Abruzzi et al., Supplemental Table 2
Motif Fold-enrichment p-value
CACGTG 2.99 3.6 x 10-192
CACGCG 1.88 3.0 x 10-39
CGCGTG 1.88 3.2 x 10-39
AACGTG 1.72 6.6 x 10-43
CACGTT 1.72 6.6 x 10-43
CACATG 1.31 1.4 x 10-15
CATGTG 1.31 1.4 x 10-15
Supplemental Table 2. CLK peaks are enriched for E-boxes. Binding sites identified as significant in one or more CLK ChIP-chips were analyzed for the presence of E-boxes. CLK peaks are enriched for canonical E-boxes (CACGTG) as well as E-boxes with a single base pair substitution. Peaks containing a canonical E-boxes cycle with an earlier phase than those containing degenerate E-boxes (14.2 versus 15.2, p-value 8x10-10).
Abruzzi et al., Supplemental Table 3
Rank Gene
1vri
2CG16721_Act5C
3Pdp1
4tim
5per
6cwo
7CG9162
8Hex-A_CG3002_CG3004
9CG6767_CG16719
10bel
11CG31324
12CG31522
13CG3842_CG3847
14CG15335_CG2116
15Sec61alpha_CG9537_CG9542
16gol
17Ald
18oaf
19CG32486
20Mnt
Supplemental Table 3. List of the top 20 genes bound by CYC at ZT14.
Abruzzi et al., Supplemental Figure 1
1523 peaks of CLK binding(p-value <10-4 for at least one timepoint; peak significant in two independent experiments )
860 peaks cycle(F24 score > 0.7)
643 peaks do not cycle(F24 score < 0.7)
319 mapped to >1 gene497
mapped to 1 gene
1503 peaks with no significant background signal
(Remove peaks present in V5 IP from wild-type)
44 mapped
to intergenic regions
(including miRNAs)
Supplemental Fig. 1. Identification of CLK direct targets. This flowchart outlines how CLK direct targets were identified from ~1500 CLK binding peaks identified by ChIP-chip and MAT analysis. First, any background binding peaks that were also identified in a V5 ChIP-chip from wild-type flies were removed (~20 peaks). Then, cycling peaks were identified using a fourier analysis and a F24 score cutoff of >0.7. Those peaks that cycled were then mapped by visual inspection to the nearest transcription start site, if possible. ~300 genes could not be mapped to one gene because the peak of CLK binding was equidistant between two different transcription start sites. Approximately 500 genes could be mapped to one unique gene.
Abruzzi et al., Supplemental Figure 2
0 5 10 15 20 25 30 35 40 45 500
20
40
60
80
100
120
CG8443_CG8445eastCG18490
B)
A)
ZT2 ZT6 ZT10 ZT14 ZT18 ZT22 ZT2 ZT6 ZT10 ZT14 ZT18 ZT22
Supplemental Fig. 2. Most CLK peaks identified as non-cycling show oscillations. To identify CLK peaks that cycle, a fourier analysis with fairly stringent cutoffs (F24>0.7 and p-value of <0.05) was applied to the data. Using these cutoffs, approximately 40% of the CLK peaks were labeled as non-cycling. However, when inspected visually, most of these peak cycle weakly and very few show equal levels of CLK binding across all six timepoints. A) CLK ChIP signal of for three randomly chosen non-cycling genes. B) Heatmap showing oscillating ClK binding on all non-cycling CLK direct targets. Data is double plotted to better visualize cycling. As seen for the cycling targets, the maximum CLK binding on these genes occurs at ZT14.
Abruzzi et al., Supplemental Figure 3
Supplemental Fig. 3. CLK, PER and Pol II binding to pdp1 across circadian tim
e. CLK, PER and Pol II ChIP-chip data is visualized in the IGB
browser (Affym
etrix). Pdp1 is on the bottom
strand and transcription is going from right to left
. This image show
s a zoomed in view
so that low
levels of binding can be seen at non-maxim
al timepoints. CLK binding begins at ZT10, increases to its m
aximum
at ZT14, and low
levels of binding are still observed at ZT18. PER binding begins at ZT14, is maxim
al at ZT18, and lower levels are observed at ZT22. Pol II
signal in the ORF (transcription) begins at ZT6, is m
aximal at ZT10 and then starts to decrease at ZT18. By show
ing all three in the same
browser one can get a sense of the tem
poral control: As soon as CLK binds, transcription begins. PER binding begins at ZT14 as transcription begins to decrease. PER binding increases further at ZT18 and then both CLK and PER are released from
the DN
A ZT22.
0.40.50.60.70.80.9
11.1
2 6 10 14 18 22 2 6 10 14 18 22
0.20.30.40.50.60.70.80.9
1
2 6 10 14 18 22 2 6 10 14 18 22
0
0.2
0.4
0.6
0.8
1
1.2
2 6 10 14 18 22 2 6 10 14 18 22
0.2
0.4
0.6
0.8
1
1.2
2 6 10 14 18 22 2 6 10 14 18 22
0.4
0.6
0.8
1
1.2
2 6 10 14 18 22 2 6 10 14 18 22
0.30.40.50.60.70.80.9
1
2 6 10 14 18 22 2 6 10 14 18 22
0.20.30.40.50.60.70.80.9
1
2 6 10 14 18 22 2 6 10 14 18 22
Abruzzi et al., Supplemental Figure 4
0.6
0.8
1
CG9894
treh
Lk6
pABP
lilli
mRN
A re
lativ
e to
RPL
32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
mRN
A re
lativ
e to
RPL
32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
dlg1
A)
B)
C)
D)
E)
F)
G)
H)
I)
J)
0.4
0.6
0.8
1
1.2
2 6 10 14 18 22 2 6 10 14 18 22
2 6 10 14 18 22 2 6 10 14 18 22
0.4
0.6
0.8
1
1.2
2 6 10 14 18 22 2 6 10 14 18 22
cbt CG32486
MESR4nap1
ZT
ZT
ZT
ZT
ZT ZT
ZT
ZT
ZT
ZT
Abruzzi et al., Supplemental Figure 5
00.10.20.30.40.50.60.7
0.20.30.40.50.60.70.80.9
1
0.20.30.40.50.60.70.80.9
11.1
0.20.30.40.50.60.70.80.9
11.1
00.10.20.30.40.50.60.70.80.9
0.20.25
0.30.35
0.40.45
0.50.55
0.6
0.4
0.6
0.8
1
0.2
0.4
0.6
0.8
1
1.2
0.2
0.4
0.6
0.8
1
1.2 nat1
JIL-114-3-3zeta
picotCG18317
dbt lim1
gol
00.20.40.60.8
11.21.41.61.8
CG31324
CG30497
mRN
A re
lativ
e to
RPL
32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
mRN
A re
lativ
e to
RPL
32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
mRN
A re
lativ
e to
RPL
32m
RNA
rela
tive
to R
PL32
A)
B)
C)
D)
E)
F)
G)
H)
I)
J)
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
2 6 10 14 18 22 2 6 10 14 18 22ZT
Supplemental Fig. 4. Only ~20% of mRNAs from non-cycling-Pol II bound CLK targets cycle. Q-RT-PCR was performed on six timepoints of total RNA extracted from wild-type (yw) fly heads and normalized relative to rpl32. Values are double plotted to better visualize cycling. To be considered cycling, mRNAs have to show cycling with an amplitude of >1.5-fold and have at least one timepoint that is high. A) CG9894 doesn’t cycle, B) lilli doesn’t cycle, C) pAbp cycles, D) cbt doesn’t cycle, E) nap1 doesn’t cycle, F) lk6 doesn’t cycle, G) dlg1 cycles, H) treh doesn’t cycle, I) CG32486 doesn’t cycle, J) mesr4 doesn’t cycle.
Supplemental Fig. 5. Most genes bound by Pol II show cycling mRNA. Q-RT-PCR was performed on six timepoints of total RNA extracted from wild-type (yw) fly heads and normalized relative to rpl32. Data is double plotted to better visualize cycling. To be considered cycling, mRNAs have to show cycling with an amplitude of >1.5-fold and have at least one timepoint that is high. A) gol cycles, B) CG31324 cycles, C) jil-1 cycles, D) picot cycles, E) dbt doesn’t cycle (amplitude only 1.4-fold), F) CG30497 cycles, G) nat1 cycles, H) 14-3-3zeta cycles, I) CG18317 cycles, J)lim1 doesn’t cycle (amplitude only 1.4-fold).
Abruzzi et al., Supplemental Figure 6
2 140
0.2
0.4
0.6
0.8
1
1.2
WTgmr-hid
Gol
iath
/RPL
32 m
RNA
A) gol
ZT
Supplemental Fig. 6. mRNA levels of gol in GMR-hid. To determine whether gene expression is eye-enriched, q-RT-PCR was used to examine mRNA levels in wild-type (CLK-V5 background) or GMR-hid (CLK-V5, GMR-hid) fly heads. gol is a CLK target primarily in eye tissue and, indeed, mRNA levels are greatly diminished in GMR-hid suggesting gol is primarily expressed in the eyes.
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