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 brow

ser (Affymetrix). Pdp1 is on the bottom

strand and transcription is going from right to left. This im

age shows a zoom

ed in view so

that low levels of binding can be seen at non-m

aximal tim

epoints. CLK binding begins at ZT10, increases to its maxim

um at ZT14, and low

levels of binding are still observed at ZT18. PER binding begins at ZT14, is m

aximal at ZT18, and low

er levels are observed at ZT22. Pol II signal in the O

RF (transcription) begins at ZT6, is maxim

al at ZT10 and then starts to decrease at ZT18. By showing all three in the sam

e brow

ser one can get a sense of the temporal 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

0.20.40.60.8

11.2

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

PL32A)

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

Golia

th/R

PL32

mRN

A

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.