036 -‐7.20852 5.34683 6.64959 -‐1.30276 -‐2.46701 Less than 01415859_at 56347 Eif3c eukaryotic translation initiation factor 3, subunit CNM_146200 0.00198584 0.463036 -‐12.5396 10.0884 10.7141 -‐0.625675 -‐1.54293 Less than 0

1439122_at 13209 Ddx6 DEAD (Asp-‐Glu-‐Ala-‐Asp) box polypeptide 6NM_001110826 /// NM_007841 /// NM_1813240.00198967 0.463036 -‐13.4326 8.11224 8.8254 -‐0.713164 -‐1.6394 Less than 01420989_at 66756 4933411K20RikRIKEN cDNA 4933411K20 geneNM_025747 0.00203649 0.466232 -‐8.0946 5.82198 7.2256 -‐1.40362 -‐2.64564 Less than 01431465_s_at 69823 Fyttd1 forty-‐two-‐three domain containing 1NM_001159349 /// NM_0272260.00209654 0.474935 -‐7.08423 5.76831 6.3973 -‐0.62899 -‐1.54648 Less than 01424675_at 106957 Slc39a6 solute carrier family 39 (metal ion transporter), member 6NM_139143 0.0023415 0.48237 -‐7.37129 7.37571 8.0029 -‐0.627189 -‐1.54455 Less than 01457281_at 78244 Dnajc21 DnaJ (Hsp40) homolog, subfamily C, member 21NM_030046 0.0024344 0.488897 -‐12.1758 7.34354 8.07158 -‐0.728036 -‐1.65638 Less than 01456316_a_at 170760 Acbd3 acyl-‐Coenzyme A binding domain containing 3NM_133225 0.00247563 0.488897 -‐12.4236 6.37963 7.14466 -‐0.76503 -‐1.69941 Less than 01431686_a_at 63985 Gmfb glia maturation factor, betaNM_022023 0.0024941 0.488897 -‐9.34869 5.20928 5.83166 -‐0.62238 -‐1.53941 Less than 01438101_at 14365 Fzd3 frizzled homolog 3 (Drosophila)NM_021458 0.00257453 0.493072 -‐6.7163 3.75187 4.39572 -‐0.643857 -‐1.5625 Less than 01427822_a_at100044236 Copg2as2 coatomer protein complex, subunit gamma 2, antisense 2NR_002845 0.00259105 0.493072 -‐13.2608 7.33528 8.65704 -‐1.32177 -‐2.49972 Less than 01438363_at 434128 Pnmal2 PNMA-‐like 2 NM_0010996360.0026402 0.493072 -‐12.9838 9.72514 10.9654 -‐1.24029 -‐2.36245 Less than 01460384_a_at 94246 Arid4b AT rich interactive domain 4B (RBP1-‐like)NM_194262 /// NM_1981220.00272016 0.496688 -‐6.7312 5.63002 6.34373 -‐0.713707 -‐1.64001 Less than 01424142_at 230233 Ikbkap inhibitor of kappa light polypeptide enhancer in B cells, kinase complex-‐associated proNM_026079 0.00281524 0.503523 -‐7.25351 6.69231 7.43614 -‐0.743824 -‐1.67461 Less than 01438476_a_at 107932 Chd4 chromodomain helicase DNA binding protein 4NM_145979 0.00287545 0.503523 -‐7.11465 7.09708 7.92828 -‐0.831197 -‐1.77916 Less than 01420402_at 11941 Atp2b2 ATPase, Ca++ transporting, plasma membrane 2NM_001036684 /// NM_0097230.00294107 0.503523 -‐9.90205 7.55284 8.76207 -‐1.20923 -‐2.31214 Less than 01426541_a_at 71946 Endod1 endonuclease domain containing 1NM_028013 0.00302446 0.503523 -‐6.42443 7.83822 8.46688 -‐0.628662 -‐1.54613 Less than 01458539_at 226412 R3hdm1 R3H domain 1 (binds single-‐stranded nucleic acids)NM_181750 0.00306733 0.503523 -‐6.46901 5.53213 6.48842 -‐0.956295 -‐1.94032 Less than 01424325_at 77805 Esco1 establishment of cohesion 1 homolog 1 (S. cerevisiae)NM_001081222 /// NM_1445420.00309521 0.503961 -‐6.38801 6.52643 7.44576 -‐0.919327 -‐1.89123 Less than 01436229_at 213056 Fam126b family with sequence similarity 126, member BNM_172513 0.00319798 0.50788 -‐8.41685 8.51144 9.31407 -‐0.80263 -‐1.74428 Less than 01455905_at 72503 2610507B11RikRIKEN cDNA 2610507B11 geneNM_0010020040.00321067 0.50788 -‐7.98834 7.82022 8.6391 -‐0.818884 -‐1.76404 Less than 01424658_at 216965 Taok1 TAO kinase 1NM_144825 0.0032724 0.50788 -‐7.91845 6.54172 7.42605 -‐0.884327 -‐1.8459 Less than 01445081_at 320271 Scai suppressor of cancer cell invasionNM_178778 0.00328951 0.50788 -‐6.28049 6.03919 6.88654 -‐0.84736 -‐1.79921 Less than 01447869_x_at 73296 Rhobtb3 Rho-‐related BTB domain containing 3NM_028493 0.00331632 0.50788 -‐7.14423 6.61191 7.36241 -‐0.750494 -‐1.68237 Less than 01438774_s_at 70974 Pgm2l1 phosphoglucomutase 2-‐like 1NM_027629 0.00336711 0.50788 -‐6.60791 8.5799 9.20057 -‐0.620671 -‐1.53759 Less than 01450093_s_at 16969 Zbtb7a zinc finger and BTB domain containing 7aNM_010731 0.00356412 0.50788 -‐7.16159 7.14616 7.93982 -‐0.793659 -‐1.73346 Less than 01439555_at 109263 Rlf rearranged L-‐myc fusion sequenceNM_0010810130.00357172 0.50788 -‐16.0932 3.92951 5.6067 -‐1.67719 -‐3.19804 Less than 01445296_at 52480 D7Ertd715e DNA segment, Chr 7, ERATO Doi 715, expressedNR_015456 0.00357992 0.50788 -‐6.2761 6.22705 6.98805 -‐0.761 -‐1.69466 Less than 01418129_at 74754 Dhcr24 24-‐dehydrocholesterol reductaseNM_053272 0.00362852 0.50788 -‐6.63792 8.20124 8.83393 -‐0.632696 -‐1.55046 Less than 01442100_at 101490 Inpp5f inositol polyphosphate-‐5-‐phosphatase FNM_178641 0.00367506 0.50788 -‐6.10057 5.44492 6.24469 -‐0.799773 -‐1.74083 Less than 01446512_at 69082 Zc3h15 zinc finger CCCH-‐type containing 15NM_026934 0.00368153 0.50788 -‐6.66769 5.75852 6.55634 -‐0.797817 -‐1.73847 Less than 01422042_at 118446 Gjc3 gap junction protein, gamma 3NM_080450 0.00376243 0.50788 -‐6.22028 5.81131 6.40237 -‐0.591063 -‐1.50636 Less than 01456337_at 212285 Arap2 ArfGAP with RhoGAP domain, ankyrin repeat and PH domain 2NM_178407 0.00379225 0.50788 -‐6.34152 3.62451 4.51998 -‐0.895466 -‐1.86021 Less than 01452479_at 100044236 Copg2as2 coatomer protein complex, subunit gamma 2, antisense 2NR_002845 0.00385484 0.50788 -‐6.63357 3.82631 4.93464 -‐1.10833 -‐2.15596 Less than 01422675_at 57376 Smarce1 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily eNM_020618 0.00391119 0.508353 -‐9.71176 6.90334 7.51276 -‐0.609421 -‐1.52565 Less than 01431024_a_at 94246 Arid4b AT rich interactive domain 4B (RBP1-‐like)NM_194262 /// NM_1981220.00408997 0.509084 -‐7.88852 4.73476 5.67892 -‐0.944156 -‐1.92406 Less than 0

1451878_a_at 57748 Jmy junction-‐mediating and regulatory proteinNM_021310 0.00411955 0.509084 -‐5.90678 3.53546 4.94513 -‐1.40967 -‐2.65676 Less than 01422834_at 16508 Kcnd2 potassium voltage-‐gated channel, Shal-‐related family, member 2NM_019697 0.00425371 0.510357 -‐14.8901 8.47153 9.28639 -‐0.814856 -‐1.75912 Less than 01439642_at 320504 5930403N24RikRIKEN cDNA 5930403N24 geneNM_177177 /// XR_1065090.00436413 0.511218 -‐7.02627 4.44526 5.44475 -‐0.999489 -‐1.99929 Less than 01437107_at 270192 Rab6b RAB6B, member RAS oncogene familyNM_173781 0.00437118 0.511218 -‐10.0886 8.9613 10.0404 -‐1.07911 -‐2.11273 Less than 01419250_a_at 18647 Cdk14 cyclin-‐dependent kinase 14NM_011074 0.00437396 0.511218 -‐10.569 6.6017 7.29958 -‐0.697873 -‐1.62211 Less than 01447723_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00440419 0.511218 -‐5.83346 6.43667 7.50909 -‐1.07242 -‐2.10296 Less than 01432558_a_at 17153 Mal myelin and lymphocyte protein, T cell differentiation proteinNM_001171187 /// NM_0107620.00453858 0.511218 -‐7.1478 8.78276 9.42077 -‐0.638005 -‐1.55618 Less than 01458147_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00459869 0.511218 -‐6.20779 5.40435 6.32042 -‐0.916073 -‐1.88697 Less than 01422910_s_at 67241 Smc6 structural maintenance of chromosomes 6NM_025695 0.00466635 0.51331 -‐6.21598 8.22115 8.9026 -‐0.68145 -‐1.60375 Less than 01426892_at 22288 Utrn utrophin NM_011682 0.00485691 0.513394 -‐6.09883 4.65364 5.51313 -‐0.859487 -‐1.81439 Less than 01425544_at 109135 Plekha5 pleckstrin homology domain containing, family A member 5NM_144920 0.00491491 0.513394 -‐6.53273 5.33528 5.92717 -‐0.59189 -‐1.50722 Less than 01419092_a_at 20874 Slk STE20-‐like kinaseNM_001164639 /// NM_0092890.0049221 0.513394 -‐13.1611 4.76434 5.56568 -‐0.801338 -‐1.74272 Less than 01428092_at 71702 Cdc5l cell division cycle 5-‐like (S. pombe)NM_152810 0.00512478 0.52282 -‐7.95767 8.10234 8.7073 -‐0.604961 -‐1.52094 Less than 01460426_at 83679 Pde4dip phosphodiesterase 4D interacting protein (myomegalin)NM_001039376 /// NM_001110163 /// NM_031401 /// NM_177145 /// NM_1780800.0052398 0.52282 -‐9.97689 6.0732 6.89936 -‐0.826165 -‐1.77297 Less than 01422206_at 26877 B3galt1 UDP-‐Gal:betaGlcNAc beta 1,3-‐galactosyltransferase, polypeptide 1NM_020283 0.0052437 0.52282 -‐6.29826 5.43803 6.15824 -‐0.720211 -‐1.64742 Less than 01438719_at 26405 Map3k2 mitogen-‐activated protein kinase kinase kinase 2NM_011946 0.00524619 0.52282 -‐9.58466 5.08427 5.82803 -‐0.743762 -‐1.67454 Less than 01417623_at 20496 Slc12a2 solute carrier family 12, member 2NM_009194 0.00563322 0.52282 -‐8.81011 7.68833 8.43884 -‐0.750508 -‐1.68239 Less than 01456656_at 108030 Lin7a lin-‐7 homolog A (C. elegans)NM_001033223 /// NM_0010393540.00564294 0.52282 -‐5.42459 7.48631 8.0743 -‐0.587992 -‐1.50315 Less than 01420947_at 22589 Atrx alpha thalassemia/mental retardation syndrome X-‐linked homolog (human)NM_009530 0.00570078 0.52282 -‐6.53787 6.42074 7.27009 -‐0.84935 -‐1.80169 Less than 01427510_at 14677 Gnai1 guanine nucleotide binding protein (G protein), alpha inhibiting 1NM_010305 0.0058313 0.52282 -‐11.4799 7.37993 8.00942 -‐0.629491 -‐1.54702 Less than 01439083_at 52906 Ahi1 Abelson helper integration site 1NM_001177776 /// NM_0262030.00583553 0.52282 -‐5.88906 8.82504 9.4386 -‐0.61356 -‐1.53003 Less than 01456187_at 241919 Slc7a14 solute carrier family 7 (cationic amino acid transporter, y+ system), member 14NM_172861 0.00583912 0.52282 -‐7.05912 5.2592 6.2321 -‐0.972897 -‐1.96278 Less than 01426168_a_at100038850 Trav9d-‐3 T cell receptor alpha variable 9D-‐3-‐-‐-‐ 0.00586904 0.524055 -‐5.49727 5.21506 5.93242 -‐0.717361 -‐1.64417 Less than 01453855_at 67622 Mxra7 matrix-‐remodelling associated 7NM_026280 0.00617814 0.53109 -‐5.44441 5.40823 6.2429 -‐0.834666 -‐1.78344 Less than 01429308_at 70673 Prdm16 PR domain containing 16NM_001177995 /// NM_0275040.00618217 0.53109 -‐5.67118 4.99135 6.06568 -‐1.07433 -‐2.10575 Less than 01440623_at 233919 Gpr26 G protein-‐coupled receptor 26NM_173410 0.00671735 0.541051 -‐9.31403 4.98377 5.64916 -‐0.665388 -‐1.586 Less than 01457040_at 246316 Lgi2 leucine-‐rich repeat LGI family, member 2NM_144945 0.00692075 0.545117 -‐5.36202 6.50926 7.22928 -‐0.720024 -‐1.64721 Less than 01430534_at 78416 Rnase6 ribonuclease, RNase A family, 6NM_030098 0.00693693 0.545117 -‐5.28748 4.57249 5.2867 -‐0.714212 -‐1.64059 Less than 01422546_at 16201 Ilf3 interleukin enhancer binding factor 3NM_001042707 /// NM_001042708 /// NM_001042709 /// NM_0105610.00697275 0.545331 -‐7.17232 6.5062 7.23389 -‐0.727687 -‐1.65598 Less than 01450530_at 26877 B3galt1 UDP-‐Gal:betaGlcNAc beta 1,3-‐galactosyltransferase, polypeptide 1NM_020283 0.007003 0.546405 -‐9.42999 4.62763 5.35606 -‐0.728429 -‐1.65683 Less than 01421905_at 116940 Tgs1 trimethylguanosine synthase homolog (S. cerevisiae)NM_054089 0.00708355 0.547985 -‐10.1173 7.10666 7.69211 -‐0.585455 -‐1.50051 Less than 01459804_at 12914 Crebbp CREB binding proteinNM_0010254320.00709576 0.54799 -‐7.72273 5.07703 6.53952 -‐1.4625 -‐2.75585 Less than 01418431_at 16573 Kif5b kinesin family member 5BNM_008448 0.00717349 0.548256 -‐9.13812 8.71626 9.38258 -‐0.666314 -‐1.58701 Less than 01451800_at 70297 Gcc2 GRIP and coiled-‐coil domain containing 2NM_027375 0.00745448 0.55235 -‐5.34644 5.17199 5.79678 -‐0.624789 -‐1.54199 Less than 0

1420911_a_at 17304 Mfge8 milk fat globule-‐EGF factor 8 proteinNM_001045489 /// NM_0085940.00746937 0.55235 -‐5.2189 9.87513 10.5682 -‐0.693097 -‐1.61675 Less than 01439605_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.00748511 0.55235 -‐7.55402 6.69513 7.46869 -‐0.773561 -‐1.70948 Less than 01432269_a_at 58194 Sh3kbp1 SH3-‐domain kinase binding protein 1NM_001135727 /// NM_001135728 /// NM_0213890.00757431 0.55235 -‐8.28449 6.40513 7.23002 -‐0.824888 -‐1.7714 Less than 01427456_at 72145 Wdfy3 WD repeat and FYVE domain containing 3NM_172882 0.00766101 0.55235 -‐6.2328 8.0485 8.67682 -‐0.628316 -‐1.54576 Less than 01420946_at 22589 Atrx alpha thalassemia/mental retardation syndrome X-‐linked homolog (human)NM_009530 0.00767979 0.55235 -‐4.97792 5.87877 6.82167 -‐0.942908 -‐1.9224 Less than 01456863_at 13838 Epha4 Eph receptor A4NM_007936 0.00769604 0.55235 -‐7.19441 5.35901 5.97871 -‐0.619699 -‐1.53655 Less than 01439566_at 243385 Gprin3 GPRIN family member 3NM_183183 0.00769622 0.55235 -‐5.38938 5.09499 5.94404 -‐0.849055 -‐1.80132 Less than 01427037_at 208643 Eif4g1 eukaryotic translation initiation factor 4, gamma 1NM_001005331 /// NM_1459410.00770086 0.55235 -‐6.97938 6.32363 7.13381 -‐0.810184 -‐1.75343 Less than 01449939_s_at 13386 Dlk1 delta-‐like 1 homolog (Drosophila)NM_001190703 /// NM_001190704 /// NM_001190705 /// NM_010052 /// NR_0338130.00788477 0.554117 -‐8.60547 9.03117 9.71637 -‐0.6852 -‐1.60792 Less than 01420935_a_at 51796 Srrm1 serine/arginine repetitive matrix 1NM_001130477 /// NM_0167990.00806011 0.558231 -‐5.517 8.3401 8.92831 -‐0.588207 -‐1.50338 Less than 01420917_at 56194 Prpf40a PRP40 pre-‐mRNA processing factor 40 homolog A (yeast)NM_018785 0.00816977 0.558231 -‐4.94226 5.92265 6.66198 -‐0.739329 -‐1.6694 Less than 01452333_at 67155 Smarca2 SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily aNM_011416 /// NM_0260030.00827968 0.562382 -‐5.12385 6.47483 7.25841 -‐0.78358 -‐1.7214 Less than 01460729_at 19877 Rock1 Rho-‐associated coiled-‐coil containing protein kinase 1NM_009071 0.00838462 0.564908 -‐7.97178 7.04196 7.68862 -‐0.646657 -‐1.56554 Less than 01417832_at 24061 Smc1a structural maintenance of chromosomes 1ANM_019710 0.00851052 0.566962 -‐6.90593 6.09303 7.04518 -‐0.952151 -‐1.93476 Less than 01430073_at 74901 Kbtbd11 kelch repeat and BTB (POZ) domain containing 11NM_029116 0.00853692 0.567881 -‐6.92592 7.05133 7.95005 -‐0.898713 -‐1.8644 Less than 01437002_at 215708 Fam73a family with sequence similarity 73, member ANM_001162375 /// NM_1748680.0085738 0.568979 -‐6.69607 7.91036 8.61954 -‐0.709183 -‐1.63488 Less than 01450035_a_at 56194 Prpf40a PRP40 pre-‐mRNA processing factor 40 homolog A (yeast)NM_018785 0.00864657 0.568979 -‐7.76323 6.38296 7.13061 -‐0.747647 -‐1.67905 Less than 01452187_at 83486 Rbm5 RNA binding motif protein 5NM_148930 0.00866696 0.568979 -‐4.93903 6.47682 7.21077 -‐0.733946 -‐1.66318 Less than 01457744_at 212880 Ddx46 DEAD (Asp-‐Glu-‐Ala-‐Asp) box polypeptide 46NM_145975 0.0086971 0.5693 -‐4.79368 6.63604 7.63842 -‐1.00238 -‐2.0033 Less than 01427488_a_at 12211 Birc6 baculoviral IAP repeat-‐containing 6NM_007566 0.00883164 0.56933 -‐4.78838 5.4691 6.37124 -‐0.902139 -‐1.86884 Less than 01437501_at 209743 AF529169 cDNA sequence AF529169NM_153509 0.0088649 0.56933 -‐4.7689 4.68165 5.37257 -‐0.690919 -‐1.61431 Less than 01455960_at 230316 Megf9 multiple EGF-‐like-‐domains 9NM_172694 0.00944369 0.580595 -‐5.92213 6.23951 7.56025 -‐1.32074 -‐2.49794 Less than 01452811_at 108147 Atic 5-‐aminoimidazole-‐4-‐carboxamide ribonucleotide formyltransferase/IMP cyclohydrolaseNM_026195 0.00952205 0.580746 -‐9.29372 6.14543 7.05095 -‐0.905518 -‐1.87322 Less than 01419256_at 20742 Spnb2 spectrin beta 2NM_009260 /// NM_1758360.00959625 0.580746 -‐8.16188 10.1395 10.8969 -‐0.75737 -‐1.69041 Less than 01438801_at 103967 Dnm3 dynamin 3 NM_001038619 /// NM_1726460.00963964 0.581074 -‐4.99337 7.04948 8.06061 -‐1.01113 -‐2.01549 Less than 01437581_at 627049 Zfp800 zinc finger protein 800NM_0010816780.00968865 0.581074 -‐7.19728 5.89162 7.16071 -‐1.26908 -‐2.41008 Less than 01435135_at 320024 Nceh1 arylacetamide deacetylase-‐like 1NM_178772 0.00979412 0.585842 -‐6.9984 9.32894 10.0571 -‐0.728117 -‐1.65648 Less than 01430974_a_at 66315 Senp7 SUMO1/sentrin specific peptidase 7NM_001003971 /// NM_001003972 /// NM_001003973 /// NM_0254830.00995044 0.586241 -‐4.68303 4.8755 5.7088 -‐0.833306 -‐1.78176 Less than 01457495_at 73040 2900052N01RikRIKEN cDNA 2900052N01 geneNR_015605 0.0103212 0.586241 -‐4.79883 6.58452 7.60786 -‐1.02335 -‐2.03263 Less than 01436311_at 216766 Gemin5 gem (nuclear organelle) associated protein 5NM_001166669 /// NM_001166670 /// NM_001166671 /// NM_1725580.0104011 0.586241 -‐5.8897 3.50192 4.24305 -‐0.741135 -‐1.67149 Less than 01428047_s_at22639 /// 22764Zfa /// Zfx zinc finger protein, autosomal /// zinc finger protein X-‐linkedNM_001044386 /// NM_009540 /// NM_011768 /// NR_0379200.0105229 0.586241 -‐8.94634 5.34648 6.07551 -‐0.729035 -‐1.65753 Less than 01416661_at 13669 Eif3a eukaryotic translation initiation factor 3, subunit ANM_010123 0.0105898 0.586241 -‐6.88536 6.79167 7.89626 -‐1.1046 -‐2.15039 Less than 01427311_at 207165 Bptf bromodomain PHD finger transcription factorNM_001080832 /// NM_1768500.0106859 0.586241 -‐4.77767 4.04456 4.89942 -‐0.854859 -‐1.80858 Less than 01417362_at 56315 Rhcg Rhesus blood group-‐associated C glycoproteinNM_019799 0.0109577 0.586241 -‐7.69601 5.24186 6.16985 -‐0.92799 -‐1.90262 Less than 0

1443069_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0110223 0.586241 -‐5.84749 4.98401 5.58184 -‐0.597827 -‐1.51344 Less than 01449999_a_at 12293 Cacna2d1 calcium channel, voltage-‐dependent, alpha2/delta subunit 1NM_001110843 /// NM_001110844 /// NM_001110845 /// NM_001110846 /// NM_0097840.011024 0.586241 -‐5.43484 8.39264 9.27768 -‐0.885034 -‐1.84681 Less than 01450174_at 19281 Ptprt protein tyrosine phosphatase, receptor type, TNM_021464 0.0111498 0.586241 -‐6.86007 7.14277 7.84864 -‐0.705874 -‐1.63113 Less than 01421978_at 14417 Gad2 glutamic acid decarboxylase 2NM_008078 0.0111714 0.586241 -‐5.06468 10.177 10.8864 -‐0.709408 -‐1.63513 Less than 01420837_at 18212 Ntrk2 neurotrophic tyrosine kinase, receptor, type 2NM_001025074 /// NM_0087450.0113727 0.586241 -‐5.42674 6.73238 8.05069 -‐1.31831 -‐2.49373 Less than 01442939_at 51869 Rif1 Rap1 interacting factor 1 homolog (yeast)NM_175238 0.0114815 0.586241 -‐6.72703 6.53987 7.15721 -‐0.617342 -‐1.53405 Less than 01437554_at 18810 Plec plectin NM_001163540 /// NM_001163542 /// NM_001163549 /// NM_001164203 /// NM_011117 /// NM_200.0116321 0.586241 -‐5.35934 5.27461 6.00325 -‐0.728645 -‐1.65708 Less than 01450906_at 54712 Plxnc1 plexin C1 NM_018797 0.011793 0.586241 -‐7.04445 4.91577 5.92947 -‐1.0137 -‐2.01908 Less than 01422741_a_at 70508 Bbx bobby sox homolog (Drosophila)NM_027444 0.0118507 0.586241 -‐4.40056 4.54502 5.37383 -‐0.828808 -‐1.77622 Less than 01416801_at 58800 Trpm7 transient receptor potential cation channel, subfamily M, member 7NM_001164325 /// NM_0214500.0120784 0.586241 -‐6.19017 4.94116 5.54481 -‐0.603642 -‐1.51955 Less than 01454198_a_at 66793 Efcab1 EF hand calcium binding domain 1NM_025769 0.0121762 0.586241 -‐8.84027 5.9891 6.69529 -‐0.706192 -‐1.63149 Less than 01457409_at 14348 Fut9 fucosyltransferase 9NM_010243 0.0123665 0.59057 -‐5.66063 6.77297 7.44848 -‐0.675512 -‐1.59716 Less than 01421144_at 77945 Rpgrip1 retinitis pigmentosa GTPase regulator interacting protein 1NM_001168515 /// NM_0238790.0127406 0.595375 -‐4.88201 8.26697 8.93949 -‐0.672527 -‐1.59386 Less than 01429665_at 230376 Haus6 HAUS augmin-‐like complex, subunit 6NM_173400 0.0129826 0.59644 -‐8.64276 3.4841 4.10664 -‐0.622539 -‐1.53958 Less than 01440227_at 53881 Slc5a3 solute carrier family 5 (inositol transporters), member 3NM_017391 0.0133887 0.599436 -‐4.7066 7.73885 8.33472 -‐0.595868 -‐1.51138 Less than 01440161_at 17389 Mmp16 matrix metallopeptidase 16NM_019724 0.0139277 0.600797 -‐4.23021 5.9193 6.71622 -‐0.796915 -‐1.73738 Less than 01442725_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0141133 0.600797 -‐7.33525 3.49357 4.87854 -‐1.38497 -‐2.61166 Less than 01455586_at 70238 Rnf168 ring finger protein 168NM_027355 0.0141614 0.600797 -‐5.81486 5.77825 6.48115 -‐0.702894 -‐1.62777 Less than 01440142_s_at 14580 Gfap glial fibrillary acidic proteinNM_001131020 /// NM_0102770.0143587 0.601418 -‐6.62807 7.74167 9.10189 -‐1.36022 -‐2.56724 Less than 01421508_at 23963 Odz1 odd Oz/ten-‐m homolog 1 (Drosophila)NM_011855 0.0145405 0.601418 -‐6.31165 7.21185 7.89699 -‐0.685137 -‐1.60786 Less than 01459310_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0147737 0.604636 -‐4.55227 5.27517 6.43559 -‐1.16042 -‐2.23522 Less than 01456495_s_at 99031 Osbpl6 oxysterol binding protein-‐like 6NM_145525 0.0151501 0.612811 -‐7.31572 6.0241 6.61814 -‐0.594038 -‐1.50947 Less than 01417043_at 16816 Lcat lecithin cholesterol acyltransferaseNM_008490 0.0153651 0.615243 -‐4.20226 7.2916 7.91594 -‐0.624341 -‐1.54151 Less than 01434374_at 319604 Fam168a family with sequence similarity 168, member ANM_178764 0.0154694 0.615243 -‐5.41012 8.61935 9.237 -‐0.617648 -‐1.53437 Less than 01450996_at 14308 Fshb follicle stimulating hormone betaNM_008045 0.0157356 0.617978 -‐4.2743 3.02166 3.61035 -‐0.588689 -‐1.50388 Less than 01449311_at 12013 Bach1 BTB and CNC homology 1NM_007520 0.0157387 0.617978 -‐5.07024 5.91085 6.62396 -‐0.713104 -‐1.63933 Less than 01444075_at 70598 Filip1 filamin A interacting protein 1NM_0010812430.0160878 0.618833 -‐5.17225 4.34135 4.9865 -‐0.645153 -‐1.5639 Less than 01439434_x_at 230863 Sh2d5 SH2 domain containing 5NM_0010996310.0162394 0.618833 -‐6.74252 5.09326 5.72788 -‐0.634628 -‐1.55254 Less than 01448885_at 74012 Rap2b RAP2B, member of RAS oncogene familyNM_028712 0.0164158 0.618833 -‐5.77843 7.34396 7.93919 -‐0.59523 -‐1.51071 Less than 01457603_at 243725 Ppp1r9a protein phosphatase 1, regulatory (inhibitor) subunit 9ANM_181595 0.0165274 0.618833 -‐5.19585 6.03874 6.77735 -‐0.738615 -‐1.66857 Less than 01456791_at 627049 Zfp800 zinc finger protein 800NM_0010816780.0165338 0.618833 -‐7.30734 6.46716 7.23342 -‐0.766264 -‐1.70086 Less than 01421955_a_at 17999 Nedd4 neural precursor cell expressed, developmentally down-‐regulated 4NM_010890 0.0165676 0.618833 -‐6.25216 8.49727 10.1927 -‐1.69544 -‐3.23875 Less than 01438271_at 210126 Lpp LIM domain containing preferred translocation partner in lipomaNM_001145952 /// NM_001145954 /// NM_1786650.0166886 0.618833 -‐5.18268 4.41781 5.30194 -‐0.884124 -‐1.84564 Less than 01417561_at 11812 Apoc1 apolipoprotein C-‐INM_001110009 /// NM_0074690.0168647 0.618833 -‐5.18061 8.74078 9.39048 -‐0.649703 -‐1.56885 Less than 0

1456807_at 105651 Ppp1r3e protein phosphatase 1, regulatory (inhibitor) subunit 3ENM_0011679080.0175712 0.622453 -‐5.30695 5.20124 5.95676 -‐0.755512 -‐1.68823 Less than 01436238_at 213469 Lgi3 leucine-‐rich repeat LGI family, member 3NM_145219 0.0176585 0.622453 -‐4.13521 6.67184 7.28915 -‐0.617306 -‐1.53401 Less than 01424077_at 66569 Gdpd1 glycerophosphodiester phosphodiesterase domain containing 1NM_025638 0.0178287 0.622453 -‐4.69849 7.2175 8.39461 -‐1.17711 -‐2.26124 Less than 01458050_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0182373 0.627674 -‐3.89998 5.25558 5.92255 -‐0.666964 -‐1.58773 Less than 01454655_at 227333 Dgkd diacylglycerol kinase, deltaNM_177646 0.0184824 0.628461 -‐3.85815 5.49193 6.1005 -‐0.608562 -‐1.52474 Less than 01459377_at 242481 Palm2 paralemmin 2NM_172868 0.0185842 0.628951 -‐4.02842 5.65128 6.25143 -‐0.600152 -‐1.51588 Less than 01454950_at 319604 Fam168a family with sequence similarity 168, member ANM_178764 0.018981 0.631049 -‐5.11581 6.15867 6.80283 -‐0.644169 -‐1.56284 Less than 01439976_at 207958 Alg11 asparagine-‐linked glycosylation 11 (alpha-‐1,2-‐mannosyltransferase)NM_001243161 /// NM_183142 /// NR_0406500.0198891 0.632006 -‐4.61679 5.82114 6.41077 -‐0.589636 -‐1.50487 Less than 01441667_s_at 12180 Smyd1 SET and MYND domain containing 1NM_001160127 /// NM_0097620.0199174 0.632006 -‐3.84799 3.3141 3.90397 -‐0.589875 -‐1.50512 Less than 01442024_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.020264 0.634286 -‐6.05598 7.14787 7.83125 -‐0.683373 -‐1.60589 Less than 01419616_at 12168 Bmpr2 bone morphogenic protein receptor, type II (serine/threonine kinase)NM_007561 0.0206284 0.634682 -‐5.71297 5.84677 6.66288 -‐0.816112 -‐1.76065 Less than 01453391_at 74062 /// 75858Speer7-‐ps1 /// Speer8-‐ps1spermatogenesis associated glutamate (E)-‐rich protein 7, pseudogene 1 /// spermatogenesNR_001584 /// NR_0015850.0209128 0.634794 -‐3.72075 5.60597 6.50659 -‐0.900622 -‐1.86687 Less than 01429517_at 78287 Zfyve20 zinc finger, FYVE domain containing 20NM_030081 0.0214375 0.63903 -‐5.70172 4.95755 5.82043 -‐0.86288 -‐1.81867 Less than 01460476_s_at 71721 Fam13c family with sequence similarity 13, member CNM_001143776 /// NM_001143777 /// NM_0242440.0216633 0.642055 -‐3.68641 5.88521 6.75823 -‐0.873028 -‐1.8315 Less than 01423325_at 18949 Pnn pinin NM_008891 0.0218283 0.644006 -‐5.65834 5.68755 6.57665 -‐0.889108 -‐1.85203 Less than 01442038_at 74213 Rbm26 RNA binding motif protein 26NM_134077 0.0220137 0.644813 -‐5.69447 7.94227 8.56153 -‐0.619265 -‐1.53609 Less than 01447231_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0224728 0.648045 -‐4.19665 5.35242 6.25644 -‐0.904019 -‐1.87127 Less than 01437018_at 239157 Pnma2 paraneoplastic antigen MA2NM_175498 0.023543 0.648545 -‐4.55529 6.48872 7.65519 -‐1.16647 -‐2.24461 Less than 01456157_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0237301 0.648545 -‐3.56409 3.8187 4.50561 -‐0.686912 -‐1.60983 Less than 01422048_at 22067 Trpc5 transient receptor potential cation channel, subfamily C, member 5NM_009428 0.0241909 0.650232 -‐5.71955 6.41887 7.09509 -‐0.676222 -‐1.59795 Less than 01436023_at 72567 Bclaf1 BCL2-‐associated transcription factor 1NM_001025392 /// NM_001025393 /// NM_001025394 /// NM_1537870.024259 0.650232 -‐5.95873 7.64728 8.72593 -‐1.07866 -‐2.11207 Less than 01450068_at 22385 Baz1b bromodomain adjacent to zinc finger domain, 1BNM_011714 0.0265258 0.658415 -‐5.36667 5.23986 6.33293 -‐1.09306 -‐2.13327 Less than 01456255_at 230249 AI314180 expressed sequence AI314180NM_172381 0.0268833 0.661661 -‐4.14122 3.77836 4.63477 -‐0.856414 -‐1.81053 Less than 01429993_s_at100039045 /// 545739 /// 664804 /// 73526Gm10471 /// Gm5862 /// Gm7347 /// Speer4bpredicted gene 10471 /// predicted gene 5862 /// predicted gene 7347 /// spermatogenesiNM_001177579 /// NM_028561 /// XM_620170 /// XM_9730240.0269137 0.661661 -‐5.02839 5.15509 5.82136 -‐0.666271 -‐1.58697 Less than 01437640_at 230235 6430704M03RikRIKEN cDNA 6430704M03 geneNM_0011429650.0286052 0.670146 -‐3.99982 9.35075 9.9613 -‐0.610556 -‐1.52685 Less than 01428045_a_at 69257 Elf2 E74-‐like factor 2NM_023502 0.0302199 0.677988 -‐5.33881 5.36074 6.07398 -‐0.713235 -‐1.63948 Less than 01438732_at 100042480 Nhsl2 NHS-‐like 2 NM_0011636100.0303407 0.678713 -‐5.32603 4.84084 6.32883 -‐1.48799 -‐2.80499 Less than 01434585_at 68842 Tulp4 tubby like protein 4NM_001033529 /// NM_001103181 /// NM_0540400.0307171 0.680144 -‐5.25922 6.44599 7.09093 -‐0.644934 -‐1.56367 Less than 01428414_at 67974 Ccny cyclin Y NM_026484 0.031426 0.680144 -‐3.86866 4.36941 5.03044 -‐0.66103 -‐1.58121 Less than 01441797_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0327735 0.685265 -‐3.92113 6.09683 6.69234 -‐0.595513 -‐1.51101 Less than 01445673_at 73040 2900052N01RikRIKEN cDNA 2900052N01 geneNR_015605 0.0334452 0.688192 -‐3.39454 6.74268 7.76178 -‐1.0191 -‐2.02665 Less than 01436231_at 73040 2900052N01RikRIKEN cDNA 2900052N01 geneNR_015605 0.0348509 0.69246 -‐3.25794 9.83333 10.6341 -‐0.800767 -‐1.74203 Less than 01459398_at 67245 Peli1 Pellino 1 NM_023324 0.0350544 0.693112 -‐4.99546 3.53945 4.19991 -‐0.660455 -‐1.58058 Less than 01418188_a_at 72289 Malat1 metastasis associated lung adenocarcinoma transcript 1 (non-‐coding RNA)NR_002847 0.0352222 0.693996 -‐3.91717 10.6148 11.559 -‐0.944205 -‐1.92413 Less than 0

1440146_at 271564 Vps13a vacuolar protein sorting 13A (yeast)NM_173028 0.0370798 0.705801 -‐4.5526 4.76762 5.5957 -‐0.828077 -‐1.77532 Less than 01431047_at 71389 Chd6 chromodomain helicase DNA binding protein 6NM_173368 0.0378303 0.709764 -‐4.86198 4.18915 5.19711 -‐1.00796 -‐2.01107 Less than 01444444_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0404362 0.718206 -‐3.96398 7.42666 8.1242 -‐0.697541 -‐1.62174 Less than 01446540_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0410373 0.71846 -‐2.9809 4.6769 5.33045 -‐0.653547 -‐1.57303 Less than 01437118_at 252870 Usp7 ubiquitin specific peptidase 7NM_0010039180.0412774 0.71846 -‐4.68891 5.34181 6.10192 -‐0.760106 -‐1.69361 Less than 01419277_at 170707 Usp48 ubiquitin specific peptidase 48NM_130879 0.0413933 0.71846 -‐3.41877 6.24071 6.92341 -‐0.682699 -‐1.60514 Less than 01441847_at -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ -‐-‐-‐ 0.0416694 0.71846 -‐3.24307 4.07565 4.86386 -‐0.788207 -‐1.72693 Less than 01444492_at 19266 Ptprd protein tyrosine phosphatase, receptor type, DNM_001014288 /// NM_0112110.0418619 0.718655 -‐3.21276 5.39311 6.68672 -‐1.29361 -‐2.45141 Less than 01460159_at 320713 Mysm1 myb-‐like, SWIRM and MPN domains 1NM_177239 0.0426826 0.718655 -‐4.63069 4.66912 5.94816 -‐1.27904 -‐2.42678 Less than 01460258_at 16840 Lect1 leukocyte cell derived chemotaxin 1NM_010701 0.0428631 0.719062 -‐3.68551 5.43432 6.0364 -‐0.602084 -‐1.51791 Less than 01423546_at 22680 Zfp207 zinc finger protein 207NM_001130169 /// NM_001130170 /// NM_001130171 /// NM_011751 /// NR_0450380.0431106 0.719272 -‐2.92395 4.15188 4.8009 -‐0.649017 -‐1.5681 Less than 01441728_at 20265 Scn1a sodium channel, voltage-‐gated, type I, alphaNM_018733 0.0431876 0.719272 -‐2.9832 5.74624 6.47315 -‐0.726911 -‐1.65509 Less than 01418189_s_at 72289 Malat1 metastasis associated lung adenocarcinoma transcript 1 (non-‐coding RNA)NR_002847 0.0454208 0.721903 -‐3.83823 11.6871 12.4456 -‐0.758491 -‐1.69172 Less than 01417645_at 16651 Sspn sarcospan NM_010656 0.048296 0.727824 -‐2.90345 4.52078 5.2152 -‐0.694416 -‐1.61823 Less than 0

Table S3, List of oligonucleotides

Primer for ChIP Sequence ChIP mFabp7_FW Real-time PCR ChIP on 5’-GGGGATCAGGATTGTGATGT-3’ ChIP mFabp7_RV 5’-AGATGGCTCCAATCCTCCTT-3’ ChIP mFabp7_TM 5’-FAM-TCCGCTAACCCAGTGGCCTGA-BHQ1-3’ ChIP mFgf21_FW Real-time PCR ChIP on 5’-CCATTGCATCATCCGTCCAGGC-3’ ChIP mFgf21_RV 5’-GTGCCCTCCCCACTCCTGAC-3’ ChIP mFgf21_TM 5’-FAM-CGCCCTGGCCACGGTGGAATTCAGG-BHQ1-3’

Primer for real-time PCR Sequence Fabp7 ms_FW 5’-AGCTGGGAGAAGAGTTTGAA-3’ Fabp7 ms_RV 5’-GAGCTTGTCTCCATCCAACC-3’ Fabp7 hs_FW 5’-AAGTCTGTTGTTAGCCTGGA-3’ Fabp7 hs_RV 5’-AGGGTCATAACCATTTTGC-3’ Gapdh_FW Real-time PCR 5’-CATGGCCTTCCGTGTTCCTA-3’ Gapdh_RV 5’-CCTGCTTCACCACCTTCTTGA-3’ Rev-erbα_FW 5’-GGGCACAAGCAACATTACCA-3’ Rev-erbα_RV 5’-CACGTCCCCACACACCTTAC-3’

Primer for cloning of ISH probe

Sequence

Fabp7 ISH_FW 5’-AGACCCGAGTTCCTCCAGTTC-3’ Fabp7 ISH_RV 5’-CCTCCACACCGAAGACAAAC-3’

Primer for cloning/ mutation of promoter

Sequence

Fabp7 prom_FW 5’-CTGCCTATTTTCAGCTGACTAGGCGGTTAAG-3’ Fabp7 prom_RV 5’-CCATACGTGTGTGCCTTCAAGTCTGAACTAC -3’ Fabp7 promΔRORE_FW 5’-GTGTGAACTGGGAGGATCTGATATCACTCCGCTAACCCAGTGGC-3’ Fabp7 promΔRORE _RV 5’-GCCACTGGGTTAGCGGAGTGATATCAGATCCTCCCAGTTCACAC-3’

FAM: 6-fluorescine; BHQ1: black hole quencher 1

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