Current Biology 20, 2199–2206, December 21, 2010 ª2010 Elsevier Ltd All rights reserved

DOI 10.1016/j.cub.2010.11.038

Acute Induction of Eya3 by Late-Night
Light Stimulation Triggers
TSHb Expression in Photoperiodism
Koh-hei Masumoto,1,3 Maki Ukai-Tadenuma,1Takeya Kasukawa,2 Mamoru Nagano,3 Kenichiro D. Uno,2Kaori Tsujino,1,4 Kazumasa Horikawa,3Yasufumi Shigeyoshi,3,* and Hiroki R. Ueda1,2,4,5,*
1Laboratory for Systems Biology
2Functional Genomics Unit
RIKEN Center for Developmental Biology, 2-2-3
Minatojima-minamimachi, Chuo-ku, Kobe,
Hyogo 650-0047, Japan
3Department of Anatomy and Neurobiology, Kinki University
School of Medicine, 377-2 Ohno-Higashi, Osakasayama City,
Osaka 589-8511, Japan
4Graduate School of Science, Osaka University,
1-1 Machikaneyama, Toyonaka, Osaka 560-0043, Japan
5Department of Mathematics, Graduate School of Science,
Kitashirakawa Oiwake-cho, Sakyo-ku, Kyoto,
Kyoto University, Kyoto 606-8502, Japan

Summary
Living organisms detect seasonal changes in day length(photoperiod) [1–3] and alter their physiological functions
accordingly to fit seasonal environmental changes. TSHb,
induced in the pars tuberalis (PT), plays a key role in thepathway that regulates vertebrate photoperiodism [4, 5].
However, the upstream inducers of TSHb expression remain
unknown. Here we performed genome-wide expression
analysis of the PT under chronic short-day and long-day
conditions in melatonin-proficient CBA/N mice, in which
the photoperiodic TSHb expression response is preserved[6]. This analysis identified ‘‘short-day’’ and ‘‘long-day’’genes, including TSHb, and further predicted the acuteinduction of long-day genes by late-night light stimulation.
We verified this by advancing and extending the light periodby 8 hr, which induced TSHb expression within one day. In
the following genome-wide expression analysis under thisacute long-day condition, we searched for candidate
upstream genes by looking for expression that preceded
TSHb’s, and we identified the Eya3 gene. We demonstrated
that Eya3 and its partner Six1 synergistically activate TSHb
expression and that this activation is further enhanced by
Tef and Hlf. These results elucidate the comprehensive
transcriptional photoperiodic response in the PT, revealing
the complex regulation of TSHb expression and unexpectedly rapid response to light changes in the mammalianphotoperiodic system.
Results and Discussion
Genome-wide Expression Analysis of the Mouse Pars
Tuberalis under Chronic Conditions
The pars tuberalis (PT) is thought to be responsible for detecting photoperiod, by integrating circadian time and environmental light/dark information [7–9]. Recently, a genome-wide*Correspondence: shigey@med.kindai.ac.jp (Y.S.), uedah-tky@umin.ac.jp(H.R.U.)

Reportexpression analysis revealed that the thyroid-stimulating
hormone (TSH) pathway triggers photoperiodic responses in
the Japanese quail [4, 5]. In mammals, nocturnal melatoninsecretion is thought to carry environmental light/dark information to the PT [10–14], where the melatonin receptor is highly
expressed [15]. However, the detailed molecular mechanismthat links melatonin signals with TSHb expression in the PT
remains unclear.
To identify the upstream inductive mechanism of TSHb
expression, we performed genome-wide expression analysesof the PT under chronic short-day and long-day conditions inmelatonin-proficient CBA/N mice, in which the photoperiodic
TSHb expression response is preserved [6] (Experimental
Procedures). The data obtained were first analyzed forcircadian gene expression (see Supplemental Experimental
Procedures available online) because PT contains circadianoscillators [16] (Figure S1A; Supplemental Results and Discussion). We identified 1000 significant 24 hr rhythmic genes in the
PT (Figure 1A; Table S1). The identified genes included several
clock and clock-controlled genes (Figure 1B; Table S2;
Supplemental Results and Discussion). Their average peaktime in the long-day condition was 7.71 hr later than in the
short-day condition (Figure 1B), suggesting that circadianclocks in the PT are entrained to the end of a light period.
The obtained data were next analyzed to identify ‘‘photoperiodic’’ genes in the PT (Supplemental Experimental Procedures). This photoperiodic expression analysis significantly
identified 246 ‘‘long-day’’ genes and 57 ‘‘short-day’’ genes in
the PT (Figure 1C; Table S3). The identified genes included
TSHb, which was further confirmed by quantitative PCR(qPCR) and radioisotope (RI) in situ hybridization (Figure 1D).
In contrast, TSH a subunit (Cga) and Tac1 [17] did not respondto the photoperiod in the mouse PT (Figures S1B and S1C; seedetails in Supplemental Results and Discussion).
Late-Night Light Stimulation Immediately Induces TSHb
Expression in the Mouse PT
We next examined the timescale of the TSHb induction after
the transition from the short-day to the long-day condition.
We transferred mice from the short-day to the long-day
condition by delaying lights-off for 8 hr (hereafter, the ‘‘delay’’condition) and sampled the PTs at zeitgeber time 16 (ZT16;
ZT0 was defined as the time of lights-on) because TSHb israpidly induced at around ZT16 in the PT of the Japanese quail[4]. However, in contrast to the previously reported immediateinduction of TSHb in the quail, TSHb expression in the mouse
PT increased gradually over the 5 days following the transition
from the short- to the long-day condition (Figure 2A). Because
the PT circadian clock was entrained to the lights-off timing(Figure 1B), we speculated that the observed slow dynamics
of TSHb induction in the mouse PT were due to the gradualentrainment of the PT circadian clock. We also hypothesizedthat the ‘‘photoinducible’’ phase (the circadian time when lightstimulation can induce TSHb expression) is in the subjective(circadian) late night (as defined in the short-day condition),and therefore entrainment over 5 days might be required forfull transition of the photoinducible phase to the photoperiod
under the long day.

Current Biology Vol 20 No 24
2200

A

B

C

D

Figure 1. Genome-wide Expression Analysis of Circadian and Photoperiodic Genes in the Mouse Pars Tuberalis under Chronic Short-Day and Long-Day
Conditions(A) Heat map of 24 hr rhythmic genes in the mouse pars tuberalis (PT) under short-day (left two panels) and long-day (right two panels) conditions. In bothconditions, time-series data of the first and second experimental replicates are plotted. In the heat maps in (A)–(C), magenta tiles indicate higher gene
expression; green tiles indicate lower expression.
(B) Peak-time difference in the circadian expression of clock and clock-controlled genes between short-day and long-day conditions. The upper panel
shows a heat map of the clock and clock-controlled genes. The middle panel shows their peak times. The lower panel indicates the difference in peaktime between the short-day and long-day conditions. The peak time in the short-day condition for each gene was set to 0. The average difference inpeak time was 7.71 hr (dashed red line).
(C) Heat map of photoperiodic genes for which the expression level changed between the short-day and long-day conditions. The location of TSHb isindicated.
(D) Confirmation of the GeneChip data for TSHb expression. TSHb expression under short-day and long-day conditions was measured by qPCR (n = 2,
top panels; TSHb expression relative to Tbp expression is plotted) and radioisotope (RI) in situ hybridization (n = 3, middle and bottom panels;
scale bars represent 300 mm). Error bars represent 6 standard error of the mean (SEM).

To confirm that the PT circadian clock was gradually shiftedin this condition, we used a molecular timetable method[18, 19], which can measure circadian phase from theexpression pattern of clock and clock-controlled genes witha single-time-point sample (Supplemental Experimental
Procedures). We found that the PT circadian time wasEya3 Triggers Photoperiodic Expression of TSHb
2201

A
Relative expression

TSHβ mRNA (qPCR)
30
25
20
15
10
5
0
Day0
Short-day

Day1

Day3
Long-day

Day5

Normalized expression

B

Relative expression

C

Day 1

Day 0
2
1
0
1
2
0

2
1
0
1
2

cor.=0.531, CT=15.7
8 12 16 20

4

0

Day 5

Day 3
2
1
0
1
2

cor.=0.370, CT=13.9
cor.=0.641, CT=11.9
8 12 16 20
40
4
8 12 16 20
Molecular peak time (hr)

2
1
0
1
2

cor.=0.831, CT=10.4
8 12 16 20
0
4

D

30
Day5

25

Day0

20

Day1

r² = 0.7569

15

Day3

10

Day3
Day1

5
0
10

11

12

Day5

Day0

13
14
Time (CT)

15

0

4

8

12
Time (ZT)

16

20

24

0

4

8

12
Time (CT)

16

20

24

16

E
TSHβ mRNA (in situ)

TSHβ mRNA (qPCR)
Advance
Relative expression

20

ZT16

ZT0

ZT4

ZT8

ZT12

Short-day

12
8

First long-day(Advance)

4
0

16

20

0
4
Time (ZT)812Advance
Relative mRNA abundance

Delay
20
Relative expression

ZT20

16

16
12
8
4
0

0

4

8
12
Time (ZT)

16

20

250

TSHβ mRNA

200
150
100
50
0
16

20

0

4

8

12 16 20
Time (ZT)

0

4

8

12

Figure 2. Late-Night Light Stimulation Immediately Induces TSHb Expression in the Mouse PT(A) TSHb expression at ZT16 on days 0, 1, 3, and 5 after the transition of light condition, in which lights-off timing was delayed by 8 hr. TSHb expression wasmeasured by qPCR (n = 2). TSHb expression relative to Tbp expression is plotted. Error bars represent 6 SEM.
(B) Circadian time measurement from the mouse PT. Colors and x values of the dots indicate the molecular peak time of individual clock and clock-controlledgenes. The peak time of the red cosine curve indicates the estimated circadian time (CT, dashed vertical line). The correlation coefficient (cor.) between the
red cosine curve and normalized expression data is also indicated in the panel.
(C) Linear regression analysis between the estimated circadian time (CT, x axis) of the PT and quantity of TSHb expression (y axis). Red dots indicate data
points. Green line indicates regression line. r2 = 0.7569.
(D) Gradual change in the estimated circadian time of the PT from day 0 to day 5. Color bars indicate the estimated circadian time (CT) of the PT on each day.
The x axis indicates the environmental zeitgeber time (ZT). The circadian time at ZT16 is indicated by a colored circle. The orange-outlined box indicates theputative photoinducible phase. The background indicates the light conditions (white, light phase; gray, dark phase).
(E) CBA/N mice kept in the short-day condition (light:dark = 8:16 hr) for 3 weeks were then transferred to a long-day condition, in which the dark period
was advanced (advance condition) or delayed (delay condition) by 8 hr. Left panels: TSHb expression on the first long day (advance and delay conditions)was measured by qPCR (n = 2). TSHb expression relative to Tbp expression is plotted. Right panels: TSHb expression in the short-day condition and on thefirst long day (advance condition) was measured by RI in situ hybridization (n = 3). Scale bars represent 300 mm. Error bars represent 6 SEM.

Current Biology Vol 20 No 24
2202

gradually shifted over 5 days (Figure 2B). We also noted thatthe circadian time in the PT correlated well with the inductionof TSHb expression (Figure 2C; r2 = 0.7569), consistent withthe hypothesis. Based on these findings, we plotted themeasured circadian time and the hypothesized photoinducible
phase (i.e., the circadian late-night period) over the 5 days after
the shift and superimposed it on the photoperiod (Figure 2D).
This plot showed that TSHb expression was not induced on
the first day as a result of the mismatch between the hypothetical photoinducible phase of the PT (Figure 2D, orange-outlined box) and the photoperiod (Figure 2D, day 1), whereas
TSHb expression was strongly induced on the fifth day
because of the match between the hypothetical photoinducible phase and the photoperiod, after gradual entrainment of
the PT over 5 days (Figure 2D, day 5). This result supports
our hypothesis that the photoinducible phase is in the circadian late-night period.
Furthermore, this hypothesis also predicted that TSHb
expression would be strongly induced on the first day in analternative long-day condition in which the lights-on timing
was advanced by 8 hr (hereafter, the ‘‘advance’’ condition).
To verify this prediction, we examined TSHb expression in
the PT under the advance condition, and we found that it
increased immediately (Figure 2E; Figure S1D). RI in situ
hybridization also confirmed this immediate TSHb expression(Figure 2E, right panels). On the other hand, TSHb expression
was not induced in the delay condition (Figure 2E, left-bottom
panel; Figure S1D). These findings suggest that the mouse PT
has a photoinducible phase during subjective late night andthat light stimulation occurring in the late night can induce
TSHb expression immediately, i.e., within one day.
Genome-wide Expression Analysis of Acute Long-Day
Genes in the Mouse PT
Given the rapid induction of TSHb, we reasoned that agenome-wide expression analysis in the advance and delay
conditions might allow us to identify the the upstream inductive mechanism of the TSHb pathway. Therefore, we performed a second set of genome-wide expression analyses
under these acute long-day conditions (Experimental Procedures). The data obtained were then analyzed to extract ‘‘acutelong-day’’ genes expressed in the PT (Supplemental Experimental Procedures). This expression analysis identified 34
acute long-day genes in the PT (Figure 3A; Table S4), which
included several transcription factors; Eya3, Rorb, Maff,
Crem, and Hdac4 (Figure S1D). We focused on Eya3 as a putative upstream activator of TSHb expression because Crem andHdac4 encode transcriptional repressors [20, 21] and because
Rorb and Maff could not activate the 7.7 kbp promoter of TSHb
(Figures S2A and S2B). We first confirmed the acute inductionof Eya3 expression in the PT under the advance condition via
qPCR and RI in situ hybridization (Figure 3B; Supplemental
Results and Discussion).
Eya3 and Six1 Synergistically Induce TSHb Expression
Eya3 is one of four mammalian homologs (Eya1–4) of eya[22, 23], a transcriptional coactivator involved in fly eye development [24, 25]. Eya family members form a complex witha DNA-binding factor of the Six family and a corepressor of
the Dach family. Six-Eya-Dach genetic interactions are reported to regulate the transcriptional activation and repressionof target genes. Of the Eya, Six, and Dach families, we foundthat the Eya3 and Six1 mRNAs were highly expressed in the
PT under the long-day condition whereas the others wereweakly or barely expressed (Figure 4A). We therefore examined whether Eya3 and Six1 activate the TSHb promoter. The
transient transfection of Eya3 or Six1 increased the TSHb
promoter activity only slightly, whereas their cotransfectionsynergistically increased its activation (Figure 4B). In contrast,
Eya3 and Six1 did not activate the SV40 promoter. We also
found that shorter versions of the TSHb promoter (Figure 4C)
were also synergistically activated by Eya3 and Six1 (Figure 4B). We thus used the shortest version of the TSHb promoter (0.1 kbp) in the following experiments unless otherwise
indicated. We also confirmed that Eya3 increased TSHb
promoter activity in a dose-dependent manner when it was
expressed alone or with Six1 (Figure 4D; Figure S2C; Supplemental Results and Discussion).
An So Site Is Important for Eya3-Six1-Dependent
Activation of the TSHb Promoter
It has been reported that Six and Eya can activate their target
genes through different consensus sequences for Six binding(MEF3 site, see [26, 27]; So site, see [27–29]). Therefore, we
searched for Six consensus sequences in the 0.1 kbp TSHb
promoter and found one MEF3 site (+1) and two So sites(245 and 252) upstream of the transcription start site (TSS).
These MEF3 and So sites in the TSHb promoter are highly
conserved among vertebrates (Figure 4C). We first deleted
and mutated the one MEF3 site in the TSHb promoter andfound that it was dispensable for the Eya3-Six-dependent
activation of the TSHb promoter (Figure 4E). We then sequentially deleted the two So sites (Figure 4C, So1 and So2).
Although deletion of the So2 site did not affect the Eya3-Sixdependent activation of the TSHb promoter, deletion of the
So1 site significantly decreased the change elicited by theEya3-Six-dependent activation (Figure 4F). These results indicate that the So1 site is essential for the full activation of the
TSHb promoter by the Eya3-Six1 complex.
Because Tef can increase TSHb promoter activity [30], we
also examined the contribution of Tef and its family member
Hlf to the 0.1 kbp TSHb promoter. We found that Tef or Hlf
synergistically increased the luciferase activity of the TSHb
promoter when cotransfected with Eya3 and Six1 (Figures
S2D–S2J; see details in Supplemental Results and Discussion).
Photoinducible Phase at Subjective Late NightIn this study, genome-wide expression analyses of the mouse
PT revealed that TSHb and Eya3 expression are induced bylate-night light stimulation. Because these expression data
might include potentially important factors besides Eya3
and TSHb, we have made them publicly available (http://
photoperiodism.brainstars.org/). We further demonstrated
that Eya3 and its partner Six1 are expressed in the mouse PT
and synergistically activate TSHb expression through an Sosite in the TSHb promoter. This activation is further enhanced
by Tef and Hlf through a D box close to the So site. Because
previous reports described Eya3 induction in the PT underlong-day conditions in birds [4, 5] and sheep [17], its induction
under long-day conditions appears to be an evolutionarily
conserved molecular mechanism in the photoperiodism ofvertebrates. Among the remaining challenges is the in vivo
functional analysis of Eya3-dependent induction of TSHb
expression.
Based on these and previous findings, we propose thefollowing hypothetical model for a gradual transition overmonths from short-day to long-day conditions in the naturalenvironment. As the photoperiod is gradually extended toEya3 Triggers Photoperiodic Expression of TSHb
2203

A

Short-day
0

4

First long-day
Delay
Advance8 12 16 20 16 20 0

-2SD

4

8 12 04

8 12 16 20

0

TSHβ
Figf
Kpna7
Rtn4rl1
Plch2
Srxn1
1456778_at
Nrp2
Slc6a4
Fam129a1436092_at
Tnfrsf12a
Hdac4
Pdzrn3
Myo1e
Tgfb2
Trhr
Ankrd44
Ppp1r3c1439944_at
Slco1c1
Lrat
Dio2
Crem
1443047_at
Eya3
Dhrs3
Man2a1
Maff
1449625_at
Rorβ
Pde10a6230424C14Rik
Dram1
Hhip

+2SD

B
Eya3 mRNA (qPCR)

Eya3 mRNA (in situ)

Advance
ZT16

Relative expression

6

ZT20

ZT0

ZT4

ZT8

ZT12

5
Short-day

4
3

First long-day(Advance)

2
1
0
16

20

0
4
Time (ZT)812Advance
Relative mRNA abundance

Delay
Relative expression

6
5
4
3
2
1
0
0

4

8
12
Time (ZT)

16

1200

Eya3 mRNA

1000
800
600
400
200

20

0
16

20

0

4

8

12 16 20
Time (ZT)

0

4

8

12

Figure 3. Genome-wide Expression Analysis of Acute Long-Day Genes in the Mouse PT(A) Heat map of photoperiodic genes whose expression was altered by the photoperiod change only in the advance condition. Magenta tiles indicate higher
gene expression in the PT; green tiles indicate lower expression. GeneChip data for TSHb expression are displayed for reference.
(B) Confirmation of the GeneChip data for Eya3 expression. Left panels: Eya3 expression on the first long day (advance and delay conditions) was measured
by qPCR (n = 2). Eya3 expression relative to Tbp expression is plotted. Right panels: Eya3 expression under the short-day condition and on the first long day(advance condition) was measured by RI in situ hybridization (n = 3). Scale bar represents 300 mm. Error bars represent 6 SEM.

completely cover the photoinducible phase (the subjective latenight, determined in the short-day condition), Eya3 is graduallyinduced, which triggers TSHb expression in the PT undernatural conditions. These natural and relatively slow expression dynamics can be speeded up by artificial light stimulation
at subjective late night, which acutely induces Eya3 expression. This artificial situation reveals that the mammalianphotoperiodic system has unexpectedly rapid dynamics andindicates that the PT of CBA/N mice is an ideal model systemfor elucidating the remaining molecular mechanisms of photoperiodism (Supplemental Results and Discussion). Identifying
the upstream inducer of the acute Eya3 elevation as well as

elucidating the signal transduction cascade from the melatonin receptor to Eya3 expression will provide further insights
into photoperiodism.
Experimental Procedures
Animals and Housing
Male CBA/N mice (Japan SLC, Shizuoka, Japan), which have normal retinas(Supplemental Results and Discussion), were purchased 3 weeks after birth.
For chronic long-day and short-day experiments, mice were first housed
under short-day conditions (light:dark = 8:16 hr, ZT0 = lights-on, ZT8 =lights-off, 400 lux), given food and water ad libitum, and maintained underthese short-day conditions for 3 weeks. The mice were then separated

Current Biology Vol 20 No 24
2204

A

Eya1

Eya2

Eya3

Eya4

Dach1

Dach2

Ski

Skil

Six1

Six2

Six3

Six4

Six5

Six6

Relative bioluminescence

B
10
8
6
4
2

0
EYA3
SIX1

- + -+- - ++- + -+- - ++- + -+- - ++- + -+- - ++- + -+- - ++

P(TSHβ-7.7kbp)

P(TSHβ-0.6kbp)

P(TSHβ-0.2kbp)

P(TSHβ-0.1kbp)

P(SV40)chr3

C

TSHβ exon1 exon2-1(TSS)-59

Vertebrates conservation

TSHβ promoter -569
0.6kbp

So1

D box

MEF3

+16

+30
-193 +30

0.2kbp-69 +30

0.1kbp

D

E
20

Relative bioluminescence

Relative bioluminescence

So2

mouse : CAGATAAGAAAGCAGCAATTCGAATGCAATTATATAAACAAGAAGATCAGAGGGGAATTATCCTGAAGGGTATAA*************** ** ** ********* ********************** *******************
human : CAGATAAGAAAGCAGTAAATCAAATGCAATTGTATAAACAAGAAGATCAGAGGG--ATTATCCTGAAGGGTATAAAGWAANYNGANA
MEF3 site consensus: ATCCTGA
So binding site
TTACATAA :D box consensus
consensus: GWAANYNGANA

15
10
5
0

EYA3 SIX1 - - - -++++ - - - - ++++

P(TSHβ-0.1kbp)

12
10
8
6
4
2
0

EYA3
SIX1

P(SV40)-+-+- - ++-+-+- - ++-+-+- - ++-+-+- - ++-+-+- - ++

P(TSHβ-0.1kbp)

P(TSHβ-ΔMEF3)

P(TSHβ-mutMEF3)

P(TSHβ-mutD)

P(SV40)Relative bioluminescence

F
20
15
10
5
0

EYA3
SIX1

-+-+
- - ++-+-+- - ++-+-+- - ++-+-+- - ++

P(TSHβ-0.1kbp)

P(TSHβ-ΔSo1)

P(TSHβ-ΔSo2)

P(SV40)Figure 4. Eya3 and Six1 Synergistically Induce TSHb Expression(A) Expression of the Eya, Six, and Dach families in the mouse PT at ZT8 in the long-day condition was detected by RI in situ hybridization. Scale bar
represent 300 mm.
(B) The TSHb promoter (27.7 kbp, 20.6 kbp, 20.2 kbp, and 20.1 kbp) is activated by EYA3 and SIX1.
(C) Left: evolutionary conservation scores among vertebrate species were obtained from the UCSC Genome Browser (http://genome.ucsc.edu/). Genomic
positions relative to the transcription start site (TSS, also designated as ‘‘21’’) of the 0.6 kbp, 0.2 kbp, and 0.1 kbp TSHb promoter constructs are indicated.
Right: the MEF3 site, D box, and So sites in the 0.1 kbp TSHb promoter are indicated. Colored letters indicate nucleotides matching the consensus sequenceof the MEF3 site, D box, and So sites.
(D) The TSHb promoter (20.1 kbp) is activated by EYA3 in a dose-dependent manner with or without SIX1 (Supplemental Experimental Procedures).
(E) The TSHb promoter (20.1 kbp) and its MEF3-deleted [P(TSHb-DMEF3)], MEF3-mutated [P(TSHb-mutMEF3)], and D box-mutated [P(TSHb-mutD)] forms
are activated by EYA3 and SIX1.

Eya3 Triggers Photoperiodic Expression of TSHb
2205

into two groups. One group was maintained under the short-day conditions
and the other was housed under long-day conditions (light:dark = 16:8 hr,
ZT0 = lights-on, ZT16 = lights-off, 400 lux) for 2 weeks. Mice in both groups
were sacrificed and their PTs were sampled every 4 hr for 1 day, startingat ZT0.
For the acute long-day experiments, mice were first housed under shortday conditions for 3 weeks as described above and then separated into
two groups. In one, the lights-on timing was advanced by 8 hr (advancecondition), and in the other, the lights-off timing was delayed by 8 hr (delay
condition). In both cases, photoperiod was extended by 8 hr. PTs from bothgroups were obtained every 4 hr for 1 day, starting at the lights-on time
(ZT16 in the advance condition and ZT0 in the delay condition, when ZT
was defined in the short-day condition).
This study was performed in compliance with the Rules and Regulations of
the Animal Care and Use Committee, Kinki University School of Medicine,and carefully followed the Guide for the Care and Use of Laboratory Animals,
Kinki University School of Medicine. Mice were also carefully kept andhandled according to the RIKEN Regulations for Animal Experiments.
Sampling of PT
Slices (0.5 mm thick) of the brain of CBA/N mice were cut on a mouse brain
matrix (Neuroscience, Inc., Tokyo) and frozen, and the PT was punched out
with a microdissecting needle (gauge 0.5 mm) under a stereomicroscope.
The samples included a small amount of the surrounding tissue, such as
the median eminence and ependymal cells. We sampled 25 mice at eachtime point. This entire procedure was repeated twice (n = 2) to obtain experimental replicates.
Microarray Analysis
Total RNA was prepared from the pooled PT samples obtained at each time
point under each condition using TRIzol reagent (GIBCO). cDNA synthesis
and cRNA labeling reactions were performed as described previously [31].
Affymetrix high-density oligonucleotide arrays for Mus musculus (GeneChip
Mouse Genome 430 2.0) were hybridized, stained, and washed according to
the Expression Analysis Technical Manual (Affymetrix). The expression
values were summarized by the robust multiarray analysis method [32].
The microarray data are available at the NCBI Gene Expression Omnibus(GSE24775) or at our website (http://photoperiodism.brainstars.org/).
Accession Numbers
Microarray data reported herein have been deposited in the NCBI Gene
Expression Omnibus with the accession number GSE24775.
Supplemental Information
Supplemental Information includes Supplemental Results and Discussion,
two figures, four tables, and Supplemental Experimental Procedures andcan be found with this article online at doi:10.1016/j.cub.2010.11.038.
Acknowledgments
We thank Joseph Takahashi for mPer2Luc mice and Yoichi Minami and
Masayuki Iigo for helpful discussions. We also thank Junko Sakai and
Yoko Sakakida for technical assistance. Some calculations were performedby the RIKEN Super Combined Cluster. This work was performed as a partof the Genome Network Project of the Ministry of Education, Culture,
Sports, Science and Technology of Japan (H.R.U.). This research was
supported by an intramural Grant-in-Aid from the RIKEN Center for
Developmental Biology (H.R.U.), the RIKEN President’s Fund (H.R.U.), anda Grant-in-Aid for Scientific Research (C) from the Ministry of Education,
Culture, Sports, Science and Technology of Japan (Y.S.).
Received: October 17, 2010
Revised: November 10, 2010
Accepted: November 12, 2010
Published online: December 2, 2010

References
1. Dawson, A., King, V.M., Bentley, G.E., and Ball, G.F. (2001).
Photoperiodic control of seasonality in birds. J. Biol. Rhythms 16,
365–380.
2. Ebling, F.J., and Barrett, P. (2008). The regulation of seasonal changes infood intake and body weight. J. Neuroendocrinol. 20, 827–833.
3. Revel, F.G., Masson-Pévet, M., Pévet, P., Mikkelsen, J.D., andSimonneaux, V. (2009). Melatonin controls seasonal breeding by
a network of hypothalamic targets. Neuroendocrinology 90, 1–14.
4. Nakao, N., Ono, H., Yamamura, T., Anraku, T., Takagi, T., Higashi, K.,
Yasuo, S., Katou, Y., Kageyama, S., Uno, Y., et al. (2008). Thyrotrophin
in the pars tuberalis triggers photoperiodic response. Nature 452,
317–322.
5. Nakao, N., Ono, H., and Yoshimura, T. (2008). Thyroid hormones andseasonal reproductive neuroendocrine interactions. Reproduction
136, 1–8.
6. Ono, H., Hoshino, Y., Yasuo, S., Watanabe, M., Nakane, Y., Murai, A.,
Ebihara, S., Korf, H.W., and Yoshimura, T. (2008). Involvement of thyrotropin in photoperiodic signal transduction in mice. Proc. Natl. Acad.
Sci. USA 105, 18238–18242.
7. Reiter, R.J. (1993). The melatonin rhythm: Both a clock and a calendar.
Experientia 49, 654–664.
8. Lincoln, G.A., Andersson, H., and Loudon, A. (2003). Clock genes incalendar cells as the basis of annual timekeeping in mammals—a unifying hypothesis. J. Endocrinol. 179, 1–13.
9. Ikegami, K., Katou, Y., Higashi, K., and Yoshimura, T. (2009).
Localization of circadian clock protein BMAL1 in the photoperiodic
signal transduction machinery in Japanese quail. J. Comp. Neurol.
517, 397–404.
10. Hoffman, R.A., and Reiter, R.J. (1965). Pineal gland: Influence on gonads
of male hamsters. Science 148, 1609–1611.
11. Bittman, E.L., Karsch, F.J., and Hopkins, J.W. (1983). Role of the pineal
gland in ovine photoperiodism: Regulation of seasonal breeding andnegative feedback effects of estradiol upon luteinizing hormone secretion. Endocrinology 113, 329–336.
12. Morgan, P.J., and Williams, L.M. (1996). The pars tuberalis of the pituitary: A gateway for neuroendocrine output. Rev. Reprod. 1, 153–161.
13. Goldman, B.D. (2001). Mammalian photoperiodic system: Formal
properties and neuroendocrine mechanisms of photoperiodic timemeasurement. J. Biol. Rhythms 16, 283–301.
14. Hazlerigg, D., and Loudon, A. (2008). New insights into ancient seasonal
life timers. Curr. Biol. 18, R795–R804.
15. Klosen, P., Bienvenu, C., Demarteau, O., Dardente, H., Guerrero, H.,Pévet, P., and Masson-Pévet, M. (2002). The mt1 melatonin receptor
and RORbeta receptor are co-localized in specific TSH-immunoreactive
cells in the pars tuberalis of the rat pituitary. J. Histochem. Cytochem.
50, 1647–1657.
16. Guilding, C., Hughes, A.T., Brown, T.M., Namvar, S., and Piggins, H.D.
(2009). A riot of rhythms: Neuronal and glial circadian oscillators in the
mediobasal hypothalamus. Mol. Brain 2, 28.
17. Dupré, S.M., Miedzinska, K., Duval, C.V., Yu, L., Goodman, R.L., Lincoln,
G.A., Davis, J.R., McNeilly, A.S., Burt, D.D., and Loudon, A.S. (2010).
Identification of Eya3 and TAC1 as long-day signals in the sheep pituitary. Curr. Biol. 20, 829–835.
18. Minami, Y., Kasukawa, T., Kakazu, Y., Iigo, M., Sugimoto, M., Ikeda, S.,
Yasui, A., van der Horst, G.T., Soga, T., and Ueda, H.R. (2009).
Measurement of internal body time by blood metabolomics. Proc.
Natl. Acad. Sci. USA 106, 9890–9895.
19. Ueda, H.R., Chen, W., Minami, Y., Honma, S., Honma, K., Iino, M., andHashimoto, S. (2004). Molecular-timetable methods for detection ofbody time and rhythm disorders from single-time-point genome-wide
expression profiles. Proc. Natl. Acad. Sci. USA 101, 11227–11232.
20. Jeong, B.C., Hong, C.Y., Chattopadhyay, S., Park, J.H., Gong, E.Y., Kim,
H.J., Chun, S.Y., and Lee, K. (2004). Androgen receptor corepressor19 kDa (ARR19), a leucine-rich protein that represses the transcriptional
activity of androgen receptor through recruitment of histone deacetylase. Mol. Endocrinol. 18, 13–25.

(F) The TSHb promoter (20.1 kbp) and its So2-deleted form [P(TSHb-DSo2)] are activated by EYA3 and SIX, whereas its So1-deleted form [P(TSHb-DSo1)] isnot.
In (B) and (D)–(F), each of the indicated promoters was fused to a luciferase reporter gene and used to transiently transfect NIH 3T3 cells. The luciferase
activity for each promoter is expressed relative to activity with an empty vector. Data are representative of two independent experiments. Error bars
represent 6 SEM (n = 3).

Current Biology Vol 20 No 24
2206

21. Gordon, J.W., Pagiatakis, C., Salma, J., Du, M., Andreucci, J.J., Zhao, J.,
Hou, G., Perry, R.L., Dan, Q., Courtman, D., et al. (2009). Protein kinase
A-regulated assembly of a MEF2,HDAC4 repressor complex controls cJun expression in vascular smooth muscle cells. J. Biol. Chem. 284,
19027–19042.
22. Xu, P.X., Woo, I., Her, H., Beier, D.R., and Maas, R.L. (1997). Mouse Eyahomologues of the Drosophila eyes absent gene require Pax6 forexpression in lens and nasal placode. Development 124, 219–231.
23. Borsani, G., DeGrandi, A., Ballabio, A., Bulfone, A., Bernard, L., Banfi, S.,
Gattuso, C., Mariani, M., Dixon, M., Donnai, D., et al. (1999). EYA4,
a novel vertebrate gene related to Drosophila eyes absent. Hum. Mol.
Genet. 8, 11–23.
24. Bonini, N.M., Leiserson, W.M., and Benzer, S. (1993). The eyes absent
gene: Genetic control of cell survival and differentiation in the developing Drosophila eye. Cell 72, 379–395.
25. Bonini, N.M., Leiserson, W.M., and Benzer, S. (1998). Multiple roles ofthe eyes absent gene in Drosophila. Dev. Biol. 196, 42–57.
26. Spitz, F., Demignon, J., Porteu, A., Kahn, A., Concordet, J.P., Daegelen,D., and Maire, P. (1998). Expression of myogenin during embryogenesis
is controlled by Six/sine oculis homeoproteins through a conserved
MEF3 binding site. Proc. Natl. Acad. Sci. USA 95, 14220–14225.
27. Li, X., Oghi, K.A., Zhang, J., Krones, A., Bush, K.T., Glass, C.K., Nigam,
S.K., Aggarwal, A.K., Maas, R., Rose, D.W., and Rosenfeld, M.G. (2003).
Eya protein phosphatase activity regulates Six1-Dach-Eya transcriptional effects in mammalian organogenesis. Nature 426, 247–254.
28. Pauli, T., Seimiya, M., Blanco, J., and Gehring, W.J. (2005). Identificationof functional sine oculis motifs in the autoregulatory element of its own
gene, in the eyeless enhancer and in the signalling gene hedgehog.
Development 132, 2771–2782.
29. Giordani, J., Bajard, L., Demignon, J., Daubas, P., Buckingham, M., andMaire, P. (2007). Six proteins regulate the activation of Myf5 expressionin embryonic mouse limbs. Proc. Natl. Acad. Sci. USA 104, 11310–
11315.
30. Drolet, D.W., Scully, K.M., Simmons, D.M., Wegner, M., Chu, K.T.,
Swanson, L.W., and Rosenfeld, M.G. (1991). TEF, a transcription factor
expressed specifically in the anterior pituitary during embryogenesis,
defines a new class of leucine zipper proteins. Genes Dev. 5, 1739–1753.
31. Ueda, H.R., Matsumoto, A., Kawamura, M., Iino, M., Tanimura, T., andHashimoto, S. (2002). Genome-wide transcriptional orchestration of
circadian rhythms in Drosophila. J. Biol. Chem. 277, 14048–14052.
32. Irizarry, R.A., Bolstad, B.M., Collin, F., Cope, L.M., Hobbs, B., andSpeed, T.P. (2003). Summaries of Affymetrix GeneChip probe level
data. Nucleic Acids Res. 31, e15.

Current Biology, Volume 20

Supplemental Information
Acute Induction of Eya3 by Late-Night
Light Stimulation Triggers
TSH Expression in Photoperiodism
Koh-hei Masumoto, Maki Ukai-Tadenuma, Takeya Kasukawa, Mamoru Nagano, Kenichiro D. Uno,
Kaori Tsujino, Kazumasa Horikawa, Yasufumi Shigeyoshi, and Hiroki R. Ueda

Supplemental Results and Discussion
Circadian Oscillators in the Mouse PT
To investigate whether PT region contains a circadian oscillator, we examined brain slices ofmPer2Luc mice [1] containing PT and found that the slice exhibited several circadian oscillationsof bioluminescence (Figure S1A). Also, it was reported that PER2::LUC rhythms were detectedin the median eminence/pars tuberalis [2]. This result and previously report indicate that the PT
contains the circadian oscillator.
Circadian Oscillations of Clock and Clock-Controlled Genes in the Mouse PT
Many known clock and clock-controlled genes exhibited 24 hr rhythmicity in the PT under both
short-day and long-day conditions (Figure 1B and Table S2). However, some clock andclock-controlled genes are not in the list of 24 hr rhythmic genes. Therefore, we checked 24 hrrhythmicity of known clock and clock-controlled genes in the short-day and long-day conditions
by using stringent criteria for 24 hr rhythmicity (correlations > 0.8 both in short-day andlong-day conditions). Per1, Clock and Tef exhibited stringent 24 hr rhythmicity in the short-day
condition (correlations > 0.8), but not in the long-day condition (correlations < 0.8). Cry2 andHlf did not exhibit stringent 24 hr rhythmicity either in the short-day or long-day conditions.
Expression of Photoperiodic Gene Candidates in the Mouse PT
The identified photoperiodic genes included TSHβ. In contrast to the β subunit of TSH (TSHβ),
its α subunit (Cga) did not respond to the photoperiod and was not identified as a photoperiodic
gene by our statistical analysis (Figure S1B). Recently, tachykinin precursor 1 (Tac1), which
encodes the tachykinin peptide hormone family members substance P and neurokinin A, wasidentified as a long-day gene in the sheep PT, and Tac1 has been suggested to regulate prolactin
release in vivo [3]. However, the Tac1 gene was not identified as a photoperiodic gene by our
statistical analysis (Figure S1B), and this finding was confirmed using quantitative PCR (qPCR)(Figure S1C).
Expression Dynamics of Eya3 and TSHβ
The induction of Eya3 was acute and transitory, whereas TSHβ expression was gradually induced.
We consider that the difference between time-scale of Eya3 and TSHβ inductions is likely due toa molecular cascade of transcriptional regulation of Eya3 followed by that of TSHβ. In general, acascade of transcriptional regulation can generate the delay in transcription [4]. Therefore, theobserved gradual induction of TSHβ after the acute induction of Eya3 could be, at least in part,
explained by the cascade of transcriptional regulation.
Activation of TSHβ Promoter by Eya3
In Figure 4B, Eya3, which is a transcriptional co-activator and does not directly bind to DNA,appears to activate the TSHβ promoter when transfected alone. We thus measured Six1 mRNA
expression in NIH3T3 cells by qPCR method and found that Six1 is expressed in the NIH3T3
cells (Figure S2C). Therefore, we speculate that transfection of Eya3 alone may be able toslightly increase the activation of TSHβ promoter through the interaction with endogenous Six1.
Tef and Hlf Enhance the Eya3-Six1-Dependent Activation of TSHβ Promoter
Since thyrotroph embryonic factor (Tef) can increase the TSHβ promoter activity [5], we
examined the contribution of Tef and its family members, hepatic leukemia factor (Hlf) and Dbp
to 7.7-kbp and 0.1-kbp TSHβ promoter. These PAR bZIP transcription factors can activate
various promoters via the D box consensus sequences [6, 7]. TSHβ promoter contains at least
three functional D boxes upstream of the TSS [5]. We first examined the ability of Tef, Hlf, and
Dbp to trans-activate the 7.7-kbp or 0.1-kbp TSHβ promoter. We found that the transfection ofTef, Hlf or Dbp significantly increased the Luciferase activity of the 7.7-kbp or 0.1-kbp TSHβ
promoter (Figure S2D, P(TSHβ-7.7k) and P(TSHβ-0.1k)), whereas neither activated the SV40
promoter activity, indicating that the Tef-, Hlf- or Dbp-dependent activation of the TSHβ
promoter was specific (Figure S2D, P(SV40)). Consistent with these results, the 0.1-kbp TSHβ
promoter possesses one of the previously identified D boxes [5], which is highly conserved
among vertebrates (Figure 4C).
We note that Dbp slightly decrease Luciferase activity of the 7.7-kbp TSHβ promoter in
combination with the Tef or Hlf. This result could be simply explained by the weaker activationof 7.7-kbp TSHβ promoter by Dbp alone than that by Tef or Hlf (Figure S2D, P(TSHβ-7.7k)). Aweaker transcriptional activator (e.g. Dbp) apparently serves as a transcriptional repressor bypartially inhibiting the binding of a stronger transcriptional activator (e.g. Tef or Hlf). Thishypothesis could be confirmed when we use the 0.1-kbp TSHβ promoter, to which Dbp, Tef or
Hlf activate almost equally. As predicted, we confirmed that Dbp does not decrease Luciferase
activity of 0.1-kbp TSHβ promoter even in combination with the Tef or Hlf (Figure S2D,
P(TSHβ-0.1k)).
In addition to Tef, Hlf, and Dbp, we also examined E4bp4 (Nfil3), which is also
rhythmically expressed in the PT (Figure 1B) because E4bp4 can function as either atrans-repressor or trans-activator of transcription in different cell types and promoter contexts [8,9]. We found that E4bp4 exhibited no significant effect on 0.1-kbp TSHβ promoter activity(Figure S2E).
The temporal expression profiles of Tef, Hlf, and Dbp under the long-day and short-day
conditions showed that Tef and Dbp exhibited 24 hr rhythmicity under both the long-day andshort-day conditions. In contrast, Hlf exhibited only slight 24 hr rhythmicity under the short-day
condition and a photoperiodic response under the long-day condition (Figure S2F).
We next investigated the spatial expressions of Tef, Hlf and Dbp genes in the mouse PT
under the long-day condition by RI in situ hybridization (Figure S2G) and found that the Tef and
Hlf mRNAs were highly expressed whereas the Dbp mRNA was expressed only weakly. Basedon these findings, we hereafter focused on the contribution of Tef and Hlf to theEya3-Six1-dependent TSHβ promoter activity. When co-transfected with Eya3 and Six1, Tef or
Hlf synergistically increased the Luciferase activity of the TSHβ promoter (Figure S2H,
P(TSHβ-0.1k)). We also confirmed that Eya3 increased the TSHβ promoter activity in adose-dependent manner when it was co-expressed with Six1 and Tef or Hlf (Figure S2I,
P(TSHβ-0.1k)).
To determine which cis-element(s) in the TSHβ promoter (P(TSHβ-0.1k)) were activatedby Tef and Hlf, we mutated the D box as well as the putative Six consensus sequences in the
TSHβ promoter, and then performed Luciferase transfection assays. We found that the mutationof the D box site in the TSHβ promoter significantly decreased the change elicited by the Tef- or
Hlf-dependent activation (Figure S2J, P(TSHβ-mutD)). In contrast, neither the mutation nor
deletion of the MEF3 site had much effect on the Tef- or Hlf-dependent activation of the TSHβ
promoter. We also noted that the mutation of the D box site in the TSHβ promoter did not affectthe change elicited by the Eya3-Six-dependent activation (Figure 4E, P(TSHβ-mutD)).
These results indicate that Tef and Hlf act additively on only one D box in the 0.1-kbp
TSHβ promoter (Figure S2D and J). Since both Hlf and Tef act as a transcriptional activator of Dbox, transfection of both Hlf and Tef into the cells seems equivalent to the increased
concentration of a transcriptional activator of D box (either Hlf or Tef). A transcriptional

activator at higher concentration can bind to a cis-element with higher probability, which will
results in the increased transcriptional activity. This simple hypothetical mechanism will explain
why co-transfection of both Hlf and Tef additively activates TSHβ promoter containing only one
D box.
This D box is located near the transcription start site in the TSHβ promoter, and looks likeand might work as a TATA box. To exclude this possibility, we examined the basal activity of
the D box-mutated TSHβ promoter (P(TSHβ-mutD)) and compared it with the wild type TSHβ
promoter (P(TSHβ-0.1k)). In fact, the basal activity of P(TSHβ-mutD) in Figure 4E was 1.16 ±0.070-fold (average ± SEM, n = 3) of that of wild type P(TSHβ-0.1k), suggesting that a
TATA-like sequence overlapping with D box in the TSHβ promoter does not serve as a TATAbox.
We next examined the effect of deleting either the So1 or So2 site in the TSHβ promoter(Figure S2J). Deletion of the So1 site slightly decreased the change elicited by the Tef- or
Hlf-dependent activation (Figure S2J). These results suggested that the D box is important for theactivation of the TSHβ promoter by Tef or Hlf and that the So1 site also, but slightly, affects theactivation of the TSHβ promoter by Tef or Hlf. We also note that both the D box and the So1 site
are important for the Eya3-Six1-Tef- or Eya3-Six1-Hlf-dependent activation of the TSHβ
promoter (Figure S2J). Deletion of So2 site increased the activation of TSHβ promoter byco-transfection with Eya3, Six1 and Tef. Therefore, So2 site may play a suppressing role inactivation of TSHβ promoter although it is unknown what molecular mechanism functions.
Since the So1 and D box are close to one another in the TSHβ promoter, and since specificcombinations of neighbouring cis-elements can exhibit qualitatively new properties from thoseof either element alone [10], we speculate that the Six consensus sequence and D box may
constitute a composite promoter and provide combinatorial regulation in the photoperiodic
response. Consistent with this hypothesis, transcriptional activators of the Six consensus
sequence (Eya3 and Six1) and of the D box (Tef and Hlf) exhibited synergistic activation of theTSHβ promoter. In addition, deletion of the Six consensus sequence affected the Tef- or
Hlf-dependent activation of the TSHβ promoter (Figure S2J). In the mouse PT, the Tef and Hlf
mRNAs exhibited circadian expression, peaking in the subjective morning under the short-day
condition (Figure S2F). Therefore, we expect that the late-night light stimulation induces theexpression of Eya3 in the PT with timing that ensures that it is co-expressed with Tef or Hlf
during the subjective morning, to synergistically trigger TSHβ expression.
CBA/N Mice as a Model System for the Study of Photoperiodism
This study provided the first genome-wide expression analysis of the CBA/N mouse PT. Ourfindings in this study, together with the previous report [11] by Dr. Yoshimura in Nagoya

University, suggest that a photoperiodic response of TSHβ and much of the photoperiodic
machinery are preserved in the PT of the CBA/N mouse. These studies have revealed theusefulness of CBA/N mouse as a model organism for the molecular study of photoperiodic
system.
Retinal Degeneration in CBA MiceIt was reported that CBA/J (rd/rd) mice have lower sensitivity to light due to the retinaldegeneration, whereas CBA/N (+/+) mice have normal light sensitivity [12, 13]. In this study, weused CBA/N mice (+/+), which have normal retina.

40000
30000
20000
10000

1

2

3

4
5
6
Time (day)B

TSHβ mRNA (GeneChip)

9

Tac1 mRNA (GeneChip)

Short
Long
14.2

15
13

Gene expression

Gene expression

8

Cga mRNA (GeneChip)

Short
Long

0

4

C

8
12 16
Time (ZT)

Short
Long
11

13.8

Short-dayLong-day11
9

7

Gene expression

Bioluminescence
(cpm)

A

13.4

20

13

Short-dayLong-day
0

4

8
12 16
Time (ZT)

Short-dayLong-day

10

20

9
8

0

4

8
12 16
Time (ZT)20

Tac1 mRNA (qPCR)

Relative expression

Short
Long
0.1
0.08

0.06
0.04

Short-dayLong-day

0.02
0

0

4

D

8
12 16
Time (ZT)20

Rorβ mRNA (GeneChip)

Maff mRNA (GeneChip)

Advance

Advance

Advance

Advance

13

Eya3

11

0 4 8
Time (ZT)

12

9
8

16 20

Delay
TSHβ

13

12

0

4

9
8

16 20

12

Eya3

11

8 12 16 20
Time (ZT)

12

0

4

Maff

7
6

16 20

11

8 12 16 20
Time (ZT)

Rorβ

9
8

0

4

8 12 16 20
Time (ZT)

Supplemental Figure 1. Masumoto et al.

0 4 8
Time (ZT)

12

Delay

10

9
8

0 4 8
Time (ZT)

8

Delay

10

12
11

Rorβ

Delay
Gene expression

Gene expression

14

0 4 8
Time (ZT)

Gene expression

16 20

11
10

10

12
11

12

Gene expression

TSHβGene expression

Gene expression

14

Gene expression

Eya3 mRNA (GeneChip)

Gene expression

TSHβ mRNA (GeneChip)

8

Maff

7
6

0

4

8 12 16 20
Time (ZT)Figure S1. Expression of Some Photoperiodic and Nonphotoperiodic Genes(Related to Figure 1)(A) Representative data of bioluminescence showing circadian profiles of mPER2 expression
from PT region from mPer2Luc knockin mouse.
(B) Expression of known photoperiodic and nonphotoperiodic genes under the short-day andlong-day conditions was measured by GeneChip. The GeneChip data for TSHβ, Cga, and Tac1
mRNA under the long-day and short-day conditions were indicated by a line plot (n = 2). The
vertical axis indicates gene expression on a logarithmic scale (base is 2). Error bars represent ±SEM.
(C) Confirmation of the GeneChip data for the Tac1 expression. Tac1 expression under the
long-day and short-day conditions measured by qPCR (n = 2). Relative expression to Tbp
expression is plotted.
(D) GeneChip data of TSHβ, Eya3, Rorβ, and Maff expression on the first long-day (advance anddelay conditions) were indicated by a line plot (n = 2). The vertical axis indicates gene
expression on a logarithmic scale (base is 2). Error bars represent ± SEM.

A

B

C

2

1

0

- +

- +

P(TSHβ-7.7kbp)

P(SV40)RORβ

3Relative expression

3
Relative bioluminescence

Relative bioluminescence

3

2

1

0

2

1

0

- +

- +

P(TSHβ-7.7kbp)

P(SV40)

MAFF

TSHβ

Eya3

Relative bioluminescence

D
60
40
20

Relative bioluminescence

0
DBP
TEF
HLF

-

+
-

+
-

+

+
+
-

+
+

+
+

+
+
+

-

+
-

+
-

-

+
-

+
-

+

+
+
-

+
+

+
+

+
+
+

-

+
-

+
-

P(TSHβ-7.7kbp)

+

+
+
-

+
+

+
+

+
+
+

+

+
+
-

+
+

+
+

+
+
+

P(SV40)

10
8
6
4
2

0
DBP
TEF
HLF

P(TSHβ-0.1kbp)

P(SV40)

Supplemental Figure 2. Masumoto et al. (1/3)

Six1

Relative bioluminescence

E

4
3
2
1

0
TEF
HLF
E4BP4

-

+
-

+
-

+

-

P(TSHβ-0.1kbp)

+
-

+
-

+

P(SV40)F
Hlf mRNA (GeneChip)

11.5

Short
Long

12

11.5

11
0

4

8
12 16
Time (ZT)

20

Short-dayLong-day

11

10.5

0

Tef

12

11.5

Hlf

12

11.5

11
16

20

0
4
Time (ZT)

8

12

11

10.5

16

20

Delay
Tef

12

11.5

0
4
Time (ZT)

8

12

11
4

Tef mRNA

10

8
12 16
Time (ZT)

Short-dayLong-day48
12 16Time (ZT)

0

20

12.5

Hlf

12

11

10.5

Hlf mRNA

0

4

20

Dbp

13
12
11
10

16

20

8
12 16
Time (ZT)

04Time (ZT)

8

12

Delay

11.5

0

11

Delay
Gene expression

Gene expression

12.5

G

20

12

Advance

12.5

Gene expression

Gene expression

12.5

10.5

8
12 16
Time (ZT)

13

Advance

Advance

10.5

4Gene expression

10.5

Short
Long
12.5

Gene expression

Short-dayLong-day12

Dbp mRNA (GeneChip)

Gene expression

Gene expression

Short
Long
12.5

Gene expression

Tef mRNA (GeneChip)20

Dbp mRNA

Supplemental Figure 2. Masumoto et al. (2/3)

Dbp

13
12
11
10

0

4

8
12 16
Time (ZT)20

Relative
bioluminescence

H

40
30
20
10

0
EYA3
SIX1
TEF
HLF

I

-

+ - +
- + +
- - - - -- + - +
- - + +
+ + + +
- - - -

P(TSHβ-0.1kbp)-- + - +
- - + +
- - - + + + +

+ - +
- + +
- - - - -- + - +
- - + +
+ + + +
- - - P(SV40)- + - +
- - + +
- - - + + + +Relative
bioluminescence

160
120
80
40

0
EYA3
SIX1
TEF
HLF

J

- - - - - - - - - -++++ - ++++
- - - - +++++- - - - - - - - P(TSHβ-0.1kbp)- ++++- - - - +++++- - - - - - - - - -++++ - ++++
- - - - +++++- - - - - - - - P(SV40)

Relative bioluminescence

50
40
30
20
10

0
EYA3
SIX1
TEF
HLF

- + - - - ++ - ++ - + - + - - - ++ - ++ - + - + - - - ++ - ++ - + - + - - - ++ - ++ - +- - + - - + - ++ - ++ - - + - - + - ++ - ++ - - + - - + - ++ - ++ - - + - - + - ++ - ++- - - + - - +++ - - - - - - + - - +++ - - - - - - + - - +++ - - - - - - + - - +++ - - - - - - + - - - - +++ - - - - + - - - - +++ - - - - + - - - - +++ - - - - + - - - - +++P(TSHβ-0.1kbp)

P(TSHβ-ΔMEF3)

P(TSHβ-mutMEF3)

P(TSHβ-mutD)

Relative bioluminescence

50
40
30
20
10

0
EYA3
SIX1
TEF
HLF

- + - - - ++ - ++ - + - + - - - ++ - ++ - + - + - - - ++ - ++ - +- - + - - + - ++ - ++ - - + - - + - ++ - ++ - - + - - + - ++ - ++- - - + - - +++ - - - - - - + - - +++ - - - - - - + - - +++ - - - - - - + - - - - +++ - - - - + - - - - +++ - - - - + - - - - +++P(TSHβ-ΔSo1)

P(TSHβ-ΔSo2)

P(SV40)

Supplemental Figure 2. Masumoto et al. (3/3)- ++++- - - - +++++

Figure S2. The Activities and the Expression Profiles of the Transcriptional Regulators of
the TSHβ Promoter (Related to Figure 4)(A and B) RORβ and MAFF had almost no effect on the TSHβ promoter activity. The TSHβ
promoter (-7.7k) was barely activated by RORβ (A) or MAFF (B).
(C) mRNA expression levels of endogenous TSHβ, Eya3 and Six1 in NIH3T3 cells. Relative
mRNA levels of each gene to Tbp expression were measured with quantitative PCR assay (n = 3,
error bars means ± SEM).
(D) The TSHβ promoter (-7.7k and -0.1k) could be activated by TEF, HLF, and DBP.
(E) The TSHβ promoter (-0.1k) was barely activated by E4BP4 (Nfil3).
(F) Gene expression of Tef, Hlf, and Dbp. GeneChip data for the Tef, Hlf, and Dbp expression
under the short-day and long-day conditions and on the first long-day (advance and delay
conditions) indicated by line plot (n = 2). The vertical axis indicates gene expression on alogarithmic scale (base is 2). Error bars means ± SEM.
(G) Tef, Hlf, and Dbp expression in the mouse PT at ZT8 under the long-day condition, detected
by RI in situ hybridization. Scale bar=300 μm.
(H) Tef and Hlf enhance the Eya3-Six1-dependent activation of TSHβ promoter. The TSHβ
promoter (-0.1k) was strongly activated by EYA3, SIX1, and TEF or HLF. Experiments of
Figure 4B and Figure S2H were performed at the same time, so Luciferase activities ofP(TSHβ-0.1k) and P(SV40) in Figure 4B were re-plotted in Figure S2H as control data.
(I) TSHβ expression is synergistically activated by Eya3 and Six1, and further enhanced by Tef
and Hlf.
(J) Role of the D box and So1 site in transcriptional activation of the TSHβ promoter by Eya3,
Six1, and Tef or Hlf. (Top panel) The TSHβ promoter (-0.1k), and its MEF3-deleted(P(TSHβ-ΔMEF3)) and MEF3-mutated (P(TSHβ-mutMEF3)) forms were strongly activated by
EYA3, SIX1, and TEF or HLF. The D box-mutated TSHβ promoter (P(TSHβ-mutD)) was not
activated by TEF and HLF. (Bottom panel) The So2-deleted form (P(TSHβ-ΔSo2)) werestrongly activated by EYA3, SIX1, and TEF or HLF. The So1-deleted TSHβ promoter(P(TSHβ-ΔSo1)) was not strongly activated.
(A, B, D, E, H-J) Each of the indicated promoters was fused to a Luciferase reporter gene andused to transiently transfect NIH3T3 cells. The Luciferase activity for each promoter isexpressed relative to the activity with an empty vector. Data are representative of two
independent experiments. Error bars means ± SEM. (n = 3).

Supplemental Tables (see accompanying Excel spreadsheet)
Table S1. List of 24 hr Rhythmic Genes in the Mouse PT under Short-Day and Long-Day
Conditions (Related to Figure 1A)
The table provides the Affymetrix probe set ID, p value, FDR, mean of the phases undershort-day and long-day conditions, phase difference between short-day and long-day conditions,
phase in the short-day condition, phase in the long-day condition, the best Pearson's correlation
to cosine curves of a 24 hr period in short-day condition, the best Pearson's correlation to cosine
curves of a 24 hr period in long-day condition, amplitude of circadian expression in short-day
condition, amplitude in long-day condition, relative amplitude in short-day condition, relative
amplitude in long-day condition, p value in short-day condition, p value in long-day condition,
description of the probe set, gene name, and gene symbol.
Table S2. List of Clock and Clock-Controlled Genes with Significant Circadian Expression
under Short-Day and Long-Day Conditions (Related to Figure 1B)
The table provides the gene symbol, Affymetrix probe set ID, phase in short-day condition, thebest Pearson's correlation to cosine curves of a 24 hr period in short-day condition, phase inlong-day condition, the best Pearson's correlation to cosine curves of a 24 hr period in long-day
condition, the minimum of the values in the 4th and 6th columns (the smaller value of two best
Pearson's correlation), and difference between the phases in short-day and long-day conditions.
Table S3. List of Long-Day and Short-Day Genes under Chronic Short-Day and Long-Day
Conditions (Related to Figure 1C)
The table provides the Affymetrix probe set ID, p value, FDR, q value, whether the expression in
long-day condition is up-regulated ('up') or down-regulated ('down') against the short-day
condition, fold change between the mean expression in short-day and long-day conditions,description of the probe set, gene name, and gene symbol.
Table S4. List of Acute Long-Day Genes (Related to Figure 3A)
The table provides the Affymetrix probe set ID, p value, FDR, q value of difference between
short-day and advance conditions, maximum fold change between the lowest and highest mean
expressions in the short-day and advance conditions, p value, FDR, q value of difference
between short-day and delay conditions, maximum fold change between the lowest and highest
mean expressions in the short-day and delay conditions, description of the probe set, gene name,and gene symbol.

Supplemental Experimental Procedures
Circadian-Time Analysis from Time-Course Expression Data
To detect how much the circadian phase differed between the short-day and long-day conditions,
we investigated the expressions of the following 20 clock and clock-controlled genes by
GeneChip: Bmal1 (Arntl), Bmal2 (Arntl2), Clock, Cry1, Cry2, Dbp, Dec1 (Bhlhb2), Dec2
(Bhlhb3), E4bp4 (Nfil3), Npas2, Per1, Per2, Per3, Tef, Hlf, Rev-Erbα (Nr1d1), Rev-Erbβ(Nr1d2), Rorα, Rorβ, and Rorγ. We used natural expression values from the GeneChip data. We
concatenated two time-courses of experimental replicates (6 time points for each replicate) andused 12 time-point dataset for the subsequent analysis. For each gene, we calculated the best
Pearson's correlation between gene expression data and cosine curves of a 24 hr period, anddetermined its phase in short-day and long-day conditions using a Fourier transformation-based
method [14]. If the GeneChip had multiple probe sets for a single gene, we first calculated, foreach probe set, two best Pearson's correlation from short-day and long-day conditions,
respectively, and then registered the smaller best Pearson's correlation. We selected the probe set
with the maximum value of the registered (i.e. smaller) best Pearson's correlation. Next, wechose genes for which the best Pearson's correlation for both the short-day and long-day
conditions was more than or equal to 0.8 (see Figure 1B). Finally, we calculated the median of
the phase difference (long-day – short-day) for these RNAs. We used this median value (~7.71
hours) as the phase delay of the long-day versus the short-day condition.
Identification of 24 hr Rhythmic Genes
As described above, we concatenated two time-courses of experimental replicates (6 time points
for each replicate) and used 12 time-point dataset for the subsequent analysis. To identify 24 hrrhythmic genes in the mouse PT, we first calculated the best Pearson's correlation between gene
expression data and cosine curves of a 24 hr period, and determined its phase in short-day andlong-day conditions using a Fourier transformation-based method [14]. We then calculated the pvalues for the best correlations of the short-day and long-day using random shuffling, and then
combined these two p values with Fisher's method ( P  P1 P2 1  logP1 P2 ). We estimated thefalse-discovery rates (FDRs) of the combined p value based on the Benjamini-Hochberg method[15], and selected significantly oscillating genes as those for which the FDRs were less than orequal to 0.05.
Identification of Photoperiodic Genes
To identify the photoperiodic genes, we used time-course expression data under chronic
short-day and long-day conditions. In order to complement the observed phase differencebetween short-day and long-day conditions (the approximately 8 hours, see above), we shifted
time points by 8 hours; that is, the short-day ZT16, 20, 0, 4, 8, 12 corresponded to long-day ZT0,4, 8, 12, 16, 20, respectively. We used log-2-transformed expression values of the GeneChip datain the following analysis. We performed a two-way ANOVA to test the difference in expression
levels between the short-day and long-day conditions for each probe set, and assigned p values
and FDRs. We selected as significantly differently expressed genes those whose FDRs were lessthan or equal to 0.05 and for which the difference between the maximum expression value in the
short-day condition and the minimum value in the long-day condition (“short-day” gene) or
between the maximum expression in the long-day condition and the minimum in the short-day
condition (“long-day” gene) was more than 2.5-fold. In this method, we did not subtract the 24
hr rhythmic genes that were identified above, and thus 38 genes were identified both in the 24 hrrhythmic and the photoperiodic genes.
Circadian-Time Estimation from Single-Time-Point Expression Data
To estimate how the circadian phases at ZT16 were changed on days 0, 1, 3 and 5 after transition
from the short-day to the delay condition, we used a previously reported molecular-timetable
method [16, 17]. To construct a molecular timetable that has the phase (peak time), mean, andstandard deviation (SD) of the gene expressions of clock and clock-controlled genes, we used theexpression values of 20 clock and clock-controlled genes from the qPCR data at every 4 hours
during 48 hours in the short-day condition. We first normalized the qPCR expression data of the
20 clock and clock-controlled genes by using Tbp expression level at the same time point. We
then calculated the best Pearson's correlation between cosine curves of a 24 hr period and theqPCR expression data. We selected genes with the best Pearson's correlation ≥ 0.8. For thesegenes, we also calculated peak time, mean and SD of the qPCR expression data. With thismolecular timetable, we determined the circadian phase of the mouse PTs at ZT16 on days 0, 1,
3, and 5. In details, we normalized each qPCR expression value obtained at ZT16 for each day
by subtracting the estimated mean and then divided by the SD in the molecular timetable. We
then plotted the data on a graph, with the phase (peak time) on the x-axis and normalized
expression values on the y-axis. From this graph, we searched for the cosine curve, y =Cos (x-b), that had the maximum correlation, and determined b as the phase of the day.

2 ×

Identification of Acute Long-Day Genes
To identify acute long-day genes whose expression patterns were changed on the first long-day
after the transition from the short-day to the long-day condition, we sought to retrieve probe sets
whose expression patterns in the short-day condition and advance condition were significantly
different but whose expression patterns in the short-day condition and delay condition were notsignificantly different. First, we estimated that the phase delay of the advance condition and thedelay condition against the short-day condition was approximately 0 hours (~0.88 hours), and 4
hours (~3.96 hours), respectively, using the method described in the above circadian-time
analysis from the time-course expression data. In order to complement these phase differences,
we set the short-day ZT0 to correspond with the advance condition ZT0, and the short-day ZT20
with the delay condition ZT0. We then performed the same statistical procedures describedabove for identifying genes whose time-course expression patterns in the short-day and long-day
condition were different. In this case, we chose genes whose expressions were significantly
different between the short-day condition and the advance condition, but were not significant
between the short-day condition and the delay condition.
Preparation of PT Region Slices from mPer2Luc Mice
The brains were removed from quickly decapitated, young (older than 4 weeks of age) mPer2Luc
mice, then 300-μm thick coronal sections containing the PT were made using a vibratome typelinearslicer (PRO7; Dosaka EM Corp) in ice cold Hanks' balanced salt solution (Invitrogen). PT
region slices included a small amount of the surrounding tissue. The slices were then placed on aculture membrane (MilliCell-CM; Millipore) and set on a dish with 1.2 mL culture medium[DMEM supplemented with 1.2 g/L NaHCO3 (Nacalai Tesque), 15 mM HEPES (Dojindo), 20
mg/L kanamycin (Invitrogen), 5 μg/mL insulin (Sigma), 20 nM putrescine (Sigma), 100 μg/mL
apo-transferrin (Sigma), 20 nM progesterone (Sigma), 30 nM sodium selenite (Sigma),
one-fiftieth part B-27 supplement (Invitrogen), and 1 mM luciferin]. The culture dishes wereplaced in a high-sensitivity bioluminescence detection system (LM-2400; Hamamatsu
Photonics), and the bioluminescence of each well was measured at 37°C.
Quantitative PCR (qPCR) of PT Samples
The total RNA was prepared from the pooled PT samples obtained at each time point under eachcondition, using Trizol reagent (Gibco BRL). qPCR was performed with the ABI Prism 7900 and
SYBR Green Reagents (Applied Biosystems). The cDNAs were synthesized from 0.25 μg of
total RNA using Superscript II reverse transcriptase (Invitrogen). Samples contained 1×SYBRGreen Master Mix, 0.8 μM primers, and 1/40 synthesized cDNA in a 10 μl volume. The PCR
conditions were as follows: 10 min at 95ºC, then 45 cycles of 15 s at 94ºC, 1 min at 59ºC. The
absolute cDNA abundance was calculated using a standard curve obtained from murine genomic
DNAs. We used Tbp as the internal control.
Oligonucleotide Sequences for qPCR of Tissue Samples
The primers (Hokkaido System Science) used in qPCR were as follows:TSHβ mRNA:
Forward primer: 5'-CTGCATACACGAGGCTGTCAG -3'
Reverse primer: 5'-CCCCAGATAGAAAGACTGCGG-3'
Eya3 mRNA:
Forward primer: 5'-TTCACAGCTCCAAGTAGAATCTGACT-3'
Reverse primer: 5'-TATGGAAGCGCCATGAGCTT-3'
Tac1 mRNA:
Forward primer: 5'-TAATGTGGTGACCTCCCCAGA-3'
Reverse primer: 5'-TCATCACTGTGCTTTGCTGAAA-3'
Tbp mRNA:
Forward primer: 5'-CCCCCTCTGCACTGAAATCA -3'
Reverse primer: 5'-GTAGCAGCACAGAGCAAGCAA -3'
In Situ Hybridization (ISH)
Mice were deeply anesthetized with ether and intracardially perfused with 10 ml saline and 20
ml of a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4. Mousebrain samples were postfixed in the same fixative for 24 hours at 4°C, soaked in PB containing
20% sucrose for 48 hours, and stored frozen at -70ºC. The ISH method was described in detailpreviously [18]. Serial coronal sections (40-µm thick) of the mouse brain were made using acryostat. Fragments of cDNA were obtained by PCR, and the products were subcloned into thepGEM-T easy vector (Promega). Radiolabelled probes were generated using 35S-UTP(PerkinElmer) via a standard protocol for cRNA synthesis. The probe sequence information ispresented in the Supplemental Experimental Procedures.
Oligonucleotide Sequences for ISH
The primers (Hokkaido System Science) used in the construction of ISH cRNA probes were as
follows:
TSHβ cRNA probe:
Forward primer: 5'-TGGGTGGAGAAGAGTGAGCG-3'
Reverse primer: 5'-ACCAGATTGCACTGCTATTG-3'
Eya1 cRNA probe:
Forward primer: 5'-GGACTTTACTACATAACTGGGTCAGCAGC-3'
Reverse primer: 5'-GGTCTCAACACTGGCCATTTCTGTCTG-3'
Eya2 cRNA probe:
Forward primer: 5'-GAATCATGCAGAGGTTTGGCCGCAAAG-3'
Reverse primer: 5'-CTCCCTTGCCAATGCCCAGAGAATGATTG-3'

Eya3 cRNA probe:
Forward primer: 5'-AGCACAAATGCCAGCCTGATACCCAC-3'
Reverse primer: 5'-CTGTTGGGTCCTTTCCATACTTCTG-3'
Eya4 cRNA probe:
Forward primer: 5'-TGCCAACAGGTGTGAGAGGAGGAGTG-3'
Reverse primer: 5'-CCAGTGCTTGATGTAGAGCCAAGAGGTCC-3'
Six1 cRNA probe:
Forward primer: 5'-CCTCCTCCTCGTCTTCTTTTAATGAACAGA-3'
Reverse primer: 5'-GAGGCAGCCTACAGATAGGAGAGTTTCTG-3'
Six2 cRNA probe:
Forward primer: 5'-CCCCGCTTTACGTTTTCTCTTTCCCTCCT-3'
Reverse primer: 5'-GCAGAAATATCTCCCGACGAACATTCACA-3'
Six3 cRNA probe:
Forward primer: 5'-ACATCGAGCGGCTGGGCCGCTTCCTCTG-3'
Reverse primer: 5'-CGATGGCCTGATGCTGGAGCCTGTTCTTG-3'
Six4 cRNA probe:
Forward primer: 5'-CGTGATCTCCTTGCACAGAATTGCAAATG-3'
Reverse primer: 5'-CATGCAGAATAACAAGGGTACATACAGAGA-3'
Six5 cRNA probe:
Forward primer: 5'-TGACAATGGTGTCAAGGTGCTAGGACAGG-3'
Reverse primer: 5'-GTTGCCCTAGGGAACAGGGAAATCTTTGG-3'
Six6 cRNA probe:
Forward primer: 5'-CATCCAGCGACAGTGAGTGCGACATCTG-3'
Reverse primer: 5'-TCACACAGAACGCGTGAGCTTGCTCATTC-3'
Dach1 cRNA probe:
Forward primer: 5'-TGGTCATGACATGGGGCATGAGTCAAAAC-3'
Reverse primer: 5'-GGGGTCAGGGAGTCATTTAAGACCCGGAG-3'
Dach2 cRNA probe:
Forward primer: 5'-GAGGGATAATAAAGAAGAAGTACCAGCTC-3'
Reverse primer: 5'-CATAGCAGCACTGTCATGCGGCGTTCCAC-3'
Ski cRNA probe:
Forward primer: 5'-CAAATTTCAGAGAAGGGAGGTGAGGTTTC-3'
Reverse primer: 5'-AGGTAGGGTAGGCATGTGGGGAGAAAC-3'
Skil cRNA probe:
Forward primer: 5'-AGATACCTCACTTGAGAATAAAGAAAGCAC-3'
Reverse primer: 5'-GGCATGAAAATGGCAAACATCTCAAAATAC-3'

Dbp cRNA probe:
Forward primer: 5'-ACCGCGCAGGCTTGACATCTAGGGAC-3'
Reverse primer: 5'-CATGACGTTCTTCGGGCACCTAGCTGG-3'
Tef cRNA probe:
Forward primer: 5'-TGGAAAGAGCCACCCTTTGGAGGAC-3'
Reverse primer: 5'-CACACACATGTAAGGCCAAAGGGCTGC-3'
Hlf cRNA probe:
Forward primer: 5'-GTGCATGACCAGAATATTCTCAGGACAGGG-3'
Reverse primer: 5'-CGGAGAAACCACAGTTTGTGACAACACC-3'
qPCR of NIH3T3 Cell Samples
NIH3T3 cells were grown in DMEM (Invitrogen) supplemented with 10% FBS (JRH
Biosciences) and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin; Invitrogen). Total
RNA was prepared from NIH3T3 cells using TRIzol reagents (Invitrogen) according to the
manufacturer's instructions. The cDNA was synthesized from 0.25 µg of total RNA with random
6 mer (Promega) and Superscript II Reverse Transcriptase (Invitrogen) according to the
manufacturer's instructions. qPCR was performed using ABI Prism 7900 and Power SYBRGreen Reagents (Applied Biosystems). Samples contained 1×Power SYBR Green Master Mix(Applied Biosystems), 0.8 µM primers and 1/40 synthesized cDNA in a 10 µl volume. PCR
conditions were as follows: 10 min at 95ºC, then 45 cycles of 15 s at 94ºC and 1 min at 59ºC.
Absolute cDNA abundance was calculated using a standard curve obtained from murine genomic
DNA. Tbp expression levels were quantified and used as an internal control.
Oligonucleotide Sequences for qPCR
The primers (Hokkaido System Science) used in qPCR were as follows:
TSHβ mRNA:
Forward primer: 5'-CTGCATACACGAGGCTGTCAG-3'
Reverse primer: 5'-CCCCAGATAGAAAGACTGCGG-3'
Eya3 mRNA:
Forward primer: 5'-TTCACAGCTCCAAGTAGAATCTGACT-3'
Reverse primer: 5'-TATGGAAGCGCCATGAGCTT-3'
Six1 mRNA:
Forward primer: 5'-TATTTTTGAAGTCTGTCACCCGAA-3'
Reverse primer: 5'-TTCGTCATATCATTAACCTAGCCACT-3'

Tbp mRNA:
Forward primer: 5'-CCCCCTCTGCACTGAAATCA-3'
Reverse primer: 5'-GTAGCAGCACAGAGCAAGCAA-3'
Transfection and Luciferase Assay
NIH3T3 cells (American Type Culture Collection) were maintained in DMEM (Invitrogen)supplemented with 10% FBS (JRH Biosciences) and antibiotics (100 U/ml penicillin and 100µg/ml streptomycin; Invitrogen). One day prior to transfection, the cells were plated onto 35-mm
dishes at a density of 2×105 cells per dish. The following day, the cells were co-transfected using
FuGene6 (Roche) with 0.4 µg of a Luciferase reporter plasmid in the presence of the following
constructs, as indicated in the respective figures: 0 or 0.4 µg of pCMVTnT-Rorβ (for Figure
S2A); 0 or 0.4 µg of pCMV-SPORT6-Maff (MGC clone # 30560, Invitrogen, for Figure S2B); 0
or 0.4 µg each of pMU2-Eya3 and pMU2-Six1 (for Figure 4B, E and F); 0, 0.133, 0.4 or 1.2 µg
of pMU2-Eya3 and 0 or 0.4 µg of pMU2-Six1 (for Figure 4D); 0 or 0.4 µg each of pMU2-Dbp[19], pMU2-Tef and pMU2-Hlf (for Figure S2D); 0 or 0.4 µg each of pMU2-Tef, pMU2-Hlf andpMU2-E4bp4 [19] (for Figure S2E); 0 or 0.4 µg each of pMU2-Eya3, pMU2-Six1, pMU2-Tef
and pMU2-Hlf (for Figure S2H and J); 0, 0.133, 0.4 or 1.2 µg of pMU2-Eya3 and 0 or 0.4 µg of
pMU2-Six1, pMU2-Tef and pMU2-Hlf (for Figure S2I). Empty vector was used to bring the total
amount of DNA to 4.0 µg per well. In addition, 50 ng of a phRL-SV40 plasmid (Renilla
Luciferase (RLuc) reporter vector, Promega) was added to each transfection as an internal control
for transfection efficiency. pGL4.13 (Promega) was used for the construction of the control
promoter (P(SV40)). Forty-eight hours after the transfection, the cells were harvested andassayed with the Dual-Luciferase Reporter Assay System (Promega). The Luciferase activity
was normalized to the RLuc activity.
Construction of Plasmids
pGL4.10-P(TSHβ-7.7k)
A 7.7-kbp promoter region of the mouse TSHβ gene was amplified from C57BL/6 genomic
DNA using PCR with the following primers (Hokkaido System Science, restriction recognition
sequences are underlined): Forward primer containing the XhoI recognition sequence,(5'-AAGCCTCGAGGTGGGTCTGGTGGATGACTGCTAAGAA-3'); reverse primer containing
EcoRV recognition sequence,(5'-TTCTGATATCGGTAGACACCTACCTTACTTTGCATGAGTG-3'). The PCR product wasdigested with XhoI and EcoRV, and cloned into the XhoI-EcoRV site of a pGL4.10 vector(Promega) immediately upstream of Luc2, to obtain pGL4.10-P(TSHβ-7.7k). TSHβ promoter
constructs were cloned at the +30 position because there is an MEF3 site at +1 position of TSHβ

gene region (Figure 4C). We thus designed TSHβ promoter constructs so that it covers asufficient flanking sequence of the MEF3 site.
pCMVTnT-Rorβ
The full-length coding sequence of mouse Rorβ was amplified from an NIH3T3 cDNA library
using
PCR
with
the
followingprimers:
Forwardprimer,5'-ATGCGAGCACAAATTGAAGTGATACC-3' (Hokkaido System Science); reverse primer,
5'-TCATTTGCAGACCGCAGCACAGTCAGG-3'. The PCR product was treated with theMighty Cloning kit (TaKaRa) for 5'-end phosphorylation, and cloned into the SmaI sites of apCMVTnT vector (Promega). The resulting construct was designated as pCMVTnT-Rorβ. In thispCMVTnT vector, the gene is regulated by a CMV promoter.
pGL4.10-P(TSHβ-0.6k), pGL4.10-P(TSHβ-0.2k), and pGL4.10-P(TSHβ-0.1k)
We used pGL4.10-P(TSHβ-7.7k) plasmid DNA as a template for cloning the 0.6-, 0.2-, and
0.1-kbp promoter regions of TSHβ. The 0.6-, 0.2-, and 0.1-kbp TSHβ promoter regions were
amplified, treated with the Mighty Cloning kit (TaKaRa) for 5'-end phosphorylation, and cloned
into the EcoRV sites of a pGL4.10 vector (Promega). The resulting constructs were designated as
pGL4.10-P(TSHβ-0.6k), pGL4.10-P(TSHβ-0.2k), and pGL4.10-P(TSHβ-0.1k).
Oligonucleotide Sequences for Constructing the Shorter TSHβ Promoters (Hokkaido
System Science)
Cloning of P(TSHβ-0.6k):
Forward primer: 5'-CCAAGACAGAATTTGATCTCAGGTCAGTT-3'
Reverse primer: 5'-CACTCTCCGTTCATTTTATACCCTTCA-3'
Cloning of P(TSHβ-0.2k):
Forward primer: 5'-GATATGTTTCAAATAGAAGAGAGGAAG-3'
Reverse primer: 5'-CACTCTCCGTTCATTTTATACCCTTCA-3'
Cloning of P(TSHβ-0.1k):
Forward primer: 5'-AGATGCTTTTCAGATAAGAAAGCAGC-3'
Reverse primer: 5'-CACTCTCCGTTCATTTTATACCCTTCA-3'
pMU2-Eya3 and pMU2-Six1
The full-length coding sequences of mouse Eya3 and Six1 were amplified from the NIH3T3
cDNA library by PCR, using forward primers containing the I-SceI recognition sequence andreverse primers containing the PI-PspI recognition sequence (Hokkaido System Science,
restriction recognition sequences are underlined). The PCR products were digested with I-SceI

and PI-PspI (New England BioLabs) and cloned into the pMU2 vector [20], and the resulting
vectors were designated as pMU2-Eya3 and pMU2-Six1. In these pMU2 vectors, the genes are
regulated by a CMV promoter.
Oligonucleotide Sequences for the Cloning of Eya3 and Six1
Cloning of Eya3:
Forward primer: 5'-ATTACCCTGTTATCCCTAATGAAGAAGAGCAAGAC
CTACCAGAGC-3'
Reverse primer: 5'-ACCCATAATACCCATAATAGCTGTTTGCCATCAGAGGAAGTCAAGCTCTAAAGCC-3'
Cloning of Six1:
Forward primer: 5'-ATTACCCTGTTATCCCTAATTCGATGCTGCCGTCGT
TTGGTT-3'
Reverse primer: 5'-ACCCATAATACCCATAATAGCTGTTTGCCAGGAACCCAAGTCCACCAAACTG-3'
pGL4.10-P(TSHβ-ΔMEF3), pGL4.10-P(TSHβ-mutMEF3), pGL4.10-P(TSHβ-mutD),
pGL4.10-P(TSHβ-ΔSo1), and pGL4.10-P(TSHβ-ΔSo2)
We used pGL4.10-P(TSHβ-0.1k) as a template. The inverse PCR products were treated with theMighty Cloning kit (TaKaRa) for 5'-end phosphorylation, and subsequently self-ligated. The
constructs were designated as pGL4.10-P(TSHβ-ΔMEF3), pGL4.10-P(TSHβ-mutMEF3),
pGL4.10-P(TSHβ-mutD), pGL4.10-P(TSHβ-ΔSo1), and pGL4.10-P(TSHβ-ΔSo2).
Oligonucleotide Sequences for Constructing Mutated or Deleted TSHβ Promoters(Hokkaido System Science)
Inverse PCR of pGL4.10-P(TSHβ-ΔMEF3):
Forward primer: 5'-AGGGTATAAAATGAACGGAGAGTGG-3'
Reverse primer: 5'-AATTCCCCTCTGATCTTCTTG-3'
Inverse PCR of pGL4.10-P(TSHβ-mutMEF3):
Forward primer: 5'-CATCTAGAGGGTATAAAATGAACGGAGAGTGG-3'
Reverse primer: 5'-AATTCCCCTCTGATCTTCTTG-3'
Inverse PCR of pGL4.10-P(TSHβ-mutD):
Forward primer: 5'-CACCCGGCACAAGAAGATCAGAGGGGAATTATCCT
Reverse primer: 5'-TTGCATTCGAATTGCTGCTTTCTTATCTG-3'

G-3'

Inverse PCR of pGL4.10-P(TSHβ-ΔSo1):
Forward primer: 5'-GCAATTATATAAACAAGAAGATCAGAGG-3'
Reverse primer: 5'-TGCTTTCTTATCTGAAAAGCATCTATCC-3'
Inverse PCR of pGL4.10-P(TSHβ-ΔSo2):
Forward primer: 5'-TTCGAATGCAATTATATAAACAAGAAGATC-3'
Reverse primer: 5'-TTATCTGAAAAGCATCTATCCTCGAG-3'
Author Contributions
H.R.U. and Y.S. designed the overall project. K.M., M.N., K.H., and Y.S. collected samples.
K.D.U. performed the GeneChip and qPCR experiments. K.M., M.N., and K.T. performed the ISH
experiments. T.K. designed and performed the computational analyses, and constructed theintegrated database. M.U. performed the promoter assay. K.M., M.U., T.K., Y.S., and H.R.U.
wrote the manuscript. All authors discussed the results and commented on the manuscript.

Supplemental References
1.
Yoo, S.H., Yamazaki, S., Lowrey, P.L., Shimomura, K., Ko, C.H., Buhr, E.D., Siepka,
S.M., Hong, H.K., Oh, W.J., Yoo, O.J., et al. (2004). PERIOD2::LUCIFERASE real-time
reporting of circadian dynamics reveals persistent circadian oscillations in mouse
peripheral tissues. Proc Natl Acad Sci U S A 101, 5339-5346.
2.
Guilding, C., Hughes, A.T., Brown, T.M., Namvar, S., and Piggins, H.D. (2009). A riotof rhythms: neuronal and glial circadian oscillators in the mediobasal hypothalamus. Mol
Brain 2, 28.
3.
Dupre, S.M., Miedzinska, K., Duval, C.V., Yu, L., Goodman, R.L., Lincoln, G.A., Davis,
J.R., McNeilly, A.S., Burt, D.D., and Loudon, A.S. (2010). Identification of Eya3 and
4.

5.

6.

7.

8.
9.

10.

11.

TAC1 as long-day signals in the sheep pituitary. Curr Biol 20, 829-835.
Alon, U. (2007). An introduction to systems biology: design principles of biologicalcircuits. In Network Motifs in Developmental, Signal Transduction, and Neuronal
Networks. (London: Chapman & Hall/CRC), pp. 97-129.
Drolet, D.W., Scully, K.M., Simmons, D.M., Wegner, M., Chu, K.T., Swanson, L.W.,
and Rosenfeld, M.G. (1991). TEF, a transcription factor expressed specifically in theanterior pituitary during embryogenesis, defines a new class of leucine zipper proteins.
Genes Dev 5, 1739-1753.
Mueller, C.R., Maire, P., and Schibler, U. (1990). DBP, a liver-enriched transcriptional
activator, is expressed late in ontogeny and its tissue specificity is determined
posttranscriptionally. Cell 61, 279-291.
Inaba, T., Roberts, W.M., Shapiro, L.H., Jolly, K.W., Raimondi, S.C., Smith, S.D., andLook, A.T. (1992). Fusion of the leucine zipper gene HLF to the E2A gene in humanacute B-lineage leukemia. Science 257, 531-534.
Cowell, I.G., Skinner, A., and Hurst, H.C. (1992). Transcriptional repression by a novel
member of the bZIP family of transcription factors. Mol Cell Biol 12, 3070-3077.
Zhang, W., Zhang, J., Kornuc, M., Kwan, K., Frank, R., and Nimer, S.D. (1995).
Molecular cloning and characterization of NF-IL3A, a transcriptional activator of thehuman interleukin-3 promoter. Mol Cell Biol 15, 6055-6063.
Kel, O.V., Romaschenko, A.G., Kel, A.E., Wingender, E., and Kolchanov, N.A. (1995).
A compilation of composite regulatory elements affecting gene transcription invertebrates. Nucleic Acids Res 23, 4097-4103.
Ono, H., Hoshino, Y., Yasuo, S., Watanabe, M., Nakane, Y., Murai, A., Ebihara, S.,
Korf, H.W., and Yoshimura, T. (2008). Involvement of thyrotropin in photoperiodic
signal transduction in mice. Proc Natl Acad Sci U S A 105, 18238-18242.

12.

13.

14.
15.
16.

17.

18.

19.

20.

Yoshimura, T., Nishio, M., Goto, M., and Ebihara, S. (1994). Differences in circadianphotosensitivity between retinally degenerate CBA/J mice (rd/rd) and normal CBA/N
mice (+/+). J Biol Rhythms 9, 51-60.
Yoshimura, T., and Ebihara, S. (1996). Spectral sensitivity of photoreceptors mediatingphase-shifts of circadian rhythms in retinally degenerate CBA/J (rd/rd) and normal
CBA/N (+/+)mice. J Comp Physiol A 178, 797-802.
Chatfield, C. (1996). The analysis of time series: An introduction, (London: Chapman &Hall/CRC).
Storey, J.D., and Tibshirani, R. (2003). Statistical significance for genomewide studies.
Proc Natl Acad Sci U S A 100, 9440-9445.
Ueda, H.R., Chen, W., Minami, Y., Honma, S., Honma, K., Iino, M., and Hashimoto, S.
(2004). Molecular-timetable methods for detection of body time and rhythm disorders
from single-time-point genome-wide expression profiles. Proc Natl Acad Sci U S A 101,
11227-11232.
Minami, Y., Kasukawa, T., Kakazu, Y., Iigo, M., Sugimoto, M., Ikeda, S., Yasui, A., vander Horst, G.T., Soga, T., and Ueda, H.R. (2009). Measurement of internal body time byblood metabolomics. Proc Natl Acad Sci U S A 106, 9890-9895.
Shigeyoshi, Y., Taguchi, K., Yamamoto, S., Takekida, S., Yan, L., Tei, H., Moriya, T.,
Shibata, S., Loros, J.J., Dunlap, J.C., et al. (1997). Light-induced resetting of a
mammalian circadian clock is associated with rapid induction of the mPer1 transcript.
Cell 91, 1043-1053.
Kumaki, Y., Ukai-Tadenuma, M., Uno, K.D., Nishio, J., Masumoto, K.H., Nagano, M.,
Komori, T., Shigeyoshi, Y., Hogenesch, J.B., and Ueda, H.R. (2008). Analysis andsynthesis of high-amplitude Cis-elements in the mammalian circadian clock. Proc Natl
Acad Sci U S A 105, 14946-14951.
Ukai, H., Kobayashi, T.J., Nagano, M., Masumoto, K.H., Sujino, M., Kondo, T., Yagita,
K., Shigeyoshi, Y., and Ueda, H.R. (2007). Melanopsin-dependent photo-perturbation
reveals desynchronization underlying the singularity of mammalian circadian clocks. Nat
Cell Biol 9, 1327-1334.