Per3. 0,0. 0,5. 1,0. 1,5. Bmal1KO. Bmal1WT. Bmal1KO. Bmal1WT relative mRNA expression. Ccnd2. Tgm3. H19. Ccna2 Ccnb1 Chek1 Rad9 Gadd45b Flg2. Lor.
SUPPLEMENTARY INFORMATION
doi:10.1038/nature10649
Supplementary Fig. 1 P19 Telogen
P22 Anagen onset
anti-GFP
Bu
Bu P25 Anagen
Bu
P31 Anagen
Bu
Supplementary Figure 1: Bulge stem cells are heterogenous in their clock. Immunohistochemistry of venus-GFP in Per1-venus mouse backskin at different hair cycle stages shows venusbright (arrow) and venusdim (arrowhead) cells in the bulge (Bu, bulge). Scale bar, 100 μm.
w w w . n a t u r e . c o m / NATURE | 1
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 2 10 4
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Supplementary Figure 2: Per1-venus expression is homogeneous in the basal epidermis during anagen. a,b, Quantification of venusbright and venusdim cells in the α6 integrinbright/CD34- epidermal population of Per1-venus mice at different stages of the hair cycle (P19, P22, P25, P28 and P31) by FACS showed no changes in the percentage of venusbright and venusdim cells in the basal epidermis (mean ± s.e.m.; n ≥ 6).
2 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 3 a
Telogen P19
Anagen onset P22
Anagen progression P27
Anagen P31
SG SG
Bu
SG
SG SG
Ist Bu
Ist
Bu
Bu
SG SG SG
Bu
SG
SG Ist Bu
Ist
Bu
Bu
Anti-GFP direct GFP fluorescence
b
UI
Ist
Ist SG
Bu
HG
Anti-GFP K14
Supplementary Figure 3: Dynamic changes of Clock gene expression during the hair cycle in Per1-GFP reporter mice. a, Whole mount immunostaining of mouse tailskin from Per1-GFP mice at different stages of the hair cycle (from P19 to P31) showed a steady increase of GFPbright cells in the bulge compartment during anagen. Upper panel, anti-GFP staining; lower panel, direct GFP fluorescence. Scale bar, 100 μm. b, Immunostaining for GFP (red) and Keratin 14 (K14; green) revealed strong GFP expression in the isthmus region, which contains epSCs that feed into the epidermis and sebaceous glands. Scale bar, 50 μm. Bu, bulge; SG, sebaceous gland; Ist, isthmus; UI, upper isthmus; and HG, hair germ.
w w w . n a t u r e . c o m / NATURE | 3
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 4 a a6-integrinbright/CD34+ LD
DD
10 4
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mean fluorescence intensity
a6-integrinbright/CD34-
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c Bulge mean fluorescence intensity
20
ZT 13
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***
p < 0.001
15
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3
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Supplementary Figure 4: GFP/venus expression changes in a circadian manner. a, b, Comparison of venus mean fluorescence intensity in α6-integrinbright/CD34+ bulge (a) and α6-integrinbright /CD34- epidermal cells (b) from P27 Per1-venus mice kept under LD and DD conditions (one representative FACS profile is shown at CT6 and CT18 from each group; the gate to separate venusbright from venusdim cells was placed in all profiles according to the time point of brightest venus fluorescence intensity at CT12). The graph in b shows quantification of α6-integrinbright/CD34- epidermal cells at 4 different time points by FACS (mean ± s.e.m.; n = 2). White and black bars represent subjective day and night. c, Timelapse confocal imaging of telogen backskin of Per1-venus mice reveals an increase in venusbright nuclei in the bulge during the subjective night (from ZT13 to ZT24; arrows) Scale bar, 25 μm. Graph shows quantification of mean fluorescence intensity of venusbright nuclei in the bulge over 24 hours (n = 5 nuclei; mean ± s.e.m.; *** p < 0.001 using Cosinor analysis) d, Whole mount immunostaining of mouse tailskin from Per1-GFP mice at Zeitgeber time ZT3 (3 hours of light) or ZT12 (12 hours of light) shows clear differences in GFP intensity in the basal epidermis (arrows and higher magnification) comparing morning (ZT3) to the evening (ZT12). Scale bars 100 μm. CT, circadian time; ZT, Zeitgeber time; LD, 12 h light/12 h dark condition; DD, 12 h dark/12 h dark condition. 4 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 5 a
Chromatin immunoprecipitation from tail epidermis 0,5
0,8
Clock
0,4
Bmal1
0,6
% of input
IgG
% of input
Clock
0,7
Bmal1
0,3
0,2
IgG
0,5 0,4 0,3 0,2
0,1
0,1
Wnt3
Lef1
Chromatin immunoprecipitation from sorted α6
bfright
Lhx2
Tcf4
/CD34+ bulge cells
Bmal1
2,0
IgG
% of input
c
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1,0
Dkk3
Sox9
KO
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WT 0,8 0,4 0,0
0h
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Sox9
Fzd2
Lhx2
Tcf4
Lef1
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Real time PCR from sorted α6bfright/CD34+ bulge cells
20 15 5
*
rel. mRNA levels
10
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Dbp
KO WT
60 40
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20
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3
KO WT
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Cdk4
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Smad7
4
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2,0
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Dab2
Smad3
KO WT
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ZT12
Tgfbr2
3 2 1 ZT0
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KO WT
Dbp Ex3
0h
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Itga6
1,6
Wnt10a
1,5 1,2
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0,9 0,6 0,3 0,0 1,2
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25 20
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rel. mRNA levels
rel. mRNA levels
KO WT
25
rel. mRNA levels
Sox9
30
WT
relative mRNA expression
Itga6
relative mRNA expression
Cdk4
rel. mRNA levels
d
Dbp
Dbp P
48h
1,0
0,0
Lefty
Dab2
1,6
1,2
0,5
Smad7
Real time PCR from cultured mouse keratinocytes
relative mRNA expression
2,5
Fzd2
relative mRNA expression
Notch2
relative mRNA expression
Cdk4
relative mRNA expression
b
0,0
Itga6
relative mRNA expression
0,0
* 0h
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Lef1
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WT
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Supplementary Figure 5: Bmal1 modulates the response of epidermal cells to activation and dormancy cues. a, ChIP from tail epidermis of Bmal1WT mice revealed binding of Bmal and Clock to E-box and E-box-like elements in the promoters of genes involved in adhesion, cell cycle, Notch, TGF-β and Wnt pathways. ChIP for Bmal1 and Clock in 2 regions of the circadian-regulated gene Dbp was used as positive (P, promoter) and negative control (Ex3, exon 3). (n = 3, mean ± s.e.m.). b, ChIP from FACS-purified bulge cells of Bmal1WT mice shows strong binding of Bmal to the promoters of genes highly expressed in the bulge SC compartment (n = 2; mean ± s.e.m.). Graphs in a and b show the percentage of immunoprecipitated DNA over the input. c, The differential expression of adhesion, Wnt and TGF-β pathway genes in primary keratinocytes isolated from newborn Bmal1WT (WT) and Bmal1KO mice (KO) was not statistically significant. Fold-change values are displayed as relative expression to Bmal1WT cells at 0h after normalization to Pumilio1 (Pum1) (n = 3). Results are shown as mean ± s.e.m., * p < 0.05 (two-way ANOVA with Bonferroni post-test). d, Real time PCR of adhesion, cell cycle, Wnt and TGF-β pathway genes in FACS purified bulge cells of 9-month-old Bmal1WT (WT) and Bmal1KO mice (KO) at ZT0 and ZT12. Expression levels are shown as relative mRNA levels after normalization to Pumilio1 (Pum1) (n = 2). Results are shown as mean ± s.e.m., * p < 0.05 (two-tailed Student’s t-test). w w w . n a t u r e . c o m / NATURE | 5
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 6 a
b 100 bp
1
2
1
3
2
Bmal1 1 Bmal1 Wt/Wt K14Cre
1 Bmal1 LoxP/LoxP 2 Bmal1 Wt/Wt K14Cre 3 Bmal1 LoxP/LoxP14Cre
(Bmal1WT)
2 Bmal1 LoxP/LoxP K14Cre (Bmal1KO)
Tubulin
c
Bmal1WT/Per1-venus 10 4
ZT12
50.6
47.8
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Supplementary Figure 6: Conditional deletion of Bmal1 in the epidermis. a, Immunostaining for Keratin 14 (green) to mark Cre-expressing cells in the epidermis. Scale bar, 50 μm. b, Efficiency of LoxP recombination in the epidermis of K14Cre/Bmal1LoxP/LoxP (Bmal1KO) mice was assessed by PCR from genomic DNA of epidermal cells (left panel; lane 1, conditional allele; lane 2, Wt allele; lane 3, recombined allele) and by western blot for Bmal1 in keratinocytes isolated from Bmal1WT or Bmal1KO mice (right panel; arrow). c, Conditional deletion of Bmal1 effects the expression of venus in the epidermis of Bmal1KO/Per1-venus mice. Quantification of venusbright and venusdim cells in the α6 integrinbright/CD34+ bulge and α6 integrinbright/CD34epidermal population by FACS revealed a loss of venus expression in Bmal1KO/Per1-venus mice at ZT0 and ZT12 as compared to Bmal1WT/Per1-venus mice at ZT12 (numbers show percentages; n ≥ 3).
6 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 7
luciferase activity (Fold increase over FOP-Flash)
TOP-Flash luciferase assay 3,0
Bmal1KO Bmal1WT
2,5
2,0
1,5
1,0
0,5
0,0
- BIO
+ BIO
Supplementary Figure 7: Primary mouse keratinocytes did not respond to activation through canonical Wnt signaling. Primary mouse keratinocytes of Bmal1WT and Bmal1KO mice transfected with TOP-Flash and FOP-Flash reporter plasmids were incubated for 24 hours in the absence or the presence of the canonical Wnt small molecular activator BIO (mean ± s.e.m.; n = 2).
w w w . n a t u r e . c o m / NATURE | 7
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 8 a
b Sox9
Lef1 WT
Wildtype
#1
#2
Per1/2dKO #1 #2
#2
Per1/2dKO #1 #2
Sox9
Tubulin
WT #1 Sox9
Lef1
Lef1
4
*
3 2
WT
**
* 1 0
0h
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Smad3
1,5
relative mRNA expression
Per1/2dKO
Per1/2dKO
1,2
WT
0,9
0,6
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Lef1
1,2 1,0
Per1/2dKO
0,8
WT
0,6
*
0,4 0,2 0,0
0h
12h
24h
36h
48h
relative mRNA expression
Dkk3
5
relative mRNA expression
relative mRNA expression
c
relative mRNA expression
Per1/2dKO
Tubulin
Itga6
1,5 1,2
Per1/2dKO
*** ***
0,9
***
***
WT
0,6 0,3 0,0
0h
12h
24h
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Tgfbr2
2,0
Per1/2dKO 1,5
WT
1,0
***
*** **
0,5
0h
12h
24h
36h
48h
Supplementary Figure 8: Per1/2dKO epidermal cells show enhanced expression of Bmal1 targets. a, Backskin of Per1/2dKO mice and wildtype control animals was stained for Lef1 and Sox9 (n ≥ 6). Scale bars, 25 μm. b, Protein extracts from tail epidermis of 6-week-old Per1/2dKO mice and wildtype control animals (WT) were analysed by western blot for the expression of Lef1 and Sox9. Lanes represent extracts from two WT and two KO animals (#1, #2). Tubulin expression was analysed as loading control. c, Real time PCR of adhesion, Wnt and TGF-β pathway genes in primary keratinocytes of Per1/2dKO and wildtype control animals (WT). Fold-change values are displayed as relative expression (mean ± s.e.m.) to WT cells at 0h after normalization to Pumilio1 (Pum1) (n = 2). Results are shown as mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 (two-way ANOVA with Bonferroni post-test).
8 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 9 a
b
Depilation Bmal1KO
Bmal1WT
Bmal1KO
LRCs
30 Number of Ki67+ cells / bulb
Bmal1WT
TPA treatment
10 weeks HF length (arbitrary units)
Bmal1WT
150 100
50
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7 days post-depilation
Bmal1KO Bmal1WT
20 15 10 5 0
Ki67
15 Number of Ki67+ cells / bulge
*
200
**
25
* Bmal1KO
10
Bmal1WT
5
0
c
Bmal1KO
Bmal1WT
5 months
Supplementary Figure 9: Clock perturbation in vivo results in changes in the number of dormant bulge stem cells and premature tissue aging. a, Depilation-induced anagen entry shows a growth delay in Bmal1KO mice (n ≥ 5). b, Wholemount immunostaining for BrdU (LRCs) and the proliferation marker Ki67 of 10-week-old Bmal1WT and Bmal1KO after 1 week of TPA treatment (n = 5). c, Histological analysis of 5 month old Bmal1WT and Bmal1KO mice. Scale bars 100 μm. Results are shown as mean ± s.e.m., * p < 0.05, ** p < 0.01 (two-tailed Student’s t-test).
w w w . n a t u r e . c o m / NATURE | 9
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 10 a
b
Bmal1WT
Wildtype Per1/2dKO
α6-integrinbright/CD34-
Bmal1KO
α6-integrinbright/CD34-
Supplementary Figure 10: Clockhigh epidermal keratinocytes have an increased clonogenic potential. a, b, Clonogenic assay of FACS-purified α6-integrinbright/ CD34- epidermal keratinocytes from the backskin of (a) Bmal1WT and Bmal1KO mice or (b) Per1/2dKO mice and wildtype control animals (1x105 cells seeded on J2 3T3 feeder cells).
1 0 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 11
10
3
Bmal1WT
CD34
4.41
10 2
10
4
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10 1
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α6-integrin
Wildtype
10 4
NS
5
0 10 0
α6-integrin
b
6
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10 2
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Bmal1KO % α6-integrinbright/ CD34+
4
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10
CD34
a
10 3
10 4
5
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Per1/2dKO WT
4 3 2 1 0
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10 3
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Supplementary Figure 11: Percentage of bulge stem cells is not altered in Bmal1KO and Per1/2dKO mice. a, b, FACS analysis of α6-integrinbright/CD34+ bulge cells of (a) Bmal1WT and Bmal1KO mice or (b) Per1/2dKO mice and wildtype (WT) control animals show no differences in the percentage of bulge stem cells in 6-week-old animals (n = 4). Results are shown as mean ± s.e.m.; statistical significance was assessed by two-tailed Student’s t-test. NS, not significant.
w w w . n a t u r e . c o m / NATURE | 1 1
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 12 Bmal1WT
Bmal1KO
Bmal1WT
Bmal1KO
Wildtype
Per1/2dKO
Ki67
Wildtype
Per1/2dKO
Ki67
Supplementary Figure 12: Bmal1KO and Per1/2dKO mice show opposite phenotypes with regard to epidermal proliferation. Whole mount immunostaining for the proliferation marker Ki67 shows lower expression of Ki67 in the epidermis of Bmal1KO mice compared to Bmal1WT mice, and enhanced expression in the epidermis of Per1/2dKO mice with regard to their wildtype controls. Scale bars, 100 μm. (Upper panel: unspecific staining in sebaceous glands was Ki67 antibody batch dependent).
1 2 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Fig. 13 Bmal1WT
p16
Bmal1KO
Bmal1KO
p19
Bmal1WT
Bmal1KO
Tunnel
Bmal1WT
Supplementary Figure 13: Analysis of aged Bmal1KO mice for the expression of p16, p19 and apoptosis. Immunohistochemistry for p16 in 10-12-month-old Bmal1WT and Bmal1KO mice revealed enhanced expression of p16 in the epidermis and hair follicles of Bmal1KO mice. Cells positively stained for p19 (black arrows) were only occasionally observed in Bmal1WT and Bmal1KO mice in similar numbers. Similarly, apoptotic cells (Tunnel; green; white arrows) were only infrequently detected and located mainly to terminal differentiated areas of the hair follicle and the interfollicular epidermis. Scale bars, 50 μm.
w w w . n a t u r e . c o m / NATURE | 1 3
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 14 a
relative mRNA expression
1,5
***
1,0
***
*
Bmal1KO Bmal1WT
0,5
0,0 Nr1d1
Dbp Bhlhe41 Per1
Per3
b GO of genes down-regulated in Bmal1KO mice
GO of genes up-regulated in Bmal1KO mice homeostatic process immune system develpment
G-protein coupled receptor signaling
hemopoiesis
sensory perception/ cognition
fatty acid metabolic process
response to organic substance
cell activation
mitosis
epidermis development
chromatin assembly
circadian rhythm
defense response to bacterium
G1/S transition of mitotic cell cycle 0
100
200 300 number of genes
400
500
0
10
15 20 25 number of genes
30
35
40
d 4,0
1,2
3,5
1,0
3,0
*
Bmal1KO
*
Bmal1WT
2,5 2,0 1,5
*
*
1,0 0,5 0,0
Ccnd2 Ccna2 Ccnb1 Chek1 Rad9 Gadd45b Flg2
Lor
Tgm3
H19
relative pri-miR expression
relative mRNA expression
c
5
*
** Bmal1KO
0,8
Bmal1WT
0,6 0,4 0,2 0,0 miR23b miR27b miR24-1
Supplementary Figure 14: Validation of microarray data comparing aged BMAL1WT and BMAL1KO mice. a, c, d, Validation of microarray data in FACS isolated α6 integrinbright/CD34- epidermal cells from Bmal1KO and Bmal1WT mice by real time PCR (n = 2-3, for each group) shows differential expression of (a) several core circadian genes, (c) genes implicated in cell cycle, DNA damage and terminal differentiation and (d) the abundance of the pri-miRNAs 23b, 27b and 24-1. Fold-change values are displayed as relative expression to Bmal1WT cells after normalization to Pumilio1 (Pum1), or 18S-rRNA. Results are shown as mean ± s.e.m., * p < 0.05, ** p < 0.01, *** p < 0.001 (two-tailed Student’s t-test). b, Graphic depicts GO enriched biological processes of genes up- and down-regulated in Bmal1KO mice. (Fold change ≥1.4 or ≤-1.4; p-value ≤ 0.05).
1 4 | WWW. NATURE . COM / NATURE
SUPPLEMENTARY INFORMATION RESEARCH Supplementary Figure 15 early stage
WT WT
KO
KO
P25
b
late stage
P41
Bmal1WT/K5-SOS
Bmal1KO/K5-SOS
Involucrin
a
WT
Ki67/ K14
Tunnel
Loricrin
WT = Bmal1WT/K5-SOS KO = Bmal1KO/K5-SOS
Supplementary Figure 15: Loss of Bmal1 reduces the development of squamous tumors. a, Bmal1KO/K5-SOS mice developed fewer neoplastic lesions compared to Bmal1WT/K5-SOS littermates at early and late stages. b, Immunostaining of tumors of Bmal1WT/K5SOS and Bmal1KO/K5-SOS mice for terminal differentiation (Involucrin, Loricrin), apoptosis (Tunnel; arrows) and proliferation (Ki67). Scale bars, 100 μm.
w w w . n a t u r e . c o m / NATURE | 1 5
RESEARCH SUPPLEMENTARY INFORMATION Supplementary Fig. 16 a
Bmal1KO/K5-SOS
β-catenin
Bmal1WT/K5-SOS
b
β-catenin
positive control (anagen HF)
Supplementary Figure 16: Absence of nuclear β-catenin in K5-SOS induced tumors. a, Immunostaining of sections from squamous tumors for β-catenin (red) shows no nuclear localization of β-catenin in K5-SOS induced tumors. b, Anagen hair follicle stained for nuclear β-catenin (arrow) was used as positive control to verify the staining protocol. DAPI (blue), scale bar, 75 μm.
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SUPPLEMENTARY INFORMATION RESEARCH
10 3
250K
200K
200K
150K
150K
FSC-H 100K
SSC-A 100K
Pacific Blue-A 10 2
50K
50K
10 5
10 5
4
10 4
10
CD34-APC-A
SSC-A
DAPI
10 4
250K
FSC-H
10 5
Epcam-APC-Cy7-A
Supplementary Fig. 17
3
10 APC-Cy7-A
10 2
10 2 0
0
0
50K
100K
150K
FSC-A
200K
250K
0
0
0 0
50K
100K
150K
FSC-A
200K
250K
0
50K
100K
150K
FSC-A
200K
250K
APC-A 10 3
0
10
2
10
3
10
4
lin neg. PE-A
10
5
0
10
2
10
3
10
4
10
5
α6-integrin-FITC-A
Supplementary Figure 17: Gating strategy for FACS analysis of K5-SOS induced tumors. Viable cells were selected by DAPI exclusion (gate 1), contaminating fibroblasts were excluded by forward and side scatter (gate 2), doublets were discarded in gate 3, epithelial cells were selected in gate 4 by a combination of a lineage negative markers in PE (CD31, CD45 and CD140a) and a positive marker (Epcam APC-Cy7), and α6bright/CD34+ tumorinitiating cells were selected in gate 5.
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RESEARCH SUPPLEMENTARY INFORMATION
Supplementary Table 2 Gene
Chromosome
Distance from transcriptional start site
Location
Motif
Validated by ChIP
TGF-β pathway Tgfb1
7
- 642
Promoter
CACGTG
no
Tgfb2
1
- 50/ + 440
Promoter/Exon1
CACGTG /AACGTG
no/ no
Smad7
18
- 4839/ -177
Promoter/ Promoter
CACGTG/ CACGTG
yes/ no
Smad9
3
- 2797/ + 262
Promoter/ Intron 1
CACCTG/ CACGCG
no/ no
Smurf2
11
- 4687/ - 3945
Promoter/ Promoter
CACGTG/ CACCTG
yes/ no
Lefty
1
- 297
Promoter
CACGTG
Dkk1
19
- 413
Promoter
AACGTG
no
Dkk3
7
- 874
Promoter
CACGTG
yes
Wnt3
11
- 2940/ - 400
Promoter/ Promoter
CACGTG/ CACGTG
no/ yes
Wnt10a
1
- 2533/ - 1410
Promoter/ Promoter
CACGTG/ CACGTG
no/ no
Lef1
3
- 4917/ - 4468/ - 4098
Promoter/ Promoter/ Promoter
CACGTG/ CACGTG/ CACGTG
yes/ no/ no
- 3243/ - 2501/ - 490
Promoter/ Promoter/ Promoter
CACGTG/ CACGTG/ CACGTG
no/ no/ no
yes
Wnt pathway
Ctnnb1
9
- 2596
Promoter
Dab2
15
- 2842/ - 1247
Promoter/ Promoter
AACGTG
Fzd2
11
- 361
Promoter
AACGTG
yes
Tcf4
18
- 2914
Promoter
CACGTG
yes
Sox9
11
- 44
Promoter
AACGTG
yes
Lhx2
2
- 4668/ -2591/ - 2040
Promoter/ Promoter/ Promoter
Notch2
3
- 2687
Promoter
Hes1
16
- 1809/ - 1014
Promoter/ Promoter
CACGTG/ CACGTG
- 4760/ - 3064
Promoter/ Promoter
AACGTG/ AACGTG
no/ no
- 1689/ + 150
Promoter/ Exon1
CACGTG/ AACGTG
no/ yes
- 251/ - 197/ -177
Promoter/ Promoter/ Promoter
- 90/ + 414
Promoter/ Intron 1
CACGTG/ AACGTG
no/ yes
- 656/ - 442
Promoter/ Promoter
CACGTG/ AACGTG
yes/ no
- 11/ + 186
Promoter/ Exon1
CACGTG/ CACGTG
no/ no
AACGTG/ AACGTG
CCCGTG/ CACGTG/ AACGTG
no no/ no
yes/ no/ no
Notch pathway CACGTG
yes no/ no
Adhesion/ Cell cycle/ others Itga6
Cdk4
Wee1
2
10
7
CACGTG/ CACGTG/ CACGTG
yes/ yes/ yes
Bmpr1a
14
- 4916
Promoter
AACGTG
no
Foxo3a
10
- 2435
Promoter
CACGTG
no
Gene promoters analyzed, but no binding sites for Bmal/ Clock found: Delta1, Filaggrin, Fzd3, Gli2, Hey1, Involucrin, Jagged1, Lgr5, Ltbp2, Ltbp3, Ltbp4, Nfatc1, Notch1, Sfrp1, Smurf1, Tcf3
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Supplementary Table 4 Real time PCR primer
Gene Forward CTGCCCGAACATCTGAAATTGA Bhlhe41 CTTTACCCGCAGCAAGAAAAC Ccna2 CTCAGGGTCACTAGGAACACG Ccnb1 CTGTGCGCTACCGACTTCAA Ccnd2 AAGGCTGGGTGAAGACCCTTA Cd34 CCCTGCCACCCTTACCAGA Cdkn2b (p15) GTTAAGCCACGAGAATGTAGTGA Chek1 AGGCCAAGCTAATCGGTATTGA Dab2 CCTGATCCCGCTGATCTCG Dbp CTCGGGGGTATTTTGCTGTGT Dkk3 GCCAACTGTCCAGTCTTGTTT Flg2 GTTCTTCTCGCAAGAGGAGAC Fzd2 CAACGCGGTTCAGAAGATGC Gadd45b AGGATGACAGGTGTGGTCAA H19 TGCAGAGGGCGAACAGAAC Itga6 TGTTTATCCCATCACGGGTGG Lef1 GATCGTCCCAACAGTATCAGTG Lor CTGATGTCCAACGCTTTGCC Ltbp2 ATCATTGACCGCTCCTTTAGGT Mki67 TACATTGGCTCTAGTGGCTCC Nr1d1 ACCAGGTCATTAAGTGTGTGC Per1 AAAAGCACCACGGATACTGGC Per3 AGGCGTTAGCATGGTGGAGTA Pum1 CACTGCAAGTATGGGGTCAAG Rad9 CCCCCACTGGATGACTACAG Smad3 GAAACCGGGGGAACGAATTAT Smad7 GAGCCGGATCTGAAGAGGGA Sox9 ACGAGCTGATCCCCTTCCA Tcf3 AGTGATGTCATGGCCAGCGAC Tgfbr2 TCAGTGCTCCCATCGGATTG Tgm3 GCTCAACGCCAACACAGTG Wnt10a CTCCTCTCGGATACCTCTTAGTG Wnt3a 48 miR primer (Sun et al., 2009) TTCTAAAGGAGGCTGCACTGC Pri-miR-23b GTTCTAAAGAGGGATTACCAC Pri-miR-27b CTAAAGGGTCCAGGTCTCCATG Pri-miR-24-1 CCTGGATACCGCAGCTAGGA 18S-RNA
Reverse GCTGCTCAGTTAAGGCTGTTAG ACGTTCACTGGCTTGTCTTCTA AGCTCTTCGCTGACTTTATTACC CACATCAGTGTGGGTGATCTTG TGAATGGCCGTTTCTGGAAGT CAGATACCTCGCAATGTCACG GATACTGGATATGGCCTTCCCT AATGTTGACCCAGATTCTTTGCT CAGGCACCTGGACTTTCCTT TCCTCCTGAGGGTAGTTGAGA GCCTTTCATTAGGGCTGAATCC TCGCTGCATGTCCACTAAATAG GGTCCACATTCATCAGTTTGGC TGAGTGAGTGGGTGGACAAT GCACACGTCACCACTTTGC CATGGAAGTGTCGCCTGACAG TGCTGAGAGGAGTAATAGCCC GTGGGGGTGAAGACGACTTT GCTCGCCTTGATGGTTCCT CAGTAGGTGATGGTGGGAAGTA CTCTCCCGGTCTTGCTTCA GGGAGGCTGTAGCTTGTCA TCCATCAAACGTACCCTTGTTC GCAATAAGTGAGGGCATGAGG TCCATCTTCACTCAGGTAGCC CGCGAGTCTTCTCCTCCCA GCTTGACGTGTGGCTTGTTC CAGGGACGACTTGACCTCAT CGCAGACTTCATGCGGCTTCTC GGCGTGGTTACTCATAAAGACAT CGAAAACCTCGGCTGAAGATG GCATGATCTCCACGTAGTTCCTG AAATCAGCATGCCAGGAACCCAAGC TGGTGAGCATCTTTGAAGGCTGTTG CTGAGCCAGTGTGTGAAATGAGAAC TCTAGCGGCGCAATACGAATG
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RESEARCH SUPPLEMENTARY INFORMATION
Supplementary Table 5 Chromatin immunoprecipitation primer
Gene Primer name Forward Dbp Dbp_P ACACCCGCATCCGGTAGC Dbp Dbp_E3 CGTGGAGGTGCTTAATGACCTTT 49 Dbp primer (Ripperger et al., 2006) Wnt3 Wnt3_UP CAACCCCTTTGCTTTCTAG Lef1 Lef1_farUP1 CAGTGTACTGTAACAGTG Dab2 Dab2_farUP CACTATGGAAAACGGGAC Dab2 Dab2_UP GAACCAGGACTGCACACT Notch2 Notch2_farUP CACTCCAACAATTCAACAG Itga6 Itga6_E1 GCTTCTAGCCCGGCTGGG Cdk4 Cdk4_UP1 CTTGTGCTCCACCCTCTC Cdk4 Cdk4_I1 GTGATCCTCTTTGTGCCTAG Lhx2 Lhx2-farUP1 CAGATCGCTGCAGGGAGAAG Tcf4 Tcf4_farUP CCAAGAAGTCTGCGGTGA Fzd2 Fzd2_farUP GCTCTTGTACCTCAGAGG Smad7 Smad7_farUP GGACATCAGCTCTGAATC Lefty Lefty_UP GCAGAGAACGTGAGACCTC Dkk3 Dkk3_UP TGCTGCAACGGACAGACC Sox9 Sox9_UP ATCCGGTCCAATCAGCGAC
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Reverse CCACTTCGGGCCAATGAG CATGGCCTGGAATGCTTGA GACACCCCCTTCCCAGTC CTTCTTCCAGCCTGCTTC CTCTTTGTTATTACACTTC GGAGGACACAAAAGCATC ACCCAACTTTAACTTCTG GAACTCACAGCCGCTTGTC CTGACGGCCACGTGACCTG GCCAACGCGATCAGCAAC GGTCATACCATACAGAGAT CCAGCGATCATTAGTTACC GCTCAGTCCGTGATTGCT AGGCTGGTATGATGATGG TGTTAGCCATATACTTCACC CTCTCCGCATCTCCTGATC CTCTCCGACTTCCAGCTCAG
