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The Journal of Neuroscience, March 1, 2000, 20(5):1746-1753
dCLOCK Is Present in Limiting Amounts and Likely Mediates Daily
Interactions between the dCLOCK-CYC Transcription Factor and the
PER-TIM Complex
Kiho
Bae1,
Choogon
Lee1,
Paul E.
Hardin3, and
Isaac
Edery2
1 Graduate Program in Microbiology and Molecular
Genetics and 2 Department of Molecular Biology and
Biochemistry, Rutgers University, Center for Advanced Biotechnology and
Medicine, Piscataway, New Jersey 08854, and 3 Department of
Biology and Biochemistry, University of Houston, Houston, Texas
77204-5513
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ABSTRACT |
In Drosophila melanogaster four circadian clock
proteins termed PERIOD (PER), TIMELESS (TIM), dCLOCK (dCLK), and CYCLE
(CYC/dBMAL1) function in a transcriptional feedback loop that is a core
element of the oscillator mechanism. dCLK and CYC are members of the
basic helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of
transcription factors and are required for high-level expression of
per and tim and repression of
dClk, whereas PER and TIM inhibit dCLK-CYC-mediated transcription and lead to the activation of dClk. To
understand further the dynamic regulation within the circadian
oscillator mechanism, we biochemically characterized in
vivo-produced CYC, determined the interactions of the four
clock proteins, and calculated their absolute levels as a function of
time. Our results indicate that throughout a daily cycle the majority
of the dCLK present in adult heads stably interacts with CYC,
indicating that CYC is the primary in vivo partner of
dCLK. dCLK-CYC dimers are bound by PER and TIM during the late evening
and early morning, suggesting the formation of a tetrameric complex
with impaired transcriptional activity. Although dCLK is present in
limiting amounts and CYC is by far the most abundant of the four clock
proteins that have been examined, PER and TIM appear to interact
preferentially with dCLK. Our results suggest that dCLK is the main
component regulating the daily abundance of transcriptionally
active dCLK-CYC complexes.
Key words:
circadian rhythms; clock proteins; Drosophila; transcription; PAS; protein-protein
interactions
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INTRODUCTION |
A transcriptional feedback loop
composed of at least four proteins termed PERIOD (PER), TIMELESS (TIM),
dCLOCK (dCLK), and CYCLE (CYC/dBMAL1) is a core element of the
circadian clock in Drosophila melanogaster (for review, see
Hardin, 1998 ; Reppert, 1998; Young, 1998; Dunlap, 1999; Edery, 1999).
dCLK and CYC are members of the basic helix-loop-helix (bHLH)/PAS
(PER-ARNT-SIM) superfamily of transcription factors (Allada et al.,
1998; Bae et al., 1998; Darlington et al., 1998; Rutila et al., 1998)
that heterodimerize to activate per and tim by
binding E-box elements (Hao et al., 1997, 1999; Allada et al., 1998;
Darlington et al., 1998; Rutila et al., 1998; Lee et al., 1999) and
that act to repress dClk transcription (Glossop et al.,
1999).
The biochemical activities of PER and TIM are less well understood than
those of dCLK and CYC, but current evidence indicates that they repress
their own transcription and activate dClk transcription (Bae
et al., 1998; Dunlap, 1999). During the late day/early evening PER and
TIM accumulate in the cytoplasm and eventually interact to form a
complex (Lee et al., 1996; Zeng et al., 1996) that enters the nucleus
in a temporally gated manner (Curtin et al., 1995). Nuclear entry of
the PER-TIM complex is accompanied by decreases in the levels of
per and tim transcripts and increases in
dClk transcripts. In Drosophila tissue culture
cells the ectopic coexpression of PER and TIM inhibit dCLK-mediated
stimulation of a reporter gene driven by E-box elements found in 5'
regulatory regions of per or tim (Darlington et
al., 1998). Furthermore, PER and TIM interact with dCLK or a
dCLK-containing complex during times in the day (Lee et al., 1998) when
the transcription rates of per and tim are
decreasing (So and Rosbash, 1997) and dClk transcripts increase (Bae et al., 1998), consistent with the suggestion that dCLK-mediated transcriptional regulation (either positive or negative) is inhibited by the binding of PER and/or TIM. Recent results using
in vitro-synthesized clock proteins suggest that PER and TIM
participate in the circadian feedback mechanism, at least partly, by
abrogating the DNA binding activity of a dCLK-CYC heterodimer (Lee et
al., 1999).
To gain further insight into the molecular circuitry underlying the
circadian transcriptional feedback mechanism in Drosophila, we biochemically characterized CYC produced in vivo and
defined its interactions with dCLK, PER, and TIM as a function of time. In addition, although previous studies have measured the relative levels of individual clock proteins, their absolute amounts are not
known. To this end we calculated the molar concentrations of dCLK, CYC,
PER, and TIM in adult fly heads as a function of time throughout a
daily cycle. We show that in adult heads the majority of dCLK stably
interacts with CYC throughout a daily cycle, strongly suggesting that
the primary or perhaps only physiologically relevant partner of dCLK in
this tissue is CYC. PER and TIM mainly interact with the dCLK-CYC
transcription factor during the late night. dCLK is present in limiting
amounts, and our findings suggest that PER and TIM preferentially
interact with dCLK as compared with CYC. Similar to recent results
obtained by using analogous proteins synthesized in vitro
(Lee et al., 1999), the binding of PER and TIM do not affect the
association of dCLK with CYC. Together, our findings strongly suggest
that dCLK is the key "molecular bridge" regulating the dynamic
interactions that activate or repress gene expression within the
Drosophila circadian feedback mechanism.
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MATERIALS AND METHODS |
Fly strains and collections. The wild-type Canton-S
(CS) flies and the mutant per01
flies used in this study were descendants of stocks originally maintained in the laboratory of Dr. M. Rosbash (Brandeis University, Waltham, MA), and were described previously (Edery et al.,
1994). The tim0 flies were
descendants of stocks originally maintained in the laboratory of Dr. A. Sehgal (University of Pennsylvania Medical School, Philadelphia, PA)
(Sehgal et al., 1994), and the cyc0
flies were described previously (Rutila et al., 1998). All flies were
grown and maintained in vials or bottles containing standard agar-cornmeal-sugar-yeast-tegosept media. Vials containing
~100 young (2- to 6-d-old) adult flies were placed in incubators
(Precision Scientific, Winchester, VA) at 25°C, exposed to at least
two cycles of 12 hr light/dark [LD; in which Zeitgeber time 0 (ZT0) is
lights-on and ZT12 is lights-off], and subsequently were maintained in
the dark (DD). At selected times during LD and DD the flies were
collected by rapid freezing in dry ice, and the heads were isolated.
In vitro transcription and translation. Recombinant plasmids
used in this study for the in vitro synthesis of PER, TIM,
dCLK, and CYC were described previously (Citri et al., 1987; Bae et al., 1998; Lee et al., 1999). To produce DOUBLE-TIME (DBT) in vitro (see Fig. 1A), we obtained a cDNA
plasmid containing the entire open reading frame (ORF) of
dbt (GenBank accession number AF055583) from Genome Systems
(St. Louis, MO). Subsequently, the plasmid was digested with
SacI and SacII, and the entire dbt ORF
was subcloned into pGEM-5Zf (+) (Promega, Madison, WI), which placed
the expression of dbt under control of the SP6 promoter (yielding a plasmid termed pGEM-5Zf dbt). In
vitro radiolabeled translation products were produced by using the
appropriate circular plasmids (described above) to prime a coupled
transcription/translation rabbit reticulocyte system (TNT; Promega) in
the presence of
L-[35S]methionine
(Amersham, Arlington Heights, IL) according to the manufacturer's
protocol. The amount of protein produced in each translation was
determined by subjecting an aliquot of the translation mixture to
trichloroacetic acid precipitation and normalizing for methionine
content. An incubation that did not contain exogenously added plasmid
served as a background control for the translation reactions. In
vitro-translated proteins were resolved by PAGE with 6 or
12% gel and visualized by either autoradiography (e.g., see Fig.
1A) or immunoblotting (e.g., see Fig.
1B).
Antibodies and immunoblotting. To generate antibodies to
CYC, we used PCR to amplify the entire open reading frame of
cyc by using a plasmid that contained a full-length
cyc cDNA (EST plasmid obtained from Genome Systems; GenBank
accession number AA695336). Then the PCR product was digested with
NdeI and EcoRI and cloned upstream of sequences
that encode a polyhistidine stretch (His) in the expression
vector pET23b (Novagen, Madison, WI). The oligonucleotide primers used
in the PCR were (cyc sequences are in italics)
5'-AAATCATATGGAAGTTCAGGAGTTCTGCG-3' and
5'-GGATAAGAACACGGAATTCTTGGCG-3'. The CYC-His fusion
protein was produced in bacteria according to the manufacturer's
recommended procedure (Novagen) and purified under
denaturing conditions (8 M urea), by using the
Talon metal affinity resin from Clontech (Palo Alto, CA). The purified
CYC-His fusion protein was used as an immunogen to produce antibodies in rats and guinea pigs (Cocalico Biologicals, Reamstown, PA). In this
study we used a guinea pig anti-CYC antibody (GP-122) that strongly
recognized in vitro-translated CYC (see Fig. 1).
Preparation of total fly head extract was essentially as described
(Edery et al., 1994; Lee et al., 1998). For each time point ~30 µl
of heads isolated from frozen flies were placed in a Microfuge tube and
homogenized at 4°C in 3 vol (relative to heads) of extraction buffer
1 [EB1; containing (in mM) 100 KCl, 20 HEPES, pH 7.5, 5 EDTA, 1 dithiothreitol (DTT), 0.25 PMSF, 5% glycerol, and 0.1% Triton
X-100, plus 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 µg/ml
pepstatin A], in a battery-operated minihomogenizer (Kontes, Vineland,
NJ). Subsequently, homogenates were centrifuged twice (12 min at
12,000 × g), and clarified supernatants were removed to new tubes. Protein concentration was determined by using a Coomassie
protein assay according to the manufacturer's instructions (Pierce,
Rockford, IL). An equal volume of 2× SDS-sample buffer was added to
the supernatant fraction, and the mixture was boiled. Equal amounts of
total protein (~20 µg total at ~4 µg/µl) from clarified
supernatant fractions were resolved by PAGE and transferred to
nitrocellulose paper; immunoblots were treated with chemiluminescence (ECL, Amersham) essentially as described (Lee et al., 1998; Sidote et
al., 1998). To visualize dCLK, PER, and TIM, we used 6%
SDS-polyacrylamide gels, whereas 12% SDS-polyacrylamide gels were used
to detect CYC, as indicated in the figure legends. Immunoblots were
incubated in the presence of anti-dCLK, anti-PER, anti-TIM, or anti-CYC antibodies at a final concentration of 1:2000. The antibodies to dCLK
(dCGP90), PER (GP73), and TIM (TR1-E3 and GP72-2) used in this study
were as described (Lee et al., 1998; Sidote et al., 1998). Bands on
autoradiographs were quantified with a densitometer (Computing
Densitometer Scan version 5.0) and ImageQuant software (Molecular
Dynamics, Sunnyvale, CA). Scanned images of autoradiographs were
manipulated with Adobe Photoshop 5.0 and Canvas 5.0.3 software.
To determine the concentrations of PER, TIM, dCLK, and CYC in extracts
prepared from adult fly heads (see Fig. 4), we resolved (1) serial
dilutions of different head extracts (4 µg of total protein/µl),
each one corresponding to a time in LD when peak levels of either dCLK
(i.e., ZT4), TIM (i.e., ZT16), or PER (i.e., ZT20) were attained, and
(2) reticulocyte lysates containing serial dilutions of known amounts
of target proteins by SDS-PAGE and immunoblotted them. The intensities
of appropriate bands were measured with a densitometer, as described
above. In each experiment the intensities of relevant immunoreactive
bands in head extracts and reticulocyte lysates yielded linear
doses-responses (data not shown). Absolute peak levels for each clock
protein in head extracts were calculated by comparing their staining
intensities with those from the relevant radiolabeled products,
followed by correction for methionine content to obtain molar
concentrations. For each protein the calculations of peak values
derived from at least two independent experiments were pooled.
Estimates of peak amounts for any given protein did not vary by >25%
when comparing values obtained from the different independent
experiments (data not shown). After the average peak levels for each of
the target proteins were determined, the rest of the daily values were
obtained by curve fitting, using the relative abundance profiles shown in Figure 3D. To ensure that our estimates of the absolute
levels of individual clock proteins as a function of time were
reliable, we also repeated the same procedure by using head extracts in which individual clock proteins attained ~25 and 50% of peak levels (i.e., ZT12 and 16 for PER; ZT12 and 20 for TIM; ZT12, 16, 20, and 23.9 for dCLK). For each clock protein the results obtained were similar to
those based on peak values (data not shown).
RNase protection assay. For each time point, total RNA was
extracted from ~10 µl of fly heads by using TriReagent (Sigma, St.
Louis, MO), as described previously (Majercak et al., 1997). The
abundance of cyc and per transcripts was
determined by RNase protection assays (Hardin et al., 1990) performed
with the modification previously described (Zeng et al., 1994). To
measure cyc RNA levels, we linearized the cyc EST
plasmid (described above) with BglII and produced antisense
radiolabeled probe in vitro by using T7 RNA polymerase. The
radiolabeled antisense probe used to determine the levels of
per RNA was as previously described (Bae et al., 1998;
Sidote et al., 1998). As a control for RNA loading in each lane, a
ribosomal protein probe (RP49) was included in each protection assay
(Hardin et al., 1990). Protected bands were quantified with a
PhosphorImager from Molecular Dynamics, and values were normalized relative to those of RP49 (Hardin et al., 1990).
Immunoprecipitation. For each time point the fly heads were
homogenized as described above, except that extraction buffer 2 (EB2)
was used instead of EB1 [containing (in mM) 5 Tris-HCl, pH
7.5, 50 KCl, 10 HEPES, pH 7.5, 1 EDTA, 1 DTT, 0.25 PMSF, 10% glycerol,
0.05% Triton X-100, plus 10 µg/ml aprotinin, 10 µg/ml leupeptin,
and 1 µg/ml pepstatin A]. To remove nonspecific interactions, we
first incubated head extracts (1.2 mg of total protein in a final
volume of 0.4 ml) with 20 µl of Gammabind Plus beads (Pharmacia, Piscataway, NJ) for 25 min at 4°C; we centrifuged and then removed the clarified supernatant to a new tube. A slurry containing either 3 µl of anti-CYC (GP-122) or anti-dCLK (dCGP90) antibody and 20 µl of
Gammabind Plus (Pharmacia) were added to the precleared head extracts
and incubated with gentle rotation for 2 hr at 4°C. Subsequently, the
beads were collected by centrifugation; in some cases (see Fig. 5)
additional rounds of immunoprecipitation were performed on the
clarified supernatants. Immune complexes were washed three times (0.5 ml of EB2 for 7 min each), mixed with 20 µl of 1× SDS-sample buffer,
boiled, and centrifuged; the resulting supernatant was resolved by
immunoblotting as described above. A similar procedure also was used to
immunoprecipitate radiolabeled target proteins synthesized in
vitro (see Fig. 1A).
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RESULTS |
The abundance of CYC is constant throughout a daily cycle
Previous work has demonstrated that in adult heads the levels of
PER, TIM, and dCLK undergo daily oscillations (Edery et al., 1994;
Hunter-Ensor et al., 1996; Myers et al., 1996; Zeng et al., 1996; Lee
et al., 1998). Heads normally are used to investigate the temporal
profiles of clock proteins in Drosophila because it is the
anatomical location of the best-characterized circadian pacemaker in
this species (Handler and Konopka, 1979; Ewer et al., 1992). To
characterize CYC biochemically in vivo, we immunized guinea
pigs and rats with bacterially produced CYC (see Materials and
Methods). The immunoreactivity and specificity of the individual antiserum were tested initially by performing Western blots
(immunoblots) of in vitro-translated CYC (Fig.
1) (data not shown).
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Figure 1.
Biochemical detection of CYC in Drosophila
melanogaster heads. Cell-free extracts were derived from either
in vitro translation reactions performed in the presence
of [35S]methionine (A,
B, lanes 1-4, and
C) or adult fly heads (A,
B, lanes 5, 6). The
radiolabeled target proteins produced in vitro (i.e.,
DBT, PER, TIM, dCLK, and CYC) and the genotype of flies that were used
to prepare head extracts (i.e., wild-type CS flies or
cyc0 mutants) are indicated on
top. Head extracts were prepared from flies collected at
time 25 (in hours since the last dark/light transition at ZT0).
C, In vitro translation products were
incubated with the indicated antibodies, and immune complexes were
recovered. Fly head extracts, in vitro translation
reactions, and immune complexes were resolved by 6 or 12% (in the case
of CYC) PAGE and either visualized by fluorography and autoradiography
(A, C) or transferred to nitrocellulose; the immunoblots
were probed with anti-CYC antibodies (B).
A, B, Two different amounts of in
vitro-translated CYC were resolved by PAGE (1× = 77 pg).
B, The arrow (left) and
asterisk (right) identify nonspecific bands that
cross-react with the anti-CYC antibody in rabbit reticulocyte lysates
(lanes 1-4) and head extracts
(lanes 5, 6), respectively.
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Figure 1B shows a typical immunoblotting result
obtained with our strongest anti-CYC antibody; in
vitro-translated CYC [Fig. 1A, lanes
3, 4; the appearance of a minor band that migrates slightly faster than the major in vitro-synthesized CYC product is
attributable to the occasional use of an internal in-frame AUG (data
not shown)], but not an equivalent molar amount of another
Drosophila clock protein of similar predicted molecular
weight, termed DOUBLE-TIME (DBT) (Kloss et al., 1998; Price et al.,
1998) (Fig. 1A, lane 1), was recognized by
the anti-CYC antibody (Fig. 1B, compare lanes 3, 4 with 1). In addition, the
anti-CYC antibody used in this study immunoprecipitates little or no
PER, TIM, or dCLOCK (Fig. 1C). When head extracts prepared
from wild-type flies were probed by immunoblotting in the presence of
this anti-CYC antibody, a strongly staining band of ~45 kDa was
detected (Fig. 1B, lane 5), in close
agreement with the predicted molecular mass of CYC (Rutila et al.,
1998). Importantly, this ~45 kDa immunoreactive band is absent in
head extracts prepared from either a presumptive null-mutant termed
cyc0 (Fig. 1B,
lane 6) or wild-type flies probed with preimmune sera (data not shown). Daily rhythms in locomotor activity and eclosion (emergence from pupal cases) as well as cycles in the protein and RNA
products from per, tim, and dClk are
abolished in cyc0 flies (Rutila et
al., 1998; Glossop et al., 1999). The
cyc0 mutation introduces a premature
stop codon that is predicted to remove the C-terminal 60% of this
protein, including most of the PAS domain, consistent with its
loss-of-function phenotype (Rutila et al., 1998). We did not observe
the appearance of novel smaller molecular weight products in the
cyc0 mutant when immunoblots were
incubated with anti-CYC antibody (data not shown), suggesting that the
putative truncated CYC protein produced in this mutant is highly
unstable. The reason or reasons for the slight variation in
electrophoretic mobility between CYC synthesized in vitro
(Fig. 1B, lane 4) and that produced
in the fly head (Fig. 1B, lane 5) are not known.
To test whether the abundance of CYC undergoes daily oscillations, we
entrained wild-type flies for 3-4 d under standard conditions of 12 hr
light/dark cycles [LD; in which Zeitgeber time 0 (ZT0) is lights-on
and ZT12 is lights-off], followed by constant darkness (DD). Flies
were collected at various times during LD and DD, and head extracts
were analyzed for the presence of CYC by immunoblotting (Fig.
2A). In sharp contrast
to PER (Edery et al., 1994), TIM (Hunter-Ensor et al., 1996; Myers et
al., 1996; Zeng et al., 1996), and dCLK (Lee et al., 1998), the
abundance of CYC remains constant during LD and DD. This result is
consistent with the observation that cyc mRNA levels are
expressed constitutively (Fig. 2B), as previously
reported (Rutila et al., 1998). We cannot rule out the possibility that
in a limited number of cells CYC undergoes daily changes in abundance
that are not detected by immunoblot analysis of total head extracts.
Furthermore, we did not detect changes in the electrophoretic mobility
of CYC as a function of time in a daily cycle (Fig.
2A). In contrast, PER (Edery et al., 1994), TIM (Zeng
et al., 1996), and dCLK (Lee et al., 1998) are modified by the addition
of phosphate moieties in a time-of-day specific manner, which results
in readily detectable changes in their electrophoretic mobilities.
Nonetheless, the different apparent molecular weights of in
vivo- and in vitro-produced CYC (see Fig. 1B) could suggest that, in Drosophila, CYC
undergoes post-translational modifications and/or that different size
variants of CYC are generated by alternative splicing or differential
usage of translation start sites.
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Figure 2.
Constitutive levels of CYC protein and RNA
throughout a daily cycle. Wild-type flies were exposed to three 12 hr
light/dark cycles (LD) and subsequently were kept in constant dark
conditions (DD). Collections were done at the indicated times (time 0 is defined as the last dark-to-light transition), and head extracts
either were analyzed by immunoblotting, using antibodies directed
against CYC (A), or were subjected to RNase
protection assays (B). A, As a
control for specificity, head extracts prepared from
cyc0 flies were included (lane
13). B, Comparison of the relative amounts of
cyc (open diamond) and per
(open square) RNA during the third day of LD and the
first day of DD. Relative RNA levels refers to ratios of
cyc or per transcripts to the
constitutively expressed RP49 RNA. Peak values for cyc
or per during a daily cycle were set to 100, and the
rest of the values were normalized. Horizontal bars
represent lights-on (open bar), lights-off
(filled bar), or subjective day (hatched
bar). Similar results were obtained in three independent
experiments; representative examples are shown.
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dCLOCK interacts with CYC throughout a daily cycle
To determine the interaction profile of CYC as a function of time
in a daily cycle, we incubated head extracts with anti-CYC antibodies
and probed the recovered immune complexes for the presence of PER, TIM,
and dCLK (Fig. 3A).
Unfortunately, we were not able to visualize immunoprecipitated CYC by
Western blotting because of the presence of strongly staining
nonspecific bands that comigrated with CYC (data not shown). To
estimate the efficiency of CYC recovery from head extracts with our
anti-CYC antibody, we added trace amounts of in
vitro-generated radiolabeled CYC to head extracts. The results
indicated that, under the conditions used in this study for
immunoprecipitation, the majority of the radiolabeled CYC protein was
recovered in the immune complex, with very little remaining in the
supernatant (data not shown). This suggests that the bulk of the
in vivo-produced CYC protein present in head extracts also
is being recovered after incubation with anti-CYC antibodies.
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Figure 3.
dCLK, PER, and TIM interact with CYC in a
time-of-day specific manner. Wild-type (CS) flies were collected at the
indicated times in LD (A, lanes
1-7; B, lanes
1-6). per01
(A, lane 8; B, lane
7) and tim0
(A, lane 9; B, lane
8) mutants were collected at time 23.5. Head extracts were
prepared and either subjected to immunoprecipitation using antibodies
against CYC (A) or analyzed directly
(B). A, Immune pellets were
divided into three equal aliquots; each fraction was probed for the
presence of dCLK (top), PER (middle), or
TIM (bottom). A, B, Twelve
percent polyacrylamide gels were used to detect CYC, whereas 6%
polyacrylamide gels were used to detect PER, TIM, and dCLK. The size
ranges of the relevant proteins are indicated (left).
The arrow (left, top panel
in B) indicates a nonspecific band recognized by the
anti-dCLK antibody that was used. C, D,
Quantitation of results shown in A and B,
respectively. Peak values for each protein were set to 100, and the
rest of the values were normalized. Horizontal bars
represent either 12 hr light (open bar) or 12 hr dark
(filled bar).
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The findings clearly indicate that, as expected, dCLK and CYC form a
partnership in vivo, an interaction that is observed throughout a daily cycle (Fig. 3A, top
panel). In agreement with previous findings that used
anti-dCLK antibodies to perform immunoprecipitations (Lee et al.,
1998), PER and TIM mainly associate with CYC (or a CYC-containing
complex) during the night (Fig. 3A, bottom two panels) when the transcription rates of per and
tim are low (So and Rosbash, 1997). Very little dCLK is
present in immune complexes recovered from head extracts prepared from
per01 and
tim0 mutant flies (Fig.
3A, top panel, lanes 8, 9), which do
not make functional PER and TIM, respectively (Konopka and Benzer,
1971; Sehgal et al., 1994). This is consistent with an earlier finding showing that the daily accumulation of dCLK requires PER and TIM (Lee
et al., 1998) (Fig. 3B, top panel, lanes 7, 8). In sharp contrast, the abundance of the CYC protein does not appear to be
regulated by PER or TIM (Fig. 3B, bottom panel, lane
8) (data not shown).
dCLOCK is present in limiting amounts in head extracts
A comparison of the curves for the daily changes in the levels of
dCLK, PER, and TIM after recovery with anti-CYC antibodies with those
obtained when total head extracts are analyzed directly revealed that
the temporal abundance profiles of dCLK are very similar in both cases
(compare Fig. 3C,D). Moreover, immune complexes recovered
with antibodies that recognize either dCLK or CYC yield essentially
identical profiles in the PER and TIM abundance rhythms (Fig.
3A) [see Lee et al. (1998), their Fig. 4]. A possible
explanation that could account for these observations is that the
interaction of PER and TIM with CYC is mediated by dCLK and that dCLK
is generally present in limiting amounts. However, molar concentrations
have not been calculated for any Drosophila clock protein.
To better understand the variables that might contribute to the dynamic changes underlying the interactions among CYC, dCLK, PER, and TIM, we
determined the absolute concentrations of the different clock proteins
in head extracts as a function of time in a daily cycle (Fig.
4; see Materials and Methods).
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Figure 4.
Average daily levels of dCLK are limiting. Shown
are the molar concentrations (10 18 mol/µg total
head protein) of dCLK, CYC, PER, and TIM during LD. For each protein
the peak amounts were calculated by pooling results obtained from at
least two independent experiments. The rest of the data points were
generated by curve fitting, using the results shown in Figure
3D (see Materials and Methods). Peak concentrations
(1018 mol/µg total head protein) for each
protein also are indicated. Note that, because PER has a very broad
electrophoretic mobility during the late night/early morning
attributable to differential phosphorylation (Edery et al., 1994), we
believe that PER levels during these times might be overestimated by up
to 50% (data not shown).
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In LD the peak molar concentration of dCLK is four- to sixfold lower
than the highest values obtained for PER and TIM, and it is ~200-fold
lower than those of CYC (Fig. 4), making CYC by far the most abundant
of the four clock proteins that have been examined. The steady-state
molar concentrations of PER and TIM are similar in head extracts,
supporting earlier calculations of the relative molar amounts of each
protein (Zeng et al., 1996; Lee et al., 1998). On the basis of the
large molar excess of CYC as compared with dCLK, we reasoned that
throughout a daily cycle the majority of dCLK might be bound to CYC.
This prediction was confirmed by the observation that even when dCLK is
at peak levels little, if any, dCLK remains in the supernatant fraction
after recovery of immune complexes with anti-CYC antibodies (Fig.
5A, top panel,
compare lanes 2 and 1, 4 and
3). That the majority of dCLK in the adult head is stably
bound to CYC also is indicated by the observation that approximately
equal amounts of dCLK are immunoprecipitated from head extracts with
antibodies against either dCLK or CYC (lanes 6, 7).
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Figure 5.
The majority of dCLK is bound to CYC during a
daily cycle. A, B, Wild-type flies were
collected at the indicated times during LD; head extracts were prepared
and subjected to immunoprecipitation (IP), using antibodies against
either CYC (A, lanes 1-4, 7, 8) or dCLK (A, lane 6;
B, lanes 2-7). In
some cases the remaining supernatant fraction subsequently was
subjected to a second round of IP, using antibodies against either dCLK
(A, lanes 2, 4, 8) or CYC
(B, lanes 3, 4, 6, 7). Finally, in some cases the supernatant resulting
from the second IP was subjected to a third round of IP, using
antibodies against PER (B, lanes
4, 7). Recovered immune complexes
were probed for the presence of dCLK, PER, or TIM as indicated
(left of panels). Control incubations
using irrelevant antibodies were used to show specificity during IP
(A, lane 5; B, lane
1) (data not shown).
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|
The results further indicate that, although significantly more PER and
TIM interact with the dCLK-CYC complex at ZT23.9 as compared with ZT4
(Fig. 5A, bottom two panels, compare
lanes 1, 3), there is no increase in the relative
amount of dCLK that is free of CYC (Fig. 5A, top
panel, compare lanes 1, 2 with 3, 4). The observation that the majority of dCLK stably
associates with CYC despite the presence of significant amounts of PER
and TIM provides biochemical evidence strongly suggesting that,
in vivo, the binding of PER and/or TIM to dCLK and/or CYC
does not disrupt the dCLK-CYC heterodimer. These in vivo
results are consistent with recent findings obtained by using the
analogous proteins synthesized in vitro (Lee et al.,
1999).
Because the molar concentrations of PER and TIM are substantially
higher than those of dCLK during the night (see Fig. 4), we wondered
whether PER and TIM interact with CYC that is not bound to dCLK. To
address this possibility, we first recovered immune complexes with
anti-dCLK antibodies (Fig. 5B, lanes 2, 5) and subjected the supernatant fraction to another round of immunoprecipitation by using anti-CYC antibodies (Fig. 5B,
lanes 3, 6). Subsequently, the supernatant
after incubation with anti-CYC antibodies was subjected to a further
incubation with anti-PER (Fig. 5B, lanes 4, 7) or anti-TIM (data not shown) antibodies to measure the
amounts of PER and TIM that are free of dCLK and CYC (Fig.
5B). The results indicate that in the late night more PER
and TIM associate with dCLK-CYC containing complexes as compared with
CYC that is relatively free of dCLK (e.g., see results obtained at
ZT20, Fig. 5B, lanes 2, 3).
Importantly, despite the large excess of CYC the overwhelming majority
of the PER and TIM proteins present in head extracts does not interact
with CYC (Fig. 5B, bottom two panels, compare
lanes 3, 4 and 6, 7). The most
parsimonious explanation that can account for these observations is
that PER and TIM preferentially interact with dCLK (or the dCLK-CYC
heterodimer) as compared with "free" CYC. Because dCLK is present
in limiting amounts (see Fig. 4) and most of it is bound to CYC (Fig.
5A), only a small fraction of the PER and TIM proteins
associates with the dCLK-CYC heterodimer (Fig. 5B). Our
findings suggest that during the night dCLK acts as a molecular bridge
or scaffold that simultaneously interacts with the PER-TIM complex and
CYC. A caveat to this contention is that, although CYC is highly
abundant in head extracts, it might be present in limiting amounts in
PER/TIM-expressing cells. The apparent in vivo preference
for dCLK is somewhat different from the situation observed in
vitro whereby PER appears to interact equally well with both dCLK
and CYC, but TIM only binds dCLK (Lee et al., 1999).
| |
DISCUSSION |
To understand further the molecular underpinnings governing the
dCLK-CYC-PER-TIM four-component transcriptional feedback mechanism, we have undertaken direct biochemical studies of these proteins. We
recently showed that in adult fly heads PER and TIM mainly interact
with dCLK or a dCLK-containing complex during the night/early morning
(Lee et al., 1998), in agreement with the observation that the
transcription rates of per and tim are low and
that dClk transcripts accumulate during these times in a
daily cycle (So and Rosbash, 1997; Bae et al., 1998). PER, TIM, or both
inhibit the DNA binding activity of dCLK-CYC in vitro (Lee
et al., 1999), suggesting a mechanism for how PER and TIM participate
in both transcriptional autoinhibition and dClk activation.
This inhibition was not accompanied by the disruption of the dCLK-CYC
heterodimer (Lee et al., 1999). In this report we biochemically
characterized in vivo-produced CYC and determined its
ability to interact with dCLK, PER, or TIM as a function of time in a
daily cycle. In addition, we also determined the molar concentrations
of the four clock proteins in adult head extracts. Throughout a daily
cycle the majority of dCLK is stably bound to the constitutively
expressed CYC protein, suggesting that in the adult fly head CYC is the major physiologically relevant bHLH/PAS partner of dCLK. Consistent with recent findings obtained in vitro (Lee et al., 1999),
PER and TIM do not disrupt the dCLK-CYC interaction. In contrast to previous models based on the ability of the PER-PAS domain to mediate
protein-protein dimerization (Huang et al., 1993), our results
indicate that PER (and TIM) does not engage in autoinhibition by
forming nonfunctional heterodimers with PAS-containing transcription factors. Furthermore, the results suggest that the PER-TIM complex mediates transcriptional autoinhibition and dClk activation
by specifically targeting dCLK, raising the possibility that the highly
abundant CYC protein interacts with other bHLH/PAS partners that
function in pathways besides the circadian clock.
By comparing the staining intensities of known amounts of in
vitro-synthesized clock proteins with their counterparts in head extracts, we were able to estimate the molar concentrations of PER,
TIM, dCLK, and CYC present in adult heads throughout a daily cycle (see
Fig. 4). The validity of our calculations is supported by the fact that
our strategy for estimating the concentrations of the various clock
proteins yielded approximately similar molar concentrations for PER and
TIM during the mid-to-late night, consistent with earlier studies that
used independent approaches (Zeng et al., 1996; Lee et al., 1998). dCLK
is almost certainly the component limiting the maximum levels of
dCLK-CYC complexes that can be assembled. Although calculations based
on total head extracts do not take into account possible differences in
spatial distributions, it is highly likely that, in all dCLK-containing
cells in the adult head, CYC is also present and at levels higher than
those of dCLK. In addition to our finding that CYC is present in at least a 200-fold molar excess relative to dCLK in head extracts (see
Fig. 4), this contention is supported further by the observations that
(1) at all times in a daily cycle essentially all of the dCLK is stably
bound to CYC (see Fig. 5A), and (2) the temporal abundance
profile of dCLK in immune complexes recovered with anti-CYC antibodies
is very similar to that of dCLK present in head extracts (see Fig.
3C,D).
PER and TIM mainly interact with dCLK/CYC during the night (see Fig.
3A), consistent with previous findings showing that PER and
TIM inhibit the activity of the dCLK-CYC heterodimer (Darlington et
al., 1998; Lee et al., 1999) and that the transcription rates of
per and tim are low during the night (So and
Rosbash, 1997). Nonetheless, during the night the majority of PER and
TIM is not associated with dCLK or CYC (see Fig. 5B). Our
findings suggest that this is because dCLK is present in limiting
amounts (see Fig. 4) and that PER and TIM (or the PER-TIM complex)
preferentially interact with dCLK (or the dCLK-CYC heterodimer) as
compared with free CYC (see Fig. 5B). In addition to
relative differences in levels, temporal changes in the subcellular
distributions of PER and TIM also might regulate their interactions
with the dCLK-CYC transcription factor. PER and TIM are detected first
in the nucleus of key pacemaker neurons in the brain at approximately
ZT17 (Curtin et al., 1995; Hunter-Ensor et al., 1996; Myers et al.,
1996), coincident with times in a daily cycle when significant amounts of PER and TIM first interact with the dCLK-CYC complex (see Fig. 3A). Immunohistochemical studies will be important in
determining whether the subcellular distributions of dCLK and CYC are
regulated and the extent to which their spatial distribution patterns
overlap those of PER and TIM. Because most of the PER-TIM complex is
not stably interacting with the dCLK-CYC heterodimer, our results raise the possibility that PER and/or TIM directly regulates other transcription factors.
Although our findings using head extracts suggest that PER and TIM
preferentially interact with dCLK as compared with CYC, results
obtained by using in vitro-generated products demonstrate that PER and CYC can interact stably (Lee et al., 1999). This difference in binding capabilities is especially curious because in
head extracts CYC is present in at least a 40- to 200-fold molar excess
relative to PER and dCLK, respectively. Although the reason underlying
the different interaction profile obtained in vitro and
in vivo is not clear, a speculative possibility is that,
in vivo, CYC interacts with other bHLH/PAS proteins besides dCLK and that these heterodimers are not recognized by PER. At the very
least, the high abundance of CYC as compared with dCLK, PER, and TIM in
conjunction with the reasonable speculation that CYC interacts with
bHLH/PAS proteins in a 1:1 molar ratio raises the strong possibility
that CYC has other functions besides its established role in circadian
clocks. In this context it is interesting to note that the putative
ortholog of CYC in mammals, termed BMAL1, has a broad expression
profile (highly expressed in skeletal muscle and brain), and
circumstantial evidence suggests it is a general partner of a number of
bHLH/PAS proteins (Hogenesch et al., 1997, 1998). This is consistent
with the observation that the PAS domain of BMAL1 identifies this
protein as a member of the ARNT-like group of bHLH/PAS factors that
behave as general partners of other more specifically regulated
bHLH/PAS proteins (Hogenesch et al., 1997). By analogy with the
mammalian system, a similar scenario also might be occurring with CYC
in flies. In agreement with this proposal, CYC is expressed
constitutively in flies (Rutila et al., 1998) (see Fig.
2A) and present in naïve
Drosophila tissue culture cells (Schneider) in contrast to
dClk, per, or tim (Darlington et al.,
1998; data not shown).
In addition to defining the dynamic interactions that occur between
clock proteins involved in generating a circadian transcriptional feedback loop in Drosophila, our data suggest that stable
structures can be assembled in vivo that comprise at least
three PAS-containing proteins (i.e., PER, dCLK, and CYC), in agreement
with recent findings obtained by using in vitro-synthesized
proteins (Lee et al., 1999). These results are surprising in light of
the well characterized ability of the canonical PAS domain to mediate
the formation of heterodimers and in some cases homodimers (for review, see Crews, 1998). What the role of the PAS region might be in promoting
the assembly of putative trimeric and tetrameric complexes comprising
the dCLK-CYC heterodimer and PER and/or TIM is not clear. Nonetheless,
findings based on determining interactions that can occur in
vitro indicate that other regions on PER besides its PAS domain
can interact stably with TIM (Saez and Young, 1996; Sangoram et al.,
1998), suggesting that at least in the case of PER it might interact
with PAS-containing proteins in a non-PAS-mediated manner.
The ability of PER and TIM to interact with a dCLK-CYC heterodimer can
account for the inhibition of per and tim
transcription and the activation of dClk transcription.
Repression of per and tim transcription results
from PER-TIM-mediated inhibition of dCLK-CYC activation (Darlington
et al., 1998; Lee et al., 1999), whereas activation of dClk
results from the release of dCLK-CYC-dependent repression of
dClk transcription (Glossop et al., 1999). Thus, the same
molecular interactions between PER-TIM and dCLK-CYC can account for
the anti-phase cycling of dClk mRNA as compared with per and tim mRNAs (Bae et al., 1998; Lee et al.,
1998). Based on the observations that dCLK is present in limiting
amounts and that the PER-TIM complex likely interacts preferentially
with dCLK as compared with CYC, our data indicate that the dynamic regulation of dCLK is a key variable governing both the
per-tim and dClk feedback loops. In
addition to our results suggesting the formation of a stable multimeric
complex containing PER, TIM, dCLK, and CYC, other studies that used
heterologous systems have shown that DBT can interact stably with PER
(Kloss et al., 1998) and that the blue-light photoreceptor CRY
can bind TIM in a light-dependent manner (Ceriani et al., 1999). Thus,
there is the possibility that in Drosophila at least six
clock proteins might interact simultaneously to form a light-responsive
multimeric complex or "clockosome" that is the key biochemical
entity in the timekeeping mechanism.
| |
FOOTNOTES |
Received Oct. 20, 1999; revised Dec. 16, 1999; accepted Dec. 23, 1999.
This work was supported by a grant from the National Institutes of
Health (I.E.). We thank Dr. Jeffrey Hall (Brandeis University, Waltham,
MA) for cyc0 flies.
Correspondence should be addressed to Dr. Isaac Edery, Department of
Molecular Biology and Biochemistry, Center for Advanced Biotechnology
and Medicine, 679 Hoes Lane, Piscataway, NJ 08854. E-mail:
edery{at}cabm.rutgers.edu.
Dr. Lee's present address: Department of Biological Sciences, 600 Sherman Fairchild Center, Columbia University, New York, NY 10027.
| |
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M. J. McDonald, M. Rosbash, and P. Emery
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M. P. Pando, A. B. Pinchak, N. Cermakian, and P. Sassone-Corsi
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TOP
ABSTRACT
INTRODUCTION
