CLOCK deubiquitylation by USP8 inhibits
CLK/CYC transcription in Drosophila
Weifei Luo,1Yue Li,2Chih-Hang Anthony Tang,2Katharine C. Abruzzi,1Joseph Rodriguez,2
Stefan Pescatore,1and Michael Rosbash1–3
1Howard Hughes Medical Institute,2Department of Biology, National Center for Behavioral Genomics, Brandeis University,
Waltham, Massachusetts 02454, USA
A conserved transcriptional feedback loop underlies animal circadian rhythms. In Drosophila, the transcription
factors CLOCK (CLK) and CYCLE (CYC) activate the transcription of direct target genes like period (per) and
timeless (tim). They encode the proteins PER and TIM, respectively, which repress CLK/CYC activity. Previous
work indicates that repression is due to a direct PER–CLK/CYC interaction as well as CLK/CYC phosphorylation.
We describe here the role of ubiquitin-specific protease 8 (USP8) in circadian transcriptional repression as well as
the importance of CLK ubiquitylation in CLK/CYC transcription activity. usp8 loss of function (RNAi) or
expression of a dominant-negative form of the protein (USP8-DN) enhances CLK/CYC transcriptional activity and
alters fly locomotor activity rhythms. Clock protein and mRNA molecular oscillations are virtually absent within
circadian neurons of USP8-DN flies. Furthermore, CLK ubiquitylation cycles robustly in wild-type flies and peaks
coincident with maximal CLK/CYC transcription. As USP8 interacts with CLK and expression of USP8-DN
increases CLK ubiquitylation, the data indicate that USP8 deubiquitylates CLK, which down-regulates CLK/CYC
transcriptional activity. Taken together with the facts that usp8 mRNA cycles and that its transcription is
activated directly by CLK/CYC, USP8, like PER and TIM, contributes to the transcriptional feedback loop cycle
that underlies circadian rhythms.
[Keywords: CLOCK; USP8; circadian rhythm; Drosophila; transcription; deubiquitylation]
Supplemental material is available for this article.
Received July 8, 2012; revised version accepted October 3, 2012.
Circadian rhythms of animal behavior, physiology, and
metabolism are an evolutionary adaptation to the rota-
tion of the earth. There is an underlying transcriptional
feedback mechanism, which is modulated by the post-
translation modification of key transcription factors. In
Drosophila, the transcription factors CLOCK (CLK) and
CYCLE (CYC) activate the transcription of period (per)
and timeless (tim) and other direct target genes during the
day. At night, PER and TIM then enter the nucleus and
inhibit their own transcription. The activity of CLK/CYC
is regulated by a direct interaction with these negative
regulators as well as by post-translational modifications,
with a focus to date on PER phosphorylation (Allada and
Chung 2010; Doherty and Kay 2010). Specifically, PER
and TIM are gradually hyperphosphorylated by different
kinases during the early night (Price et al. 1998; Martinek
et al. 2001; Lin et al. 2002; Chiu et al. 2011) and then
interact with and/or sequester the CLK/CYC complex,
which inhibits its transcription activity (Menet et al.
2010). In the morning after exposure to light, PER and
TIM are degraded, which liberates and reactivates CLK/
CYC. In addition, hyperphosphorylation of CLK appears
to coincide with low transcription activity, suggesting
that direct phosphorylation–dephosphorylation may also
modulate CLK/CYC activity.
Other modifications also occur on core clock proteins.
BMAL1, the mammalian ortholog of CYC, is acetylated
by its partner, CLK, at the single lysine residue K572 with
a timing corresponding to the repression phase of circa-
dian transcription of clock-controlled genes (Hirayama
et al. 2007; Nakahata et al. 2008). BMAL1 also exhibits
a circadian pattern of sumoylation and ubiquitylation,
which parallels its activation in mouse livers (Cardone
et al. 2005; Lee et al. 2008); these dual modifications are
essential for CLK/BMAL1 transcriptional activation.
Ubiquitylation of a protein substrate results from the
antagonistic functions of ubiquitin ligases and deubiqui-
tylating enzymes. Polyubiquitylation is usually associ-
ated with proteasome-mediated proteolysis, whereas
monoubiquitylation can regulate the cellular localiza-
tion, trafficking, and even transcriptional activity of
a protein (Komander and Rape 2012). Proteolysis is in-
timately connected tothe circadianmolecular cycle due to
the daily degradation that many circadian proteins un-
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.200584.112.
2536 GENES & DEVELOPMENT 26:2536–2549 ? 2012 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/12; www.genesdev.org
dergo. Indeed, work over the last two decades has identi-
fied specific E3 ligases required for this proteasome-
mediated degradation. For example, Drosophila SLIMB
and JETLAG are involved in the degradation of PER and
TIM, respectively (Grima et al. 2002; Ko et al. 2002; Koh
et al. 2006). The mouse F-box protein FBXL3, a compo-
nent of the SKP1–CUL1–F-box-protein (SCF) E3 ubiquitin
ligase complex, mediates the degradation of transcription
repressors PER1 and PER2 and/or CRY1 and CRY2 (Busino
et al. 2007; Godinho et al. 2007; Siepka et al. 2007). Unlike
their mammalian orthologs, the protein levels of CLK and
CYC do not oscillate across a circadian cycle, and there
are no reports of post-translational modifications of these
proteins other than phosphorylation.
In contrast to ubiquitin E3 ligases, deubiquitylating
enzymes are less well studied. This protease family
removes the ubiquitin moiety from substrates, which
are generally linked to their stabilization and/or recy-
cling. A large number of these proteins belong to the
ubiquitin-specific protease (USP) family, which can rec-
ognize a variety of substrates and therefore function in
many essential pathways within cells, such as protein
degradation, localization, and transcriptional regulation.
In response to oxidative stress, deubiquitylation of the
transcription factor FOXO4 by USP7 represses FOXO
transcriptional activity in mammals without affecting
its degradation (van der Horst et al. 2006). In Drosophila,
USP7 contributes to the Pc-mediated silencing of the
homeotic genes through modulation of histone H2 de-
ubiquitylation (van der Knaap et al. 2010). In contrast,
UBP8, a component of the yeast SAGA complex, deubiq-
uitylates H2B and stimulates transcription by enhancing
Lys4 trimethylation of H3. Also notable is mammalian
usp2; its mRNA cycles, and its transcription is under
CLOCK/BMAL1 control (Oishi et al. 2003, 2005). USP2
was shown more recently to stabilize BMAL1 and CRY1
by deubiquitylation in mouse livers (Scoma et al. 2011;
Tong et al. 2012). USP8/UBPY, another deubiquitylating
enzyme in the USP family, functions in the endocytosis
and endosome sorting of ligand-activated receptor tyro-
sine kinases (RTKs) in mammals (Mizuno et al. 2005;
Wright et al. 2011), and a knockout of the usp8 gene
causes developmental lethality in Drosophila as well as
in mammals (Niendorf et al. 2007; Mukai et al. 2010).
usp8 mRNA always appeared near the top of the lists of
cycling mRNAs in several early microarray studies of
Drosophila head RNA (Claridge-Chang et al. 2001;
McDonald and Rosbash 2001; Ueda et al. 2002) and has
been shown more recently to also cycle within circadian
neurons (Kula-Eversole et al. 2010).
In this study, we address the function of USP8 in
Drosophila circadian rhythms and show that it deubiqui-
tylates CLK in a circadian manner and contributes to
cyclical repression of core clock transcription. Loss of
function (RNAi) or expression of a dominant-negative
form of the protein (USP8-DN) not only enhances CLK/
CYC transcriptional activity,but also alters fly locomotor
activity rhythms. This is especially prominent in circa-
dian neurons within which clock protein and mRNA
molecular oscillations are virtually absent in the USP8-
in wild-type flies and peaks coincident with maximal
CLK/CYC transcription. As USP8 interacts with CLK and
expression of USP8-DN increases CLK ubiquitylation,
the data indicate that USP8 deubiquitylates CLK, which
down-regulates CLK/CYC transcriptional activity. Taken
together with the facts that usp8 mRNA cycles and that
its transcription is activated directly by CLK/CYC, the
data indicate a new function for USP8: It negatively
regulates its own transcription and that of other direct
target genes, thereby contributing to the circadian tran-
scriptional feedback cycle.
The usp8 short isoform is a CLK direct target
As described above, usp8 mRNA is one of the very few
Drosophila mRNAs other than core clock gene mRNAs
that cycle robustly in fly heads as well as in purified
circadian neurons. For this reason as well as the lack of
a robust role for ubiquitylation in the fly circadian system,
we decided to investigate the function and regulation of
usp8. We first asked whether usp8 is a direct target of the
CLK/CYC complex by assaying CLK occupancy by chro-
matin immunoprecipitation (ChIP) and tiling arrays
(ChIP–chip). The chromatin was made from fly heads
collected at different times during a light–dark (LD) cycle
(Abruzzi et al. 2011).
usp8 is a very robust CLK target gene and manifests
canonical CLK cycling on its target sites exactly like core
clock genes (rank #639). Moreover,there is a cycling RNA
polymerase II (Pol II) signal near the promoter of the short
isoform of usp8 mRNA. In contrast, there is a noncycling
Pol II and an absence of CLK signal near the promoter of
the long isoform (Fig. 1A). Consistent with these isoform
differences, RNA sequencing (RNA-seq) (Fig. 1B) and
quantitative RT–PCR (qRT–PCR) of mRNA from both
LD and constant darkness (DD) conditions (Fig. 1C) show
that only the short isoform is cycling, with a temporal
profile similar to those of per and tim mRNAs (Menet
et al. 2010).
The short isoform of usp8 mRNA is most abundant in
head RNA; its expression level is approximately fourfold
to fivefold higher than the long isoform at times of
maximal expression level; e.g., at Zeitgeber time 10
(ZT10). Importantly, levels of this isoform are high in
mutants of the repressor genes tim and per and low in
clkjrkand cyc01mutants; levels of the long isoform are
relatively unaffected (Fig. 1D). Taken together, the results
indicate that CLK/CYC specifically regulates the tran-
scription of the short form of usp8 mRNA, resulting in
a cycling mRNA.
USP8 is important for locomotor activity rhythms
To investigate the contribution of USP8 to circadian
rhythms, we took advantage of the GAL4-UAS system
and several usp8 RNAi strains (Dietzl et al. 2007; Mukai
et al. 2010) to selectively knock down the expression of
CLOCK deubiquitylation by USP8
GENES & DEVELOPMENT2537
USP8 within circadian neurons and analyze the resulting
usp8-RNAi R2, encodes a dsRNA construct against both
usp8 isoforms (Mukai et al. 2010) and consistently ex-
hibited long periods when expressed with a pdfgal4 driver
(;1 h longer than control flies) (Fig. 2A). This strategy
restricts expression of the RNAi construct to PDF neurons,
which include the principal circadian pacemaker neurons
(Stoleru et al. 2004). With the timgal4 driver, expression of
the usp8-RNAi R2 construct in all circadian neurons as
well as additional locations (Kaneko and Hall 2000)
caused premature mortality (data not shown), consistent
with the essential nature of the gene (Mukai et al. 2010).
To overcome this developmental lethality, we took ad-
vantage of a timgal4, tubgal80ts driver to repress UAS
expression at a permissive temperature (18°C) until the
adult stage, when the temperature was raised to the non-
et al. 2003). Almost 50% of the flies were arrhythmic with
this strategy compared with 96%–100% rhythmicity of
control flies. The remaining rhythmic flies have long
periods, an average of 1 h longer thancontrol flies (Fig. 2A).
To further examine the effect of USP8 knockdown, we
examined fly locomotor activity with another USP8
RNAi line (Bloomington HMS01898). This construct
was expressed in a somewhat broader set of circadian
neurons with the dvpdfgal4 driver; i.e., all PDF cells,
LNds, and the PDF-negative fifth s-LNv (Bahn et al. 2009).
These flies also exhibit long periods, 1.3-h longer than
control flies (Supplemental Fig. 1).
To confirm the importance of USP8 to fly locomotor
activity rhythms, we constructed a dominant-negative
form of USP8, USP8-DN. It harbors a point mutation
within its catalytic domain (C572A), which was reported
to render it inactive in mammalian systems (Mizuno et al.
2005; Mukai et al. 2010). We first restricted expression of
USP8-DN to PDF neurons. Most pdfgal4; uas-usp8-DN
flies showed robust rhythms with a 25-h period, ;1.5 h
longer than the uas-usp8-DN and pdfgal4 control flies
(Fig. 2A,B, top panels). This result was similar to but
stronger than the RNAi result shown above.
Expression of USP8-DN with the dvpdfgal4 driver
either significantly reduced rhythmicity (58.3% at 25°C)
or almost completely abolished it (8% at 29°C) (Fig. 2A,B,
performed as in Abruzzi et al. (2011). The CLK and Pol II occupancy at the usp8 promoter regions were visualized using the integrated
gene browser (IGB; Affymetrix). RA and RB are two isoforms of the usp8 gene. USP8-PA contains 896-amino-acid residues; USP8-PB has
367-amino-acid residues and a unique 29-amino-acid N terminus. Both isoforms share the same enzymatic domain. The black arrows
above the usp8 gene diagram indicate the start codon. (B) RNA-seq of usp8 mRNA at six time points from fly heads was performed as in
Rodriguez et al. (2012). (C) Real-time PCR analysis of RA and RB isoforms of usp8 from head total RNA. The Y-axis represents the
relative value of usp8 mRNAs normalized to that of rpl32 mRNA. (D) RNA-seq of usp8 mRNA at ZT16 in yw as well as the clock
mutants tim01, per01, cyc01, and clkjrk.
usp8 transcription is under clock control. (A) CLK and Pol II ChIP at six time points (ZT2–ZT22) from fly heads were
Luo et al.
2538GENES & DEVELOPMENT
bottom left panel). We attempted to do similar USP8-DN
experiments with the even broader timgal4 driver, but a
preliminary experiment indicated that expression in all
clock neurons once again conferred developmental lethal-
ity (data not shown). To circumvent this issue, we again
used the timgal4, tubgal80ts driver combination and
In contrast to the permissive temperature in which
timgal4, tubgal80ts; uas-usp8-DN flies exhibited normal
locomotor activity rhythms and period length, raising the
temperature had profound effects on fly behavior. For
example, a temperature shift to 25°C or 27°C dramati-
cally reduced the percentage of rhythmic flies from 80%–
90% to 65% (25°C) or 35% (27°C) (Fig. 2A,B, bottom right
panel). Furthermore, the remaining rhythmic flies had
very weak rhythms. Somewhat surprisingly, overexpres-
sion of wild-type USP8 in either pacemaker neurons
(pdfgal4) or all clock neurons (timgal4) had no effect on
behavior (data not shown). Taken together, the data
indicate that USP8 contributes to circadian rhythms.
USP8 is required for molecular oscillations of PER
Since the timgal4, tubgal80ts; uas-usp8-DN (tim>DN)
flies are arrhythmic or have weak rhythms, we hypoth-
esized that molecular oscillations might be disrupted and
even the cause of the disrupted rhythms under free-
running conditions (DD). Under normal LD conditions,
however, we did not observe dramatic changes in CLK,
PER, and TIM protein levels or cycling in USP8-DN flies,
most likely because light can trigger efficient TIM and
PER degradation and override or compensate for a modest
issue with transcriptional regulation. To circumvent this
light issue and address molecular oscillations that might
be more relevant to the behavioral phenotypes described
above (in DD), we assayed these clock proteins by
Western blotting during the first day of DD (DD1).
As previously reported (Zeng et al. 1996), control flies
have very low levels of PER and TIM between circadian
time 12 (CT2) and CT10, which increase dramatically
usp8-DN constructs by different gal4 drivers. Flies were entrained for 3–4 d in LD cycles and released into DD for at least 5 d at 25°C
unless indicated. (%R) Percentage of rhythmic flies; (N) number of flies assayed. (B) Actograms and average actograms under DD were
shown for flies expressing USP8-DN driven by pdfgal4, dvpdfgal4 and timgal4, tubgal80ts drivers (Levine et al. 2002).
usp8 mutant flies exhibit abnormal locomotor activity. (A) Behavioral analysis of fly strains expressing usp8 RNAi or uas-
CLOCK deubiquitylation by USP8
GENES & DEVELOPMENT 2539
throughout the subjective night in head extracts (Fig.
3A,B). Strikingly, PER and TIM did not manifest these
low levels in the early–mid-subjective day (CT6–CT10) in
the tim>DN strain, resulting in much less cycling (Fig. 3A
[cf. lanes 8,9 and 2,3], B [cf. lanes 5 and 1,3]). Consistent
with its normal behavior phenotype (data not shown),
overexpression of wild-type USP8 has little effect on PER
cycling (Supplemental Fig. 2). In contrast, CLK protein
levels as well as its phosphorylation remain quite similar
to the control strain (Fig. 3A,B; Supplemental Fig. 3).
To determine whether this change of protein expression
is due to effects of the tim>DN on CLK/CYC transcrip-
tion, we examined per, tim, and vri pre-mRNA as well as
mRNA expression during the first subjective day of DD.
Previous work has demonstrated that pre-mRNA profiles
are quite similar to other transcriptional assays, such as
nuclear run-ons, and can be used to approximate tran-
scription (Menet et al. 2010).
Expression of USP8-DN caused a twofold to fourfold
increase in pre-mRNA levels of all threeCLK direct target
circadian genes relative to the control strain (Fig. 3C–E,
top). The mRNA levels of per and vri also increased in the
tim>DN flies (Fig. 3C,E, bottom). For unknown reasons,
the increase in tim mRNA levels is less consistent and
can only be reliably seen at certain time points (Fig. 3D,
bottom). The results indicate that reducing USP8 activity
increases the transcription of many CLK/CYC direct
target genes and suggests that USP8 functions to repress
USP8 represses CLK/CYC transcriptional activation
by direct deubiquitylation of CLK
To address more directly the role of USP8 in modulating
CLK/CYC transcription, we used three different S2 cell
assays. First, we examined the extent towhich fourdifferent
USP8 dsRNAs affect endogenous TIM expression in a stable
cell line expressing CLK (pMT-3XFlag-clk-HBH; leaky ex-
efficiently knocked down endogenous USP8 (ds2–4) (Fig.
4A), and these three most strongly increased endogenous
TIM expression. In contrast, there was no strong or system-
atic effect on the levels of CLK and USP7, the latter known
for its effect on H2B deubiquitylation (Fig. 4A, lanes 3–5).
Second, we tested whether this effect of USP8 knock-
down on endogenous TIM expression reflects an increase
and tim>DN flies were entrained for three LD cycles at 29°C and collected at the first day of DD (DD1). Protein levels from head
extracts at six time points were analyzed by Western blotting. Note: The lower USP8 band is the endogenous wild-type (WT) USP8, and
the higher band is the USP8-DN band. (B) tim>DN flies were entrained for three LD cycles at permissive (18°C) and nonpermissive
(29°C) temperatures and collected at DD1. As a control, the uas-usp8-DN flies (uas-DN) were entrained at 29°C. Proteins from head
extract at CT10 and CT22 were analyzed as in A. (C–E). Real-time PCR analysis of per (C), tim (D), and vri (E) mRNA and pre-mRNA in
tim>DN and control (uas-DN) flies. Flies were entrained as in A and B. Total RNA was prepared from six time points on DD1 and
analyzed by real-time PCR with specific primers for per, tim, and vri genes.
The levels of PER and TIM and their RNA were increased in the tim>DN flies. (A) Control driver flies (timgal4, tubgal80ts)
Luo et al.
2540 GENES & DEVELOPMENT
in tim transcription and whether PER can still repress
CLK/CYC transcriptional activation under these condi-
tions. To this end, we assayed CLK/CYC-mediated lu-
ciferase expression from a reporter construct expressing
luciferase under the control of the tim promoter (tim-luc)
(Darlington et al. 1998; Nawathean and Rosbash 2004). S2
cells were cotransfected with pAct-clk as well as dsRNA
for either USP8 (ds3) or GFP (control). Indeed, transfection
compared with control cells (Fig. 4B, blue), and increasing
amounts of a PER-expressing plasmid still gave rise to
a dose-dependent reduction in luciferase levels.
Third, we assayed the effect of USP8-DN on CLK/CYC
transcription. Increasing amounts of a USP8-DN-express-
ing plasmid were transfected with pAct-clk and either of
two circadian reporters in S2 cells: the tim-luc reporter
described above or a reporter that expresses luciferase
under the control of a minimal per promoter (3x69-luc)
(Hao et al. 1997). With both reporters, USP8-DN led to
a dose-dependent increase in luciferase activity (Fig. 4C).
This increased CLK/CYC transcription activity is also
sensitive to PER repression (Fig. 4D). The three S2 cell
transcription activation assays indicate that USP8 func-
tions to repress CLK/CYC activity, at least in S2 cells.
four dsRNAs against USP8 (ds1, ds2, ds3, and ds4) for 2 d. The presence of TIM, USP8, CLK, and USP7 in whole-cell extract was shown
by Western blotting. Antibody for USP8 was raised to recognize the full-length (long) isoform only (Mukai et al. 2010). Note that ds2
and ds3 were able to knock down both the long and short isoforms of usp8 mRNA. dsRNA for GFP (dsGFP) was used as the control. (B)
Repression of PER on CLK/CYC transcription activation of a luciferase gene fused to the timeless promoter (tim-luc) when cells were
incubated with USP8 dsRNA (ds3). The values at the Y-axis are the normalized luciferase activity. Four different amounts of pAct-per
plasmids (0, 10, 25, and 50 ng) were cotransfected with 5 ng of pAct-clk plasmid after 2 d of incubation with dsRNA. Cells were
harvested 2 d after transfection. Results were averaged from two experiments with duplicates. (C) The effect of USP8-DN on CLK/CYC
transcription activation of tim-luc and 3X69-luc reporters. Different amounts of pAct-usp8-DN (0, 50, 100, and 150 ng) were
cotransfected as in A. (D) Normalized luciferase activity of tim-luc and 3X69-luc when pAct-per and pAct-usp8-DN were cotransfected.
(E) Coimmunoprecipitation of USP8 with CLK. pAct-usp8-V5 or pAct-usp8-DN-V5 plasmids (150, 300, and 600 ng) were transfected
into pMT-3XFlag-clk-HBH S2 stable cells. CLK was immunoprecipitated using anti-Flag beads. The presence of USP8 in the input and
immunoprecipitation was visualized by anti-V5 Western blotting. A negative immunoprecipitation control without CLK expression is
shown in the right panel. (F) The effect of USP8 knockdown on CLK ubiquitylation. pMT-3XFlag-clk-HBH cells were incubated with
USP8 dsRNAs (ds2 and ds3) for 2 d. Cells were harvested 4 h after addition of 50 uM MG132 or DMSO. CLK was immunoprecipitated
as in E. Ubiquitylated CLK was visualized by an FK2 monoclonal antibody recognizing both mono- and polyubiquitylated proteins
(Enzo Life Sciences). The same blot is shown as both a short and long exposure. (U) USP8 dsRNA; (C) control GFP dsRNA; (mono-Ub-
CLK) monoubiquitylated CLK; (poly-Ub-CLK) polyubiquitylated CLK.
USP8 inhibits CLK/CYC transcription activity in S2 cells. (A) The pMT-3XFlag-CLK-HBH S2 stable line was incubated with
CLOCK deubiquitylation by USP8
GENES & DEVELOPMENT 2541
Since USP8 is a ubiquitin-specific protease, we designed
two experiments to test whether CLK is ubiquitylated
and a substrate of USP8. We first assayed whether USP8
and CLK can interact in vitro. Plasmids expressing V5-
tagged USP8 (both wild-type and DN versions) were
transfected into the pMT-3XFlag-clk-HBH stable S2 cells.
CLK was immunoprecipitated using anti-Flag beads
(Sigma), after which both the input and the immunopre-
cipitates were examined via anti-V5 Western blotting for
USP8. Both wild-type and DN forms of USP8 were
present in the CLK immunoprecipitates (Fig. 4E, lanes
1–6, left panel), indicating that USP8 can interact with
CLK at least in S2 cells.
Next, we tested whether CLK ubiquitylation is affected
by depletion of USP8. pMT-clk stable S2 cells were
transfected with either USP8 dsRNAs or control dsRNAs
(GFP). CLK was immunoprecipitated as in Figure 4E, and
the pellets analyzed by Western blotting with anti-ubiq-
uitin or anti-Flag (CLK) reagents.
CLK protein levels are not affected by USP8 knock-
down (Fig. 4F, bottom panel), whereas CLK ubiquitylation
(most likely mono-Ub) is increased dramatically (Fig. 4F,
top, lanes 1,2, arrow). Furthermore, addition of USP8
dsRNA as well as MG132, a broad proteasome inhibitor,
led to the accumulation of more mono-Ub as well as poly-
Ub CLK compared with treatment by USP8 dsRNA or
MG132 alone (Fig. 4F, top, cf. lanes 4 and 2,3). The results
indicate that CLK is ubiquitylated and that Ub-CLK is
turned over by the proteasome as well as deubiquitylated
by USP8 in S2 cells.
CLK ubiquitylation in fly heads
Is CLK also ubiquitylated and regulated by USP8 in flies?
To address these questions, we generated a transgenic fly
strain expressing a 3XFlag tagged clk14.8 transgene that
contains the full-length CLK genomic sequence (3XFlag-
CLK fly) and functions identically to the wild-type gene
(see the Materials and Methods; data not shown). Fly head
extracts were prepared from flies entrained in 12:12 LD
cycles, and CLK was immunoprecipitated by anti-Flag
beads. The resulting pellets were analyzed via Western
blotting with either an FK2 anti-ubiquitin antibody or
anti-Flag to detect Ub-CLK or CLK, respectively.
Maximal CLK ubiquitylation occurs at ZT10–ZT14
(Fig. 5A, lanes 3,4, left panel), a time coincident with
maximal CLK/CYC transcription activation and CLK
binding to chromatin (Abruzzi et al. 2011). We also
assayed USP8, and it is associated with CLK at all six
time points. There appears to be a higher ratio of USP8 to
CLK at ZT18–ZT2 (Fig. 5B; Supplemental Fig. 4), approx-
imately coincident with maximal transcriptional repres-
sion. In contrast, PER appears not to interact with USP8
(Supplemental Fig. 5).
time points. CLK was precipitated by anti-Flag beads. CLK and ubiquitylated CLK present in the immunoprecipitates were visualized
by Western blotting with an anti-Flag and an FK2 ubiquitin antibody, respectively. (Ub-CLK) Ubiquitylated CLK. (B) USP8 was
coimmunoprecipitated with CLK. CLK immunoprecipitation from 3XFlag-CLK flies was performed as in A. The presence of USP8 in
the CLK precipitates is shown by anti-USP8 Western blotting. (C) CLK ubiquitylation was increased in the tim>DN flies. tim>DN and
uas-DN control flies were entrained for three LD cycles at 29°C. Extracts from fly heads collected at ZT10 and ZT22 were subjected to
CLK immunoprecipitation with guinea pig anti-CLK antibody (Houl et al. 2008). Ubiquitylated CLK was shown by anti-ubiquitin
Western blotting. (D) CLK binding at the E-box of the tim gene in tim>DN flies. CLK ChIP in tim>DN and uas-DN control flies at ZT10
and ZT22 was performed with guinea pig anti-CLK antibody. The occupancy of CLK at the tim E-box was measured by qPCR with
primers within the E-box. The Y-axis value was calculated by the ratio of chromatin immunoprecipitated by CLK to the input. Results
were averaged from two experiments.
(A) CLK ubiquitylation is cycling in fly head extract. 3XFlag-CLK flies were entrained for three LD cycles and collected at six
Luo et al.
2542 GENES & DEVELOPMENT
Because CLK ubiquitylation is relatively low in the late
night to early morning (ZT18–ZT2) (Fig. 5A), we specu-
late that USP8 functions to remove ubiquitin from CLK
and contributes to transcriptional inhibition at these
times. Therefore, inhibition of USP8 may give rise to
increased CLK ubiquitylation. To test this hypothesis, we
immunoprecipitated CLK with a CLK polyclonal anti-
body (Houl et al. 2006) and probed the Western blot with
a ubiquitin antibody using tim>DN and uas-usp8-DN
(control) fly head extracts.
Only low levels of CLK ubiquitylation were detected in
the control flies at both ZT10 and ZT22 (Fig. 5C, lanes
1,2), most likely due to the relatively inefficient immu-
noprecipitation with the polyclonal antibody. More im-
portantly, CLK immunoprecipitates from tim>DN head
extract had much higher levels of ubiquitylated CLK (Fig.
5C, lanes 3,4), with stronger signals at ZT22 than at
ZT10. This indicates that USP8 indeed deubiquitylates
CLK in fly heads.
To address whether expression of USP-DN affects CLK
binding to core clock gene promoters, we performed an
anti-CLK ChIP with the same strains. CLK binding to the
tim E-box does not change significantly (Fig. 5D), suggest-
ing that CLK ubiquitylation impacts transcriptional ac-
tivation rather than DNA binding.
To further characterize CLK ubiquitylation, we per-
formed a genome-wide ubiquitylated CLK binding on
chromatin at ZT10 (approximate peak of maximal clock
gene transcription) and ZT22 (approximate peak of min-
imal transcription) by sequential ChIP (seq-ChIP) in ex-
tracts from 3XFlag-CLK fly heads. Seq-ChIP has been
successfully used to show the co-occupancy of two tran-
scription factors and the modification of a transcription
factor or histone at a specific gene locus (Geisberg and
Struhl 2004; Huang et al. 2006; Metivier et al. 2008;
Katan-Khaykovich and Struhl 2011). First, CLK-bound
chromatin was precipitated with anti-Flag beads. Proteins
were eluted from the beads and subjected to anti-Ub
immunoprecipitation, and both chromatin fractions ana-
lyzed with Drosophila Tiling 2.0 arrays (Affymetrix).
As expected, there was a relatively high CLK signal at
the promoters of four canonical clock genes (per, tim, vri,
and pdp1) at ZT10, withmuch less signal at ZT22. Despite
the difference in the tagged gene and antibody, the CLK
patterns on these genes are similar to those previously
reported with a V5-CLK transgene (Abruzzi et al. 2011).
The ubiquitylated patterns are also similar, including
much more signal at ZT10 than at ZT22 (Fig. 6), suggest-
ing that CLK is broadly ubiquitylated on clock gene
A more detailed comparison of the ZT10 Ub-CLK
peaks with the CLK peaks indicates little or no difference
on tim (Fig. 6A), whereas the Ub-CLK distribution on per
is somewhat shifted toward the transcribed region (Fig.
6B). There is, however, a more striking difference be-
tween the CLK and Ub-CLK patterns on vri (Fig. 6C). The
most upstream peak of CLK binding, which is the
strongest and derives principally from the eye (Abruzzi
et al. 2011), is essentially absent in the Ub-CLK-binding
profile. The middle Ub-CLK peak is comparable with its
relative strength as a CLK peak. In contrast, the Ub-CLK
signal is strongest at the most downstream CLK peak. Its
position coincides well with the start site of the short vri
isoform, which is under CLK control in circadian neurons
(Cyran et al. 2003).
A similar situation occurs at the promoter of pdp1.
The expression of the pdp1-D and pdp1-J isoforms is
cyclic and important for maintaining rhythms in clock
neurons (Cyran et al. 2003; Zheng et al. 2009). The peaks
of CLK that correspond to these two transcription start
sites have a substantial Ub-CLK signal. In contrast, the
two more upstream peaks of CLK on the pdp1 promoter
are more prominent and eye-specific but with rather little
Ub-CLK signal (Abruzzi et al. 2011). Based on the vri and
pdp1 patterns, Ub-CLK binding may be important for
isoform-specific gene expression within clock neurons.
USP8 is essential for molecular oscillations within
Clock gene molecular oscillations in pacemaker neurons
underlie rhythmic behavior. Does USP8 have a particu-
larly strong effect on molecular oscillations within these
flies are largely arrhythmic, we used immunostaining to
examine TIM and PER levels in PDF neurons at peak and
trough times, CT10 and CT22, respectively. As expected,
we observed normal TIM and PER oscillations in uas-
usp8-DN control flies (Curtin et al. 1995; Shafer et al.
2002): TIM and PER proteins are undetectable at CT10,
whereas high TIM and PER signals are observed at CT22
(Fig. 7A,B, right panels). In contrast, expression of USP8-
DN dramatically increases TIM and PER levels at CT10
within PDF neurons; they are comparable with the
signals observed in tim>DN flies at CT22 (Fig. 7A, arrows
point to PDF neurons). This indicates that expression of
USP8-DN virtually eliminates the normal oscillations of
TIM and PER in PDF neurons even more dramatically
than what is observed by Western blotting of extracts
from the same flies (Fig. 3).
To determine whether the effect of USP-DN on TIM
and PER oscillations in PDF neurons is due to transcrip-
tion, we purified GFP-labeled PDF neuron RNA from
mCD8-GFP; pdfgal4, uas-usp8-DN (pdf>DN) flies as pre-
viously described (Nagoshi et al. 2009; Kula-Eversole et al.
2010). PDF neurons expressing USP8-DN are morpholog-
ically normal (Fig. 8A) and appropriately enriched in pdf
mRNA (Fig. 8B). Consistent with published results from
LD conditions (Kula-Eversole et al. 2010), tim mRNA
levels at CT11 are four to five times of those at CT23
within wild-type PDF neurons. In striking contrast, tim
mRNA from pdf>DN neurons are almost at the same
levels at these same circadian times. This indicates that
expression of USP8-DN within PDF neurons disrupts tim
RNA cycling, suggesting that this is the cause of the
increased PER and TIM levels at CT10 as well as the poor
rhythms of these files. We conclude that USP8 contributes
to the temporal regulation of CLK/CYC transcriptional
activity, which is particularly important for molecular
oscillations within clock neurons.
CLOCK deubiquitylation by USP8
GENES & DEVELOPMENT2543
In this study, we addressed the function of the deubiqui-
tylating enzyme USP8 in Drosophila circadian rhythms.
Although usp8 mRNA was one of the top cyclers identi-
fied in head RNA more than a decade ago (McDonald and
Rosbash 2001), it was only recently identified as one of
the few non-clock gene mRNAs that cycles in circadian
neuron RNA as well as in head RNA (Kula-Eversole et al.
2010). This revived our interest in understanding its role
in circadian rhythms. The data here indicate that USP8
deubiquitylates CLK, which likely contributes to inhib-
iting clock gene transcription at the appropriate trough
times in the circadian cycle. This regulation appears
particularly important for clock neurons because inter-
ference of USP8 function by either RNAi knockdown or
expression of USP8-DN dramatically alters fly locomotor
activity, ranging from long periods to complete arrhyth-
micity (Fig. 2).
The expression of USP8-DN also had dramatic effects
on the oscillations of both tim and per mRNAs and
proteins. For example, expression of USP8-DN under
the control of the tim driver increases the RNA levels
of per, tim, and vri at almost all times of the circadian
cycle. Effects on pre-mRNA were similar and included
the usual trough times, from late night to early morning
(CT22–CT6) (Fig. 3C–E). The increased levels of mRNA
presumably contribute to the increase in PER and TIM
in head extracts at CT6–CT10, a time when there is no
more than a trace visible in wild-type extracts (Fig. 3A,B).
However, we note that per and tim RNAs and proteins are
still cycling in the USP8-DN-expressing flies, in apparent
conflict with the stronger behavioral phenotypes of these
mutant flies. We suspected that this inconsistency reflects
at least in part the heterogeneity of fly head tissues; i.e.,
a substantial circadian contribution from the eyes might
mask a quantitatively more important contribution of
USP8 within circadian neurons. Indeed, expression of
USP8-DN has a much more dramatic effect on the levels
of PER and TIM at CT10 and tim mRNA at CT22 in PDF
neurons(Figs.7, 8), reflecting andperhapsevencausingthe
long period or arrhythmicity of these flies (Fig. 2).
Most USP family proteins stabilize their substrates by
preventing proteasome-mediated degradation and there-
fore enhancing protein levels (Reyes-Turcu et al. 2009).
Yet USP8-DN increases CLK activity without affecting
CLK levels. The same conclusion results from the S2 cell
experiments: RNAi knockdown of endogenous USP8 or
chromatin was measured by seq-ChIP. CLK ChIP was performed at ZT10 and ZT22 in 3XFlag-CLK flies. The elution from CLK ChIP
was subjected to anti-ubiquitin immunoprecipitation. The precipitated DNA was analyzed by Drosophila Tiling 2.0 array (Affymetrix).
The CLK and ubiquitylated CLK binding on the promoters of per (A), tim (B), vri (C), and pdp1 (D) were visualized by IGB browser.
Thick black arrows indicate the peaks of ubiquitylated CLK binding near the promoter region of vri and pdp1 cycling isoform. Asterisk
indicates the RD and RJ isoforms of pdp1, which share the same transcription start site and promoter and only differ by one small exon
(42 base pairs). Thin black arrows indicate 59-to-39 transcription direction.
Ubiquitylated CLK binding on the promoter regions of per, tim, vri, and pdp1 genes. Ubiquitylated CLK binding on
Luo et al.
2544 GENES & DEVELOPMENT
transfection of USP8-DN enhanced CLK/CYC transcrip-
tion activity, which is still sensitive to PER repression
(Fig. 4B,C). Based on the results from flies as well as S2
cells, USP8 probably functions as an inhibitor of CLK/
CYC transcriptional activation.
Inhibition of USP8 causes an increase in CLK ubiqui-
tylation, mostly monoubiquitylation (Figs. 4, 5). This
presumably reflects the interaction of CLK with USP8
in S2 cells and fly heads (Fig. 5B); i.e., the direct deubiqui-
tylation of CLK. Interestingly, CLK ubiquitylation levels
cycle and peak at ZT10–ZT14, when maximal transcrip-
tion occurs (Fig. 5A). Moreover, expression of USP8-DN
dramatically increased the level of CLK ubiquitylation at
ZT10 and ZT22, especially at ZT22 (Fig. 5C). Because CLK
binding at the E-box of the tim promoter did not change
dramatically between the two strains (and perhaps even in
the opposite direction) (Fig. 5D), it is likely that CLK
monoubiquitylation does not enhance DNA binding, but
tion of a transcription factor has been shown to help recruit
other transcription factors and even Pol II to a promoter
(Salghetti et al. 2001; Kodadek 2010).
Ubiquitylation may be mechanistically related to the
‘‘black widow’’ model of transcription activation, in which
transcription factors on chromatin are actively degraded
by the proteasome (Tansey 2001). This may apply to
mammalian BMAL1, the ortholog of CYC, suggesting
that the same turnover mechanism may be relevant to
CLK/CYC in flies (Lee et al. 2008; Stratmann et al. 2012).
A maximally active chromatin-bound CLK may therefore
be monoubiquitylated, which can then experience two
inhibitory fates. It can be further ubiquitylated, perhaps
mediated degradation to allow replacement by another
maximally active monoubiquitylated CLK, or it can be
deubiquitylated by USP8. The former may predominate at
times of maximal transcription so that rapid recycling of
CLK/CYC by the proteasome maintains maximal levels of
monoubiquitylated CLK and maximal transcription rates.
In the late night–early morning, deubiquitylation by USP8
may predominate and help minimize clock gene transcrip-
tion. It is notable that these are circadian times when
cycling usp8 mRNA levels are maximal. However, it is
possible that regulation of an E3 ligase also contributes to
the cycling of CLK ubiquitylation.
The importance of differential ubiquitylation is rein-
forced by the different ubiquitylated CLK-binding pat-
terns on some clock genes; i.e., vri and pdp1. The fact that
ubiquitylated CLK occurs preferentially close to the tran-
scription start sites of these genes (Fig. 6; data not shown)
suggests that CLK monoubiquitylation may help recruit
factors to drive transcription; Pol II or factors associated
with transcription initiation are good candidates. In this
view, the eye-specific vri and pdp1 CLK-binding sites
with poor ubiquitylation may reflect CLK-binding sites
without these cofactors, allowing deubiquitylation to
predominate. The relatively poor CLK ubiquitylation at
these sites may also indicate a relationship with other
transcription factors. Put otherwise, deubiquitylated CLK
may play a more modest role at these sites and partner
with (unidentified) factors that contribute most of the
transcriptional activation potential, perhaps within cer-
tain eye cell types.
Nonetheless, the fact that CLK deubiquitylation by
USP8 appears maximal at the end of the transcriptional
cycle suggests that deubiquitylated CLK is associated
rons are disrupted in tim>DN flies. Representative
staining results of TIM (A) and PER (B) in uas-usp8-
DN (uas-DN) control and tim>DN brains. Fly heads
were collected at CT10 and CT22 of DD1 after 3 d of
LD entrainment at 29°C. Brains were immuno-
stained by anti-PDF (green), anti-TIM (magenta),
and anti-PER (blue) antibodies. PDF neurons were
labeled by PDF (green). At CT10 in the tim>DN
brains, TIM and PER staining signals remained high.
Bar, 50 mm. Yellow arrows point to PDF neurons.
PER and TIM oscillations in PDF neu-
CLOCK deubiquitylation by USP8
GENES & DEVELOPMENT2545
with changes in chromatin structure and/or transcription
complexes at this time. Indeed, most CLK is sequestered
by PER in an off-DNA inhibitory complex at the end of
thecycle (Menetet al. 2010), suggesting thatthis complex
contains substantial levels of deubiquitylated CLK. PER
may enhance USP8 activity on CLK or inhibit CLK
monoubiquitylation within the PER–CLK complex. Alter-
natively, PER may function strictly to inhibit DNA
binding, suggesting that CLK monoubiquitylation and
even deubiquitylation are predominantly on-DNA events.
It is interesting that the original analysis of cycling
mRNAs within PDF neurons indicated that the circadian
amplitudes of most clock gene mRNAs were dramati-
cally enhanced compared with their amplitudes in head
RNA (Kula-Eversole et al. 2010). This is consistent with
the enhanced effect of USP8 inhibition of mRNA cycling
in PDF neurons compared with the effects on head RNA,
suggesting that USP8 function may contribute to this
enhanced amplitude of clock gene cycling. A challenge
for the future will be to understand how and why USP8
functions differentially within circadian neurons. It
will also be important to integrate this goal with the
ability to assay CLK/CYC binding and even ubiquityl-
ated CLK binding as a function of circadian time within
Materials and methods
All gal4 and gal80ts drivers used in this study have been
described previously: timgal4 (Kaneko et al. 2000), pdfgal4
(Stoleru et al. 2004), dvpdfgal4 (Bahn et al. 2009), tubgal80ts
(McGuire et al. 2003), usp8-RNAi R2 (Mukai et al. 2010), and
usp8-RNAi (HMS01889, Bloomington). uas-usp8-DN transgenic
flies were generated by injecting yw embryos with pUAST-usp8-
DN plasmid (BestGene, Inc.). The pUAST-usp8-DN plasmid was
generated by cloning usp8 coding sequence with cysteine at
residue 572 mutated to alanine into pUAST plasmid. uas-usp8
wild-type and uas-usp8-RB-DN (short isoform) transgenic flies
were generated by injecting yw embryos with pUAST-uas-usp8
flies were generated by injecting yw embryos with pCasPer4.0
3XFlag-clk14.8 plasmid. The pCasPer4.0 3XFlag-clk14.8 plas-
mid was constructed previously (Kadener et al. 2008) with the
following modification: Sequence encoding 3XFlag peptides
was inserted in-frame before the ATG codon of clk 14.8-kb
Young male flies were entrained and monitored for 3–4 d in LD
conditions, followed by at least 5 d in DD using Trikinetics
brains. Dissected brains were directly visualized by GFP microscope. (B) PDF mRNA is highly enriched in PDF neurons from pdf>DN
brains. pdf>DN flies were entrained for three LD cycles at 25°C and collected at CT11 and CT23 of DD1. PDF neurons were manually
sorted from papain-treated brains. PDF mRNA was analyzed by qPCR. The enrichment was calculated by PDF mRNA signals
normalized to rpl32 signals. (C) tim mRNA from PDF neurons remained high at CT11 in pdf>DN brains. Control flies and pdf>DN flies
were entrained, and PDF neurons were isolated as in B. tim mRNA level was assayed by qPCR. The Y-axis values are relative tim
mRNA signals normalized to rpl32. Results were averaged from three experiments.
tim mRNA in PDF neurons is not cycling in tim>DN flies. (A) PDF neurons are GFP-labeled in the control and pdf>DN
Luo et al.
2546 GENES & DEVELOPMENT
Drosophila activity monitors. Behavior analyses were performed
with a signal processing toolbox (Levine et al. 2002) using
ChIP–chip and seq-ChIP
Anti-CLK and anti-Pol II ChIPs were performed as previously
described (Abruzzi et al. 2011) with slight modifications: Fly
heads were quickly ground using a mortar on dry ice. The
resulting powder was transferred to a glass dounce homogenizer
and homogenized in 5 vol of homogenization buffer (10 mM
Hepes-KOH at pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 0.8 M
sucrose, 0.5 mM EDTA, 1 mM DTT, 13 protease inhibitor, 13
phosphatase inhibitor cocktail). Homogenates were loaded on
equal volumes of sucrose cushion buffer (with 1.0 M sucrose and
10% glycerol in the homogenization buffer) and centrifuged in
a HB-6 rotor (Sorvall) at 11,000 rpm for 10 min at 4°C. Pelleted
nuclei were cross-linked in 1% formaldehyde for 15 min at room
temperature and sonicated in 0.5 mL of lysis buffer (20 mM Tris-
HCl at pH 7.5, 150 mM NaCl, 10% glycerol, 1% NP-40, 1 mM
DTT, PI and PPase inhibitors).
Seq-ChIP was performed according to published protocols
(Geisberg and Struhl 2004) with the following modification:
Chromatin immunoprecipitated by anti-Flag beads was eluted
by 150 mL of ChIP elution buffer (50 mM Tris–HCl at pH 7.5, 10
mM EDTA, 1% SDS) for 10 min at 65°C. Elutes (135 mL) were
adjusted to 1.5 mL by adding lysis buffer (20 mM Tris-HCl at pH
7.5, 10% glycerol, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM
DTT, 10 mM N-ethylmaleimide) and incubated with 5 mL of FK2
ubiquitin antibody and 30 mL of protein G magnetic beads
(Invitrogen) for 2 hat room temperature inthe presence of 5 mg/mL
BSA and 50 mg/mL Escherichia coli tRNA. Precipitated chromatin
was eluted by ChIP elution buffer, decross-linked for 6 h at 65°C,
analysis were performed as previously described (Abruzzi et al.
Western blotting and immunoprecipitation
S2 cell and fly head extracts were prepared in lysis buffer (20 mM
Tris-HCl at pH 7.5, 10% glycerol, 1% NP-40, 150 mM NaCl,
1 mM EDTA, 1 mM DTT, 10 mM N-ethylmaleimide) supple-
mented by protease inhibitor cocktail and phosphatase inhibitor
cocktail. Extracts were subjected to mild sonication. For exper-
iments of phosphatase treatment, phosphatase inhibitor cocktail
was omitted. Clear and denatured lysates were resolved by
NuPAGE Novex 3%–8% Tris-Acetate gel (Invitrogen). Protein
transfer was performed by using the iBlot dry blotting system
(Invitrogen). Protein bands were visualized by an ECL reagent kit
according to the manufacturer’s manual. For CLK immunopre-
cipitation, 25 mL of M2 anti-Flag beads (Sigma) were incubated
with extracts for 2 h at 4°C. Proteins were eluted by 13 SDS
loading buffer for 5 min at 95°C. Antibodies used for Western
blotting were as follows: anti-CLK (Houl et al. 2008), anti-PER
(Dembinska et al. 1997), anti-TIM (Zeng et al. 1996), anti-USP8
(Mukai et al. 2010), anti-USP7 (van der Knaap et al. 2005), anti-
Flag (Sigma), anti-V5 (Invitrogen), and Fk2 anti-ubiquitin (Enzo
S2 cell transfection, dsRNA systhesis, RNAi treatment,
and luciferase activity assay
S2 cells were maintained in insect tissue culture medium
(HyClone) supplemented with 10% fetal bovine serum and
antibiotics. Transfection was performed at 60%–70% confluence
with 10 mL of cellfectin II (Invitrogen) per well in a six-well plate
or 75 mL of cellfectin in a 75-cm2flask. We performed dsRNA
synthesis and RNAi treatment of S2 cells according to the
protocol described previously (Nawathean et al. 2005). For
luciferase activity experiments, 50 ng of luciferase firefly re-
porter and 100 ng of pCopia-Renilla luciferase reporter (trans-
fection control) were always included in the transfection.
RNA analysis by real-time PCR
Total RNA was prepared from adult heads using Trizol reagent
(Invitrogen) and was DNase-treated using RQ1 DNase (Promega)
according to the manufacturer’s protocols. Two micrograms of
RNA was used for RT–PCR using SuperScript II (Invitrogen) and
random primers (Promega). The resulting cDNA was used as
a template in qPCR using the Syber Master Mix (Qiagen) and
a Rotorgene qPCR machine (Qiagen). Primer sequences used for
qRT–PCR are available on request. Total RNA purification and
amplification from PDF neurons were performed according to
published protocols (Nagoshi et al. 2009).
Immunostaining of Drosophila brains using anti-PER, anti-PDF
(Shafer et al. 2002), and anti-TIM (Tang et al. 2010) was
performed as described previously. Dissected brains were in-
cubated with PER antibody (1:1000) for two overnights at 4°C or
with rat anti-TIM (1:1000) overnight at 4°C.
We thank Paul Hardin for generously providing the CLK anti-
body, and Satoshi Goto for providing the USP8 antibody and usp8
RNAi strain. We thank Sebastian Kadener, Sean Bradley, Jerome
Menet, and members of the Rosbash laboratory for valuable
advice and comment. This research was supported by the Howard
Hughes Medical Institute and NIH grants (P01 NS44232 to M.R.).
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