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.

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Supplemental Figure 1. Masumoto et al.

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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.

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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.

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