supplemental online material - genes & developmentgenesdev.cshlp.org › content › suppl ›...
TRANSCRIPT
Supplemental Material
Items in Supplemental Materials and their relationship to main text and main figures
Figure S1 relates to Figure 1 and shows the phase relationship of different circadian
transcripts.
Figure S2 relates to the Result section Low-amplitude temperature fluctuations can
synchronize circadian gene expression over a wide temperature range and shows that within
the examined range of temperatures (30°C and 40°C) circadian clocks can be phase-entrained
to low amplitude temperature oscillations.
Figure S3 relates to Figure 5 and shows the phase relationship between the bioluminescence
cycles produced by different reporter genes in cells exposed to T-cycles with different period
lengths.
Figure S4 relates to Figures 5 and S3 and shows the behavior of luminescence cycles
entrained to a 12-hour T-cycle after release into a constant temperature
Figure S5 relates to Figure 5 and shows the phase relationship of various rhythmically
expressed transcripts in cells entrained to a very short T-cycle of only 10 hours.
Figure S6 relates to Figure 6 and shows the downregulation of HSF1 and HFS2 expression
by RNA interference using shRNAs.
Table 1 lists the DNA primers and Taqman probes used in quantitative RT-PCR experiments
2
Legends to Supplemental Figures
Figure S1. The expression of various mRNAs in NIH3T3 cells exposed to simulated
body temperature rhythms.
(A) Real-time RT-qPCR (TaqMan) was used to quantify mRNAs in whole cell RNAs
prepared from NIH3T3 cells at four-hour intervals during the sixth day of temperature
entrainment (orange) or during the sixth day at constant temperature (blue). The primer- and
probe-sequences used for measuring Bmal1, Rev-erbα, Per2, Per3, Cry1 mRNAs and Hsp90
pre-mRNA levels are given in Supplemental Table 1. Two different biological samples per
time point were analyzed. For graphical purposes, the same values are plotted twice.
Bioluminescence profiles of NIH3T3/Bmal1-luc cells subjected to the same temperature
conditions (B) and corresponding temperature recordings (C) are shown for comparison.
Figure S2. Temperature cycles can synchronize circadian clocks within a broad
temperature range.
NIH3T3/Bmal1-luc cells whose circadian oscillators were transiently synchronized by a
serum shock are efficiently re-synchronized by temperature cycles with different magnitudes
but with the same relative amplitudes. Note the different phase relationships between the
peaks of Bmal1-luciferase expression and the peak of temperature (blue curves), which likely
reflects an over-compensation of period length at reduced temperatures. Indeed, in cells
exposed to the indicated constant temperatures, free-running periods shortened when the
incubation temperatures were lowered (grey curves). Thus, the shorter period length at lower
temperatures elicits phase advances under phase-entrained conditions. All bioluminescence
data were filtered by moving average transformation.
Figure S3. The phase relationship of circadian gene expression in cells exposed to T-
cycles of different lengths.
PER2::luciferase immortalized tail fibroblasts (turquoise), NIH3T3/DBP-luciferase
fibroblasts (pink), NIH3T3 fibroblasts transfected with a Bmal1-luciferase (blue), or a CMV-
luciferase reporter (grey, used as a non-circadian control) were exposed to temperature T-
cycles (35.5-38.5°C) with the period lengths indicated above the panels. Bioluminescence
data were filtered by moving average transformation on 24h intervals. For cells subjected to
temperature cycles of period lengths shorter than 24h, an enlarged panel on the right shows
3
the 60 last hours of monitoring, whose bioluminescence data are filtered by moving average
transformation on 6, 10, 14 and 18h intervals respectively, allowing a better comparison of
the phases of each reporter. Note that under normal conditions Per2 and Dbp expression are
nearly antiphasic to Bmal1 expression.
Figure S4. Circadian oscillators of NIH3T3 cells entrained to 12-hour temperature T-
cycles revert to approximately 24-hour low amplitude cycles after release into constant
temperature.
(A). Left panel. NIH 3T3 cells transiently transfected with a pBmal1-luciferase (pBmal1-luc)
reporter plasmid or stably transfected with a Dbp-luciferase (Dbp-luc) reporter gene were
incubated with fresh medium and then immediately entrained to square-wave T-cycles of 12
hours (6 h 38°C/6h 34°C) during the time period indicated on the x-axis and then released to
a constant temperature of 34°C. Note that the bioluminescence cycles generated by the two
transgenes are almost antiphasic under both entrained and free-running conditions. Right
panel. Same as in left panel, but the T-cycles were initiated after a pre-run of six hours at
37°C after the medium change. (B) Same data as in (A), but shown individually for pBMAL1
and Dbp-luc, to illustrate the phase differences for the same reporters caused by the different
entrainment protocols (i.e. with or without a 6-hour pre-run). Note that the first valleys and
peaks at constant temperature are observed after 12 to 14 hours, before the oscillators resume
an approximately 24-hour cycle.
Figure S5. The expression of different genes in NIH3T3 cells exposed to temperature
square cycles of 10 hours.
(A) Programmed (blue) and measured (yellow and turquoise) temperature square cycles of
10h period (5h at 34°C, 5h at 39°C) used to entrain NIH3T3 fibroblasts. RNAs were prepared
from cells at two-hour intervals during the tenth and eleventh cycles (vertical grey arrows).
(B) Real-time RT-qPCR (SYBR Green) was used to quantify pre-mRNA of Hsp105, and
mRNAs of Per2, Bmal1, Rev-erbα and Dbp in whole cell RNAs prepared from these cells.
The primer-sequences used are given in Supplemental Table 1. Four biological samples per
time point were analyzed (each curve represents a pool of two biological samples).
4
Figure S5. The accumulation of HSF1 and HSF2 mRNAs and proteins is down-
regulated in the presence of the corresponding shRNAs.
(A) Western-blots of total cellular proteins with antibodies against HSF1 and U2AF65
(loading control). Note the slower migration of HSF1 proteins upon a 2-hour heat shock at
42°C. The more slowly migrating proteins probably represent the hyperphosphorylated
(activated) forms of HSF1. Protein extracts were prepared form primary tail tip fibroblasts
(from wild-type and Hsf1 knockout mice) or from NIH3T3 fibroblasts transfected with
different shRNA vectors (pSUPER-empty, pshHSF1, pshHSF2 or pshHSF1+2) or
untransfected NIH3T3 fibroblasts (NT). (B) Western-blot of total cellular proteins with
antibodies against HSF2 and U2AF65 (loading control). (C) Real-time RT-PCR (TaqMan)
assays performed on mRNAs prepared from same cells that were used for the data presented
in (A) and (B), after a 2-hour heat shock at 42°C. The cells transfected with shRNA plasmids
also expressed green fluorescent protein (GFP) from a PGK promoter and could thus be
FACS-sorted before RNA and protein extracts were prepared. This ensured that only shRNA
expressing cells were included for the Western-blot and RT-qPCR experiments. The primer-
and probe-sequences used for measuring Hsf1 and Hsf2 mRNAs levels are given in
Supplemental Table 1.
5
Supplemental Table 1. DNA sequences of primers and probes used for the real-time RT-
PCR experiments. TaqMan
mBmal1-F 5′-CCAAGAAAGTATGGACACAGACAAA-3′
mBmal1-R 5′-GCATTCTTGATCCTTCCTTGGT -3′
mBmal1 probe 5′-FAM-TGACCCTCATGGAAGGTTAGAATATGCAGAAC-TAMRA-3′
mRev-erbα-F 5`-TGAATGGCATGGTGCTACTG-3`
mRev-erbα-R 5`-CAGGCGTGCACTCCATAGT-3`
mRev-erbα probe 5`- FAM-GTAAGGTGTGTGGGGACGTG-TAMRA-3`
mPer2-F 5`-GTAGCGCCGCTGCCG-3`
mPer2-R 5`-GCGGTCACGTTTTCCACTATG-3`
mPer2 probe 5`-FAM-CGCGTCGCCCTCCGCTGT-TAMRA-3`
mPer3-F 5'-CCAGGTCACGTGGGACACA-3'
mPer3-R 5'-TCAATGACTGGCGTTTCAGAGT-3'
mPer3 probe 5'-FAM-AGCCGTGCGTTACCTCACCACAGTC-TAMRA-3'
mCry1-F 5'-CTGGCGTGGAAGTCATCGT-3'
mCry1-R 5'-CTGTCCGCCATTGAGTTCTATG-3'
mCry1 probe 5'-FAM-CGCATTTCACATACACTGTATGACCTGGACA-TAMRA-3'
pre-mHsp90-F 5'-CAGGACCTGGGCTAGGAAAT-3'
pre-mHsp90-R 5'-CAGCTAACCTTCCCCACAAA-3'
pre-mHsp90 probe 5'-FAM-AAGCTTTGGAGACCTTTGCA-TAMRA-3'
mHSF1-F 5'-GGACACAGACGCGCTCAT-3'
mHSF1-R 5'-CCTGGTCAAACACGTGGAAG-3'
mHSF1 probe 5'-FAM-GAGCCCGAGTGGGAACAG-TAMRA-3'
mHSF2-F 5'-GGCGAGCTTTGTGAGACAACT-3'
mHSF2-R 5'-CCATCTCTTTCCTGTTTGATAATTCC-3'
mHSF2 probe 5'-FAM-TGTATGGCTTCCGAAAAGTAGTGCATATCGAA-TAMRA-3'
GAPDH-F 5`-CATGGCCTTCCGTGTTCCTA-3`
GAPDH-R 5`-CCTGCTTCACCACCTTCTTGA-3`
GAPDH probe 5`-FAM-CCGCCTGGAGAAACCTGCCAAGTATG-TAMRA-3`
pre-m45S-F 5`-ACACCCGAAATACCGATACG-3'
pre-m45S-R 5'-TAGCTGCGTTCTTCATCGAC-3'
pre-m45S probe 5'-FAM-TCTTAGCGGTGGATCACTCG-TAMRA-3'
SYBR Green
mRps9-F 5′-GACCAGGAGCTAAAGTTGATTGGA-3′
mRps9-R 5′-TCTTGGCCAGGGTAAACTTGA-3′
mCyclophillin-F 5′-GGAGATGGCACAGGAGGAA-3′
mCyclophillin-R 5′-GCCCGTAGTGCTTCAGCTT-3′
pre-mHsp105-F 5′-GTGTCAGGGTCCTGTGGAGT-3′
pre-mHsp105-R 5′-GACCCCACCTCCTCAGTGTA-3′
mBmal1-F 5′-CCAAGAAAGTATGGACACAGACAAA-3′
mBmal1-R 5′-GCATTCTTGATCCTTCCTTGGT-3′
6
mPer2-F 5′-ATGCTCGCCATCCACAAGA-3′
mPer2-R 5′-GCGGAATCGAATGGGAGAAT-3′
mDbp-F 5′-TGGCCCGAGTCTTTTTGC-3′
mDbp-R 5′-GCGTCCAGGTCCACGTATTC-3′
mRev-erbα-F 5′-TGAATGGCATGGTGCTACTG-3′
mRev-erbα-R 5′-CAGGCGTGCACTCCATAGT-3′
Saini_FigS1
A
B C
biol
umin
esce
nce
(filte
red
valu
es)
tem
pera
ture
mR
NA
leve
l
mR
NA
leve
l
126 130 134 138 142 146 126 130 134 138 142 146 126 time (hours)
mRev-erbα
mR
NA
leve
l
126 130 134 138 142 146 126 130 134 138 142 146 126 time (hours)
NIH3T3/Bmal1-luc cells
mCry1
mR
NA
leve
l126 130 134 138 142 146 126 130 134 138 142 146 126
time (hours)
mPer3
126 130 134 138 142 146 126 130 134 138 142 146 126 time (hours)
mPer2
126 130 134 138 142 146 126 130 134 138 142 146 126
mBmal1
mR
NA
leve
l
time (hours)
0.2
0
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
126 130 134 138 142 146 126 130 134 138 142 146 126 time (hours)
mpreHsp90
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
mR
NA
leve
l
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
00.10.20.30.40.50.60.70.80.9
1
time (hours)
0.6
0.8
1
1.2
1.4
126 130 134 138 142 146 150 154 158 162 166 170 174
constant 37°C temperature cycles (35.5-38.5°C)
time (hours) 126 130 134 138 142 146 150 154 158 162 166 170 174
constant 37°C temperature cycles (35.5-38.5°C)
34
35
36
37
38
39
40
30
35
40
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 12 24 36 48 60 72 84 96 108
constant 38.9°C temperature cycles 37.3-40.5°C
Saini_FigS2bi
olum
ines
cenc
e (fi
ltere
d va
lues
)
tem
pera
ture
30
35
40
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 12 24 36 48 60 72 84 96 108
temperature cycles 35.5-38.5°C
tem
pera
ture
30
35
40
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 12 24 36 48 60 72 84 96 108
temperature cycles 30-32.5°C
biol
umin
esce
nce
(filte
red
valu
es)
tem
pera
ture
30
35
40
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 12 24 36 48 60
time (hours)
72 84 96 108
temperature cycles 31.8-34.5°C
30
35
40
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 12 24 36 48 60 72 84 96 108
constant 33.15°C
constant 31.25°C
biol
umin
esce
nce
(filte
red
valu
es)
constant 37°C
temperature cycles 33.7-36.5°C constant 35.1°C
time (hours)
72 96 120biol
umin
esce
nce
(filte
red
valu
es)
biol
umin
esce
nce
(filte
red
valu
es)
biol
umin
esce
nce
(filte
red
valu
es)
Saini_FigS3
time (hours) time (hours) time (hours)
Tem
pera
ture
Tem
pera
ture
Tem
pera
ture
35
37
39
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 24 48 72 96 120
pCMV-luc pBmal1-luc Per2-luc
DBP-luc
6h period 10h period
14h period
24h period 30h period 34h period 40h period
18h period
temperature
35
37
39
0.4
0.6
0.8
1
1.2
1.4
72 96 12035
37
39
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 24 48 72 96 12035
72 96 12072 96 12035
37
39
0.4
0.6
0.8
1
1.2
1.4
35
37
39
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 24 48 72 96 12035
37
39
0.3
0.5
0.7
0.9
1.1
1.3
1.5
35
37
39
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 24 48 72 96 120
37
390.4
0.6
0.8
1
1.2
1.4
1.6
35
37
39
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0 24 48 72 96 120 0 24 48 72 96 120 0 24 48 72 96 120
time (hours)
0 24 48 72 96 12035
37
39
0
0.5
1
1.5
2
35
37
39
0
0.5
1
1.5
2
35
37
39
0
0.5
1
1.5
2
0 12 24 36 48 60 72hours
84 96 108 132120 144 156
0 12 24 36 48 60 72hours
84 96 108 132120 144 156
0.8
1.0
1.2
1.4
0 12 24 36 48 60 72hours
84 96 108 132120 144 156
0.8
1.0
1.2
1.4
biol
umin
esce
nce
(filte
red
valu
es)
0.8
1.0
1.2
1.4
biol
umin
esce
nce
(filte
red
valu
es)
biol
umin
esce
nce
(filte
red
valu
es)
Saini_FigS4
A
B
34
38
34
38
0 12 24 36 48 60 72hours
84 96 108 132120 144 156
0.8
1.0
1.2
1.4
biol
umin
esce
nce
(filte
red
valu
es)
34
38
34
38
tem
pera
ture
tem
pera
ture
tem
pera
ture
34
38
tem
pera
ture
34
38
pBmal1-luc, 0 hours prerunDBP-luc, 0 hours prerun
pBmal1-luc, 0 hours prerunpBmal1-luc, 6 hours prerun
DBP-luc, 0 hours prerunDBP-luc, 6 hours prerun
pBmal1-luc, 6 hours prerunDBP-luc, 6 hours prerun
Saini_FigS5
A
B
mR
NA
leve
lm
RN
A le
vel
time (hours)
time (hours) time (hours)
tem
pera
ture
tem
pera
ture
tem
pera
ture
33353739
0
0.2
0.4
0.6
0.8
1
94 96 98 100 102 104 106 108 110
pool1 pool2 temperature
preHsp105
33353739
0.2
0.4
0.6
0.8
1
94 96 98 100 102 104 106 108 110
33353739
0
0.2
0.4
0.6
0.8
1
1.2
94 96 98 100 102 104 106 108 110
33353739
0
0.2
0.4
0.6
0.8
1
94 96 98 100 102 104 106 108 11033353739
0.4
0.6
0.8
1
94 96 98 100 102 104 106 108 110
Per2 Bmal1
Rev-erbα Dbp
30
32
34
36
38
40
42
44
0 24 48 72 96
temperature program liquid probe 1 liquid probe 2
Saini_FigS6
Temperature °C WT
U2AF65
115 37 42
82
64
48
HSF1 (~85 and 95 KDa)
Temperature °C
U2AF65
HSF2 (~70 KDa)
115 kDa
82
64
A
B
C
HSF1 KO
37 42
3T3 NT
37 42
emptyV
37 42
shHSF1
37 42
shHSF2
37 42
shHSF1+2
37 42
WT
37 42
HSF1 KO
37 42
3T3 NT
37 42
emptyV
37 42
shHSF1
37 42
shHSF2
37 42
shHSF1+2
37 42
0
0.2
0.4
0.6
0.8
1
1.2 1.2
WT
HSF1
KO
3T3
NT
pSUP
-em
pty
pSUP
-shH
SF1
pSUP
-shH
SF2
pSUP
-shH
SF1+
2
pSUP
-shH
SF1+
2
HSF2 probe
0
0.2
0.4
0.6
0.8
1
WT
HSF1
KO
3T3
NT
pSUP
-em
pty
pSUP
-shH
SF1
pSUP
-shH
SF2
HSF1probe