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The Journal of Neuroscience, February 1, 2000, 20(3):958-968
Altered Entrainment and Feedback Loop Function Effected by a
Mutant Period Protein
Peter
Schotland,
Melissa
Hunter-Ensor,
Todd
Lawrence, and
Amita
Sehgal
Howard Hughes Medical Institute, Department of Neuroscience,
University of Pennsylvania School of Medicine, Philadelphia,
Pennsylvania 19104
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ABSTRACT |
The period (per) and timeless (tim)
genes encode interacting components of the circadian clock. Levels and
phosphorylation states of both proteins cycle with a circadian rhythm,
and the proteins drive cyclic expression of their RNAs through a
feedback mechanism that is, at least in part, negative. We report here that a hypophosphorylated mutant PER protein, produced by creating a
small internal deletion, displays increased stability and low-amplitude oscillations, consistent with previous reports that phosphorylation is
required for protein turnover. In addition, this protein appears to be
defective in feedback repression because it is associated with
relatively high levels of RNA and high levels of TIM. Transgenic flies
carrying the mutant PER protein display a temperature-dependent shortening of circadian period and are impaired in their response to
light, particularly to pulses of light in the late night that normally
advance the phase of the rhythm. Interestingly, per RNA is induced by light in these flies, most likely because of the removal of the light-sensitive TIM protein, thus implicating a more
direct role for TIM in transcriptional inhibition. These data have
relevance for mechanisms of feedback repression, and they also address
existing models for the differential behavioral response to light at
different times of the night.
Key words:
circadian rhythms; Drosophila; per; tim; entrainment to light; feedback
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INTRODUCTION |
In Drosophila, the period
(per) and timeless (tim) genes are interacting
components of the circadian clock (Sehgal et al., 1996 ). Mutations in
these genes affect the periodicity of eclosion and locomotor activity
rhythms, and a lack of either gene product results in complete loss of
circadian rhythms (Konopka and Benzer, 1971; Konopka et al., 1994;
Sehgal et al., 1994; Rutila et al., 1996; Ousley et al., 1998). RNA and
protein levels of per and tim cycle with a
circadian period, and the two proteins form a heterodimer that
negatively regulates the synthesis of both mRNAs (Hardin et
al., 1990; Zerr et al., 1990; Sehgal et al., 1995; Hunter-Ensor et al.,
1996; Myers et al., 1996; Zeng et al., 1996). The feedback loop thus
generated is thought to constitute the molecular basis of behavioral
rhythms. Other components of this feedback loop are the products of the
dclock (clk) and cycle
(cyc) genes, both of which are required for transcriptional
activation of per and tim (Allada et al., 1998;
Bae et al., 1998; Darlington et al., 1998; Rutila et al., 1998), and
the double-time (DBT) kinase that phosphorylates PER (Kloss et al.,
1998; Price et al., 1998). Both PER and TIM are cyclically
phosphorylated (Edery et al., 1994; Zeng et al., 1996), a modification
that appears to be required for protein turnover (Price et al., 1998;
Naidoo et al., 1999).
Levels of TIM are rapidly decreased by light treatment, thereby
providing a mechanism for photic entrainment of this circadian clock
(Hunter-Ensor et al., 1996; Myers et al., 1996; Zeng et al., 1996).
Pulses of light in the early night, which delay the phase of the overt
rhythm, as well as those in the latter half of the night, which advance
the phase, reduce TIM levels (Suri et al., 1998; Yang et al., 1998).
PER levels are unchanged, although PER phosphorylation is delayed in
the former case and advanced in the latter (Lee et al., 1996). A number
of models have been proposed to explain how a unidirectional response
of a clock protein (TIM) is converted to a bidirectional effect on the
overt rhythm. One possible explanation is that delays versus advances
are determined by the level of tim RNA, such that high RNA
levels produce delays because they can replace the degraded protein,
and low RNA levels produce advances because the protein is not replaced
(Myers et al., 1996; Zeng et al., 1996). Other models invoke the
phosphorylation state of the proteins or perhaps their subcellular
localization (Lee et al., 1996).
We report here that deletion of a segment of PER that is downstream of
the PAS protein-protein interaction domain affects the rescue of
rhythms in transgenic per01 flies.
In addition to aberrant circadian periodicity, transgenic flies show
altered responses to pulses of light. The molecular deficits underlying
these behavioral phenotypes include an increase in protein stability
and a defect in negative feedback. Apart from identifying an important
functional domain in PER, these studies suggest novel aspects of
feedback regulation and shed light on possible resetting mechanisms.
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MATERIALS AND METHODS |
Activity assays. Three to 7-d-old flies were
entrained on a 12 hr light/dark cycle at 25°C for 3 d,
after which time they were placed in constant darkness (DD) at
the appropriate temperature (20, 25, or 27°C) to monitor locomotor
activity. Locomotor activity was monitored using the TriKinetics Inc.
(Waltham, MA) system (Konopka et al., 1994). Periodogram
analysis was performed on 7 consecutive days of data using the tau
analysis package from Mini-Mitter Co. (Sunriver, OR) (Sehgal et al.,
1994). For temperature compensation assays, some animals were
monitored for 1 week each at both 20 and 27°C, whereas others
were monitored at one temperature only. Data from both procedures were
pooled into a 20°C pool and 27°C pool, and statistical comparison
of means was performed using Student's t test.
Resetting experiments. Three to 7-d-old flies were entrained
on a 12 hr light/dark cycle for 3 d. An Aschoff TypeII phase response curve (PRC) was used to facilitate comparison between animals of different period (Aschoff, 1965). In this modification of
the standard PRC, light pulses normally delivered during the subjective
night [CT12-CT0 (CT, or circadian time, denotes time of day under
free-running conditions)] are instead delivered during the last
night of light-dark conditions (LD) [ZT12-ZT0 (ZT, or zeitgeber time, refers to the entrainment regimen with ZT0 being lights
on and ZT12 being lights off in a 12 hr light/dark cycle)]. Subjective day pulses were delivered during the first day of DD. At the
times tested, 23 flies were treated with light (2000 lux, 10 min) and
placed into constant darkness at 27°C for monitoring of locomotor
activity (described above). Seven consecutive days of activity records
were analyzed for period and activity offsets using the Circadia
software package (Mistlberger et al., 1996). Flies that survived 7 d and were deemed rhythmic by  2
periodogram analysis were used to compute phase shifts; the number of
such flies varied between 14 and 20, except the  C2
population pulsed at ZT6 of which 11 flies were used. Phase shifts were
computed as the difference in average activity offset between
light-treated populations and an unpulsed control. Statistical
significance of phase shifts was determined by comparing average
activity offsets of light-treated populations with an unpulsed control
using Student's t test. For the experiment shown in Figure
6, the temperature throughout the experiment was kept at 27°C to
facilitate comparison between the two genotypes because the period
difference is smaller at that temperature (see Table 2). However, a
similar PRC for both genotypes was generated at 25°C.
Generation of constructs. A 159 bp in-frame deletion
(per-C2) was made by excising sequences between
restriction sites EcoRV (at bp 4572) and
XbaI (at bp 4630) of the per gene. An
Xba-Xho fragment containing this deletion was
cloned in the context of a genomic per construct in CasPer 4 (Chen et al., 1998)
Western analysis. Three to 7-d-old flies were placed in
bottles containing a 5% sucrose-2% agar medium, entrained for 3 d at 25°C on a 12 hr light/dark cycle, and collected at the indicated times on dry ice (see Figs. 2, 3). Heads were fractionated on stacked,
liquid N2-chilled sieves, and homogenized in
lysis buffer (150 mM NaCl, 50 mM Tris-HCl, pH7.6, 10 mM
EDTA, 0.1% Triton X-100, 10 mM DTT, and protease
inhibitors) with a Kontes motorized pestle. Homogenates were spun two
times at 4°C, 15,000 × g for 5 min. Supernatant protein concentrations were assayed using the Bio-Rad (Hercules, CA) DC protein assay. Total protein (50 µg) from
each sample were run on a 6% SDS-PAGE gel and transferred to a
nitrocellulose membrane. Immunological staining was visualized using
the ECL chemiluminescence detection system (Amersham, Arlington
Heights, IL) according to the manufacturer's instructions. For
detection of the PER and PER-C2 proteins, we used a rabbit anti-PER
antibody kindly provided by M. Rosbash (Brandeis University, Waltham,
MA) at a dilution of 1:20,000. For detection of TIM, a rat anti-TIM antibody was used at a dilution of 1:500 (Hunter-Ensor et al., 1996).
RNase protection assays. Three to 7-d-old flies were placed
in bottles containing a 5% sucrose-2% agar medium, entrained for 3 d at 25°C on a 12 hr light/dark cycle, and collected at the indicated times on dry ice (see Figs. 4, 5). Heads were fractionated on
stacked, liquid N2-chilled sieves and homogenized
in RNA extraction buffer (50 mM Tris-HCl, pH 9, 1% SDS, 150 mM NaOAc, 5 mM
EDTA, and 0.5 vol of Tris-HCl-saturated phenol, pH 9) at 60°C.
Extracts were spun at 15,000 × g for 10 min at 4°C, and the supernatants were removed to another tube. The
supernatants were phenol-chloroform extracted, then chloroform
extracted, and finally ethanol precipitated. RNase protection assays
were performed as described by Sehgal et al. (1995). per RNA
levels were assayed with a 32P-labeled
riboprobe antisense to nucleotides 123-438 of per, and tubulin RNA levels were assayed with a
32P-labeled riboprobe antisense to
nucleotides 1-142 of d-tubulin. Total head RNA (10 µg) was used per RNase protection assay. Protected bands were
quantitated with a phosphorimager (Molecular Dynamics, Sunnyvale, CA).
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RESULTS |
A mutated C domain (C2) affects function of a
per transgene
Based on the analysis of different Drosophila species,
per was found to contain six conserved regions (C1-C6)
(Colot et al., 1988) (Fig. 1). Of these,
the C2 region contained the largest block of conserved sequence and was
also well conserved in silkmoth and mammalian per homologs
(Reppert et al., 1994; Albrecht et al., 1997; Shearman et al., 1997;
Sun et al., 1997; Tei et al., 1997; Takumi et al., 1998; Zylka et al.,
1998). The C2 region contains the PAS domain, which consists of two
repeats of 51 amino acids each: the cytoplasmic localization
domain (amino acids 448-512), which retains PER in the
cytoplasm in the absence of TIM, and the
pers domain, which when mutated
produces short period rhythms (Baylies et al., 1992; Saez and Young,
1996). The perl mutation also maps
to this region (Baylies et al., 1987; Yu et al., 1987; Hamblen et al.,
1998). In addition, a large segment of the C2 region called the C
domain (amino acids 524-686) binds the PAS domain through an
intramolecular interaction that was proposed to account for
temperature-compensation of circadian period (Huang et al., 1995b).
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Figure 1.
Map of PER protein showing the region deleted in
this study. The map shows Drosophilid-conserved regions
C1-C6, the PAS A and PAS B repeats, and the nuclear localization
signal (NLS). The positions of pers
and perl mutations are also
indicated. Transgenes with and without the C2 region (amino acids
515-568) were made in a full-length per genomic
construct (Baylies et al., 1992). The constructs included 4 kb of
upstream promoter sequence and several hundred base pairs downstream of
the transcription stop site.
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To address possible functions of the C2 region, we made small deletions
in a full-length per genomic construct containing the
per promoter (Chen et al., 1998). Resulting constructs were introduced into per01 flies and
tested for their ability to rescue behavioral rhythms. As one might
predict, based on its requirement for the interaction of PER
with TIM, a deletion in the PAS domain failed to rescue rhythms (data
not shown). We focused instead on a per transgene carrying a
deletion of amino acids 515-568, including 11 serine and threonine
residues (per-C2) (Fig. 1). This transgene rescued rhythms at ambient temperature with periods that were shorter than
those produced by the full-length control per construct
(per-con) (Table 1).
Data in Table 1 were obtained from four per-C2 transgenic lines (4a and 4e represent lines that were maintained independently but
derived from the injection of a single egg) and one per-con line. However, consistent data from three per-con lines,
including the one shown here, have been reported previously (Chen et
al., 1998).
per-C2 transgenic flies fail to
compensate circadian period with alterations in temperature
Because the C2 domain is included in the larger region implicated
previously in temperature compensation (Huang et al., 1995b), we tested
per-C2 flies for activity rhythms at different
temperatures. As shown in Table 2, these
flies displayed an increase in period of as much as 5 hr when moved
from 20 to 27°C compared with a 0.9 hr increase in Canton S flies.
The effect was especially pronounced (5 hr) with flies that carried two
copies of the mutant transgene. For comparison, the period increased by
~2 hr in control per transgenics carrying two copies of
the transgene, whereas there was no significant difference in period in
per-con flies carrying one copy of the transgene. The small
loss of temperature compensation in two-copy per-con flies
may reflect the consequence of increasing doses of the per
gene (the endogenous per gene is present although it does
not make protein). However, the C2 mutant flies are
significantly impaired compared with controls in their ability to
maintain period length at different temperatures. In a wild-type
background, the per-C2 transgene does not affect
temperature compensation, but it does shorten period (Table 2).
PER-C2 is hypophosphorylated and cycles with
reduced amplitude
To determine the molecular basis for the behavioral phenotype of
per-C2 flies, we assayed levels of PER protein at
different times of day. In per-con flies, levels of PER
cycle as they do in wild-type flies, with peak levels reached in the
middle-end of the night. Thus, levels are high at ZT20 and low at ZT8
(Fig. 2A). In
per-C2 flies, PER cycles with reduced amplitude, such that trough levels (at ZT8 and even ZT2) are higher than those seen in
per-con flies. Consistent with higher trough levels,
immunocytochemistry experiments indicated that PER is expressed in
nuclei at all times in per-C2 flies as opposed to its
cyclic expression in wild-type and per-con flies (data not
shown).
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Figure 2.
Effect of the C2 mutation on PER
expression in LD and LL. A, Western blot of head protein
extracts from per-con and per-C2 flies
collected at 6 hr intervals during a 12 hr light/dark cycle. The blot
was probed with an anti-PER antibody (see Materials and Methods). The
numbers at the top of the
lanes denote ZT. PER-C2 is hypophosphorylated
and cycles with reduced amplitude compared with wild-type PER. Samples
were run on the same gel to allow comparison of relative mobilities.
Note that we do not observe a significant difference in mobility
between control and mutant PER because of the high molecular weight of
PER compared with that of the deleted region. B, After
3 d in LL, per-con and per-C2
flies were transferred to DD. PER expression was assayed before the
transfer to DD and at 1.5 hr intervals thereafter. The Western blot
shown here indicates that PER-C2 is more abundant than PER in
LL (top). Stripping the blot and staining with an
anti-TIM antibody revealed that TIM levels are equivalent in the wild-type
and mutant backgrounds (bottom). Thus, the
C2 mutation increases PER stability. All
lanes were equivalently loaded as determined by
measuring protein concentrations using the Bio-Rad DC assay and also as
reflected by nonspecific bands that cross-react with both antibodies
(see top band in A and also the
top band in the TIM blot in B). These
bands were also present in per0 and
tim0 controls that were loaded on the
gels shown in A and B, respectively (data
not shown). C, Flies were collected as in
B and treated for RNase protection assays.
D, Two C2 transgenic lines,
C2.7j and C2.3f, were placed in a
tim0 background, and head extracts
were Western blotted and stained with an anti-PER antibody. PER is more
abundant in the
tim0,C2 lines than
in the yw;tim0 line, further
supporting an effect of the C2 mutation on PER stability.
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We also noticed that, although the PER protein in per-con
flies underwent mobility shifts over the course of the day/night cycle,
it did not do so in per-C2 flies. These mobility shifts are characteristic of wild-type PER (and TIM) and are the result of
cyclic phosphorylation (Edery et al., 1994; Zeng et al., 1996). Alterations of these shifts is routinely taken as a measure of altered
phosphorylation (Rutila et al., 1996; Dembinska et al., 1997; Price et
al., 1998). In the per-C2 mutant, the maximally phosphorylated forms of PER that appear at the end of the
night-beginning of the day are absent (Fig. 2A,
compare ZT2 in per-con and per-C2). Given that
the deletion removes 11 serines and threonines, this is perhaps not
surprising and could account for lower amplitude cycling of PER (see
below). We cannot preclude a constant, hyperphosphorylated state (which
is also consistent with the absence of gel shifts in PER-C2), but we
believe this is unlikely given the increased stability that mimics the
dbt phenotype (see below), as well as the fact that multiple
serines and threonines were deleted. PER expression data were based on
the analysis of two independent lines (per-C2.2c
and per-C2.3f), which gave the same results. All
subsequent characterization was done in the per-C2.2c
line, which contained a high percentage of rhythmic flies (Table
2).
The C2 mutation increases the stability
of PER
Phosphorylation of PER by the DBT protein renders it
unstable in the absence of TIM (Price et al., 1998). Increased
stability of the hypophosphorylated PER-C2 protein could accelerate
its rate of accumulation and delay its disappearance, thereby producing low-amplitude oscillations. To test the rate of accumulation of PER-C2, we first placed flies under constant light conditions (LL)
for 3 d to promote the turnover of TIM and PER (light degrades TIM, and PER is unstable in the absence of TIM). After 3 d of LL,
we transferred the flies to DD and assayed PER levels at regular intervals (Fig. 2B).
In per-con flies, levels of PER were extremely low at the
end of the constant light treatment (Fig. 2B). This
is similar to what is seen in wild-type flies (Price et al., 1995).
However, the PER-C2 protein continued to be expressed at high levels
(Fig. 2B). To confirm that the light treatment had
been effective in decreasing expression of TIM, we stripped and
reprobed the blot with an anti-TIM antibody. As expected, TIM levels
were equivalent at the light-to-dark transition in both
per-con and per-C2 flies. per and
tim mRNA levels were also measured to address the
possibility that the PER-C2 protein is higher in constant
light because of elevated mRNA levels (Fig.
2C). The difference in mRNA levels was not sufficient to
explain the elevated PER-C2 protein.
To further test the effect of the C2 mutation on PER stability, we
assayed PER-C2 levels in a tim0
background. Because all of our per-C2 transgene
insertions map to the same chromosome as tim, we used
meiotic recombination to place the per-C2 transgene in a
tim0 background. Head extracts from
these lines were Western blotted and stained with an anti-PER antibody
(Fig. 2D). Both the C2.7j and
C2.3f lines show increased PER abundance in a
tim0 background compared with the
per+,tim0
control in which PER levels are low because of the absence of TIM (we
were unable to generate a
C2.2c,tim0 recombinant
line). Thus, the PER-C2 protein is less dependent on TIM for stability.
TIM is expressed at high levels and cycles with reduced amplitude
in per-C2 flies
If PER-C2 levels are high and nuclear at all times, one would
expect increased feedback repression, resulting in lower levels of RNA
and, therefore, lower levels of TIM. To determine whether this was the
case, we examined the expression of TIM in per-C2 flies.
Figure 3A demonstrates that
these flies actually express high levels of TIM. In addition, as seen
for PER, TIM cycling is of reduced amplitude with peak levels
equivalent to those in wild-type flies but with higher trough
levels.
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Figure 3.
Effect of the C2 mutation on TIM
expression in LD and DD. A, TIM expression was assayed
at the time points indicated in per-C2 and
per-con flies. The blot used here was the same as that
probed with an anti-PER antibody in Figure 2A. As
seen for PER, the amplitude of TIM oscillations is reduced, and it
cycles around a higher level. B, per-con
and per-C2 flies were entrained to a 12 hr light/dark
cycle for 3 d, then transferred to DD, and collected at 44 and 56 hr after transfer. The Western analysis indicates that TIM continues to
be expressed at high levels in per-C2 flies in DD.
The blot was stripped and probed with an anti-PER antibody
(bottom). PER-C2 is high at 44 and 56 hr, consistent
with its increased stability, and yet TIM is also expressed at high
levels, suggesting a defect in feedback. Equivalent loading was based
on the criteria mentioned in the legend to Figure 1.
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dbtP flies, like
per-C2 flies, express a highly stable form of PER (Price
et al., 1998). In these flies, TIM expression oscillates in the
presence of light/dark cycles because cyclic feedback is driven, to
some extent, by light effects on PER-TIM oscillations. However, in
constant darkness, TIM levels are greatly reduced (undetectable by
immunocytochemistry) because of constitutive feedback from the
stabilized PER (Price et al., 1998). To determine whether high levels
of TIM persist in per-C2 flies in the absence of light,
we transferred flies to DD and assayed TIM expression at 44 and 56 hr
(second day of DD). Under these conditions, TIM levels were still high
in per-C2 flies, and the oscillation was further dampened
such that levels were equivalent at 44 and 56 hr. The increased
dampening is typical for flies kept in constant dark and also occurs in
per-con flies, although an oscillation is still evident in
these flies (Fig. 3B). The higher levels in per-C2 suggest that expression of tim RNA, and
ultimately TIM protein, is not being effectively repressed by the more
abundant mutant PER (Fig. 3B).
The C2 mutation decreases the repression of
per RNA
The higher levels of TIM in per-C2 flies could not
be explained by increased PER stability because TIM does not depend on PER for stability. In addition, increased stability of the PER-C2 protein was not sufficient to explain the behavioral phenotype of
per-C2 flies. The prediction for a more stable PER would
be a longer, not a shorter, period than per-con. If the
increased stability occurred only during the rising phase of the
protein profile, i.e., in the early night, perhaps a shorter period
would be generated. However, this is clearly not the case for this
mutant. Its expression persists well into the day and even after
several days in constant light (Fig. 2B). As
mentioned above, the most likely explanation was that feedback was
affected in the per-C2 mutant.
To address the issue of feedback, we studied the temporal expression
pattern of per RNA in per-C2 flies and
compared it with that of per-con flies using RNase
protection assays. In per-con flies, the RNA cycled as
expected, peaking at ZT14, falling off at ZT20, and decreasing still
further until ZT2 (Fig.
4A). In per-C2 flies, on the other hand, the RNA was low as
expected at ZT20, but it rose again after that so it was close to peak levels at ZT2. Throughout this time, the PER-C2 protein is still expressed at high levels (Fig. 2A), suggesting that
the protein is ineffective in repressing its own transcription. We
examined the phase of feedback repression in these flies more closely
by determining RNA levels at 1.5 hr intervals. As shown in Figure 4B, the RNA levels are never reduced to the same
extent as in wild type, and they start to rise again at ZT0.5, with a
dramatic increase seen at ZT2.
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Figure 4.
Effect of the C2
mutation on per RNA expression. A, RNase
protection assay of per RNA from fly heads collected at
6 hr intervals during a 12 hr light/dark cycle. A probe for tubulin RNA
was used for normalization of per RNA. Bands were
quantitated on a phosphorimager, and per/tubulin ratios
were expressed as a percentage of the maximum value (which corresponds
to the ZT20 time point in per-con) and graphed
(bottom). Note that per-C2 RNA cycles
with reduced amplitude compared with per RNA from
per-con flies. B, RNase protection assay
of per RNA from fly heads collected at 1.5 hr intervals
from ZT20 to ZT2 during a 12 hr light/dark cycle. Procedures and data
analysis were identical to those in Figure 4A.
Note that per-C2 RNA levels increase rapidly between
ZT0.5 and ZT2. C, RNase protection assay of
per and tim RNA under free-running
conditions. per-C2 and per-con flies
were collected at 3 hr intervals starting 12 hr after transfer to
constant darkness. This was selected as the first time point because
the first 12 hr of DD correspond to the dark phase of the last LD
cycle. Levels of per and tim RNA were
assayed as indicated in A and B. All
values are expressed relative to the maximum per/tubulin
(top) or tim/tubulin
(bottom) ratio in per-con flies.
Statistical analysis of these data indicate the RNA values cycle.
One-way ANOVA performed on per-C2 mRNA values yields
p = 0.0327 and F = 4.108. Furthermore, post hoc planned comparison analysis shows
significant differences between the following time points (in DD): 12 versus 21, 27, and 33 (p < 0.006, 0.012, and 0.03, respectively); 15 versus 21, 27, and 33 (p < 0.008, 0.016, and 0.041, respectively); 18 versus 21 and 27 (p < 0.011 and 0.023, respectively). Additionally, planned comparison
analysis shows no significant differences between 21, 24, 27, 30, and
33 in any combination. This indicates a trend toward lower RNA in the
night and higher RNA in the subjective day, i.e., the RNA cycles.
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We also assayed the temporal profiles of per and
tim RNA in per-con and per-C2 flies
in constant darkness. Entrained flies were collected every 3 hr on the
first day of DD, and head RNA was processed for RNase protection assays
in which levels of per and tim RNA were assessed
simultaneously. As reported previously , oscillations of both RNAs
dampened (Hardin et al., 1990; Sehgal et al., 1995) in both genotypes,
although the amplitude of the oscillation was significantly lower in
per-C2 flies (Fig. 4C). Consistent with the LD
experiments, trough levels in per-C2 flies were higher
than those in per-con flies. However, unlike the increase at
ZT2 in the LD data, there was no increase of RNA levels at the 15 hr DD
time point (which would correspond to CT3 for a 24 hr rhythm) or even
at the 18 hr point. Note that, because these flies have a longer period
than 24 hr under free-running conditions, we would have expected the
ZT2 equivalent rise to occur a little later in DD.
per RNA is induced by light in
per-C2 flies
We were intrigued by the relatively large increase in
per RNA levels at ZT2 in C2 flies. The DD data
argued against a clock-controlled event that coincided with the advent
of light, suggesting instead an induction of per RNA by
light, reminiscent of the mPer1 and mPer2 RNA
induction seen in mammals (Albrecht et al., 1997; Shearman et al.,
1997; Shigeyoshi et al., 1997). To test this idea, we treated
per-con and per-C2 flies with bright light
(~2000 lux, 30 min) at ZT15, ZT16, ZT17, and ZT21 and assayed
per RNA levels 2 hr after the initiation of the light pulse.
At all these times, we observed an induction of per RNA in
per-C2 flies with light treatment (Fig.
5). The unpulsed controls in Figure 5
were collected at the same time as the light-treated populations. This
was done to exclude any clock-regulated changes within the 2 hr
period.
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Figure 5.
Response of per RNA to
light in per-C2 flies. A, A composite
image of RNase protection assays of per RNA from
light-treated flies. per-con and
per-C2 flies were treated with light of 2000 lux
intensity for 30 min at the zeitgeber times indicated. Pulsed and
unpulsed flies were collected 2 hr after the initiation of the light
pulse. Probes used were identical to those in Figure 4.
B, The gels shown in A were quantitated
on a phosphorimager, and the data were graphed as percent induction by
light compared with unpulsed controls. As in Figure 4,
per RNA was normalized relative to tubulin. Significant
induction of per RNA in per-C2 flies
was seen at ZT16, ZT17, and ZT21, whereas induction at ZT15 was not
reliable, resulting in the large error shown. Error bars represent one
SE.
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The highest level of induction was seen at ZT17, with slightly less at
ZT16 and ZT21 (note that in each case the samples are being examined 2 hr later, i.e., at ZT18 and ZT23). As indicated by the size of the
error bars in the ZT15 sample, the data at this time point were quite
variable. This temporal pattern of RNA induction correlates with the
phase of feedback repression, suggesting that light is perhaps
relieving transcriptional inhibition (further discussed in Discussion).
The variability at ZT15, therefore, may reflect the fact that it is on
the cusp between active transcription and repression. Small changes
from one experiment to another, such as the time of collection, or even
some period differences between individual flies could account for this
variability. Experiments at ZT21, which actually reflect a collection
at ZT23, lie at the end of the repression phase in per-C2
flies and also shows less RNA induction. Finally, we examined the
response of tim mRNA to light at ZT16, ZT17, and ZT21 and
found that it was induced in a parallel manner, which further supports
the hypothesis that light removes a transcriptional repressor (data not shown).
per-C2 alters the PRC of
behavioral rhythms
As discussed earlier, several models have been proposed to account
for the differential behavioral response to light at different times of
the night. Because the C2 mutation affected some of the
parameters that were thought to be critical for resetting (phosphorylation and RNA levels), we reasoned that this mutation could
be informative in addressing current resetting models. Thus, we
generated a PRC for this line using 10 min pulses of light and again
compared it with that of per-con flies. The resetting assays
were performed as described previously (Yang et al., 1998), except that
they were conducted at 27°C. This was done because per-con
and per-C2 flies have similar periods at this temperature (Table 2), thus reducing period-influenced changes in the PRC.
As shown in Figure 6, per-con
flies displayed a PRC that one would predict for flies with 27 hr
periods. In wild-type flies, relatively little resetting occurs in
response to pulses during subjective day, delays occur in the first
half of the night, and advances occur in the second half. In
per-con flies, consistent with their longer period, the
switch from delay to advance occurs later in the night and advances
occur up to CT2. Thus, delays are observed at ZT15, ZT17, and ZT20 and
advances at ZT22 and CT2. per-C2 flies display a very
different PRC despite the similarity of their periods. Delays do not
occur until ZT17 (the delay at ZT15 is statistically insignificant),
and advances do not occur at all at the times predicted by the
per-con PRC (ZT22 and CT2). In fact, a pulse at ZT22 elicits
a large delay in per-C2 flies but produces the maximal
advance in per-con flies. Advances of very low magnitude are
observed in per-C2 flies during the day, although only
one of these (ZT10) was statistically significant.
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Figure 6.
Behavioral response of per-C2
flies to light. per-con and per- C2
flies were exposed to light at the times indicated, and activity
rhythms were assayed using the Aschoff Type II PRC (Aschoff, 1965). The
experiment was performed at 27°C to facilitate the comparison of the
two genotypes because the period difference is smaller at that
temperature (Table 2). Phase shifts were computed as the difference in
average activity offsets between light-treated populations and
untreated controls. Note that per-C2 flies are
deficient in advances at all times tested, with the exception of ZT10.
Student's t test was used to compare average activity
offsets of light-treated populations with untreated controls (table of
p values at right). Similar PRCs for both
genotypes were obtained at 25°C.
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DISCUSSION |
Although per has been the subject of intense research
for ~14 years, major aspects of its regulation and function remain
unresolved. Available data indicate that cyclic expression of PER can
be achieved in the absence of per RNA cycling (Frisch et
al., 1994; Cheng and Hardin, 1998), but RNA cycling requires the
protein, suggesting that the feedback loop is driven primarily
by the cycling of the protein (Hardin et al., 1990; Sehgal et al.,
1995). This makes it all the more important to address the mechanisms
that control protein turnover and the effects of protein on
transcription. Traditional methods to dissect the regulation of a
protein involve structure-function studies, which allow specific
functions to be assigned to distinct domains. In the case of PER, these
studies have been limited. We show here that a deletion within the
conserved C2 region in PER does not prevent rescue by a per
transgene but affects the properties of the behavioral rhythm.
Behavioral phenotypes of the per-C2 flies have their
basis in specific underlying molecular defects that we discuss below.
We believe that the direct effects of the C2 mutation are
twofold: (1) to increase PER stability and (2) to decrease its ability
to effectively feedback repress transcription. Thus, the effect on RNA
cycling is probably secondary and caused by inadequate feedback by the
protein. A direct effect on RNA stability is unlikely given that the
deletion is in coding regions and has a similar effect on the cycling
of tim transcripts as it does on the transgenic RNA (Fig.
4C). Consistent with this idea, the effect on RNA cycling is
to reduce amplitude via an increase in trough levels, i.e., decrease
repression during the trough. An effect on feedback is also supported
by the fact that the protein itself is expressed at high levels and is
constitutively nuclear. Normal negative feedback should lead to a
reduction of RNA expression and, ultimately, protein expression, making
this phenotype (of high RNA and high protein levels) highly unlikely.
The question then is the following: what causes the increased stability
and decreased feedback associated with the protein? The increased
protein stability is almost certainly attributable to the lack of
phosphorylation events. A hypophosphorylated, more stable protein is
also produced by a truncated form of per fused to
 -galactosidase (Dembinska et al., 1997). The latter protein retains
the region deleted in this study, indicating that more than one region
of PER can confer this phenotype. Either one or both regions may be
targets of the DBT kinase, which phosphorylates and destabilizes PER
monomers. If the DBT sites are also responsible for the reduced
feedback in per-C2 flies, then one would predict that DBT
mutants themselves must be defective in feedback. This does not appear
to be the case because, although PER does not cycle in larvae of
strongly hypomorphic dbt mutants, tim RNA and protein do so in a robust manner (although absolute levels could not be
quantitated) in the presence of LD cycles (Price et al., 1998).
Moreover, expression of tim gene products in dbt
mutants is greatly reduced in DD compared with wild type, which is
inconsistent with reduced feedback. In contrast, tim RNA, as
well as TIM, continue to be expressed at high levels in DD in
per-C2 flies (Figs. 3B, 4C). We
propose that the effect of the PER-C2 protein on feedback is caused
by an additional defect, such as in DBT-independent phosphorylation
sites or in the region per se.
The current model for how PER and TIM feedback to inhibit their own
transcription is that they bind transcriptional activators dCLK and
CYC, both of which contain PAS domains, as does per (Allada et al., 1998; Darlington et al., 1998; Lee et al., 1998; Rutila et al.,
1998). Although homotypic interactions between PAS-containing proteins
are common, the PAS domain can also engage in heterotypic interactions.
For instance, the PAS A repeat in PER binds a part of TIM through a
heterotypic association (Gekakis et al., 1995). In addition, a larger
region encompassing the C2 sequence was shown previously to interact
with the PAS domain (Huang et al., 1995a). Although it was thought to
engage in an intramolecular interaction, association with a different
molecule, possibly a different protein, cannot be excluded. Thus, this
domain may be required for the interaction of PER with a PAS-containing
transcription factor.
The defect in feedback can explain the short periodicity displayed by
per-C2 flies. As discussed earlier, increased stability of the protein, in particular during the falling phase after lights are
turned on, is inconsistent with a shorter period. In fact, the
dbtL allele, which reduces PER
phosphorylation and increases PER stability, produces a long period
(Price et al., 1998). Moreover, the
pers mutant, which accelerates decay
of the protein in a day/night cycle (Marrus et al., 1996), shortens
circadian period. Thus, feedback is truncated and the RNA levels rise
earlier. In case of the per-C2 flies, the protein remains
but is impaired in feedback inhibition. Note that the phenotype of
these flies ranges from short periods to arrhythmia. Reduction of
feedback below a certain threshold level could result in arrhythmia.
Relevant to this issue is the phenotype of flies kept in altered
light/dark cycles. When the length of the night is reduced to <8 hr,
flies are arrhythmic (Qiu and Hardin, 1996). Thus, a minimum night
length is critical to maintain circadian cycles, perhaps by ensuring
adequate feedback. Note that the defect in feedback in
per-C2 flies also elevates levels of tim RNA
and protein (Fig. 3A), thereby leading to accelerated accumulation of PERC2-TIM dimers and contributing to a shorter circadian cycle.
The response of the per-C2 flies to pulses of light is
intriguing. Induction of per and tim RNA by light
has never been seen in wild-type flies, although it is a characteristic
of some of the mammalian per homologs (Albrecht et al.,
1997; Shearman et al., 1997; Shigeyoshi et al., 1997). In case of the
C2 mutant, it is most likely because of the removal of
transcriptional repression, supported by the fact that best induction
is observed at times of maximal feedback. Because PER appears to be
defective in feedback inhibition in per-C2 flies, we
believe that repression is mediated primarily by the light-sensitive
TIM protein. We examined the response of PER and TIM proteins in
response to light at ZT21, using the same pulse that induced the two
RNAs, and found, as expected, that TIM levels were reduced, but PER
levels were constant (data not shown). The apparent increased
dependence on TIM in per-C2 flies may indicate that TIM
compensates for the defect in PER or it may reflect the normal role of
TIM, which is usually masked by PER, i.e., in wild-type flies PER
continues to repress after TIM is degraded by light. In either event,
these data suggest that TIM can participate in feedback repression.
Note that TIM can bind the PAS domain of PER (Gekakis et al., 1995) and
was shown recently to interact with dCLK in a per null
background (Lee et al., 1998). The hypothesis that TIM can mediate
feedback is also consistent with the report of tim mRNA and
protein cycling in dbtP flies in LD
but not DD (Price et al., 1998). Light-driven cycling of tim
gene products in dbtP flies
indicates that light-dependent TIM degradation can drive rhythmic
feedback, even when PER does not cycle.
The behavioral response to resetting pulses of light is also altered. A
model that incorporates increased stability of PER-C2, together with
reduced feedback, can account for these resetting defects (Fig.
7). We propose that the lack of advances
is attributable to the high RNA levels in per-C2 flies,
which are further increased in response to light. As proposed (Myers et
al., 1996), these could replace the TIM protein lost by light and
thereby delay the cycle instead of advancing it to the following dawn
(Fig. 7). The reduced delays can be explained on the basis of the RNA induction, as well as by the generally elevated levels of
tim RNA, both of which allow rapid resynthesis of TIM. The
large pool of stable PER monomers may also contribute by allowing
PER-TIM heterodimers to accumulate rapidly after a light pulse
degrades TIM. Thus, the time required to get back to the same point of the cycle is shortened.
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Figure 7.
Model for the resetting response in
wild-type and per-C2 flies. In wild-type flies,
delays occur in response to light in the early part of the night when
both genes are being actively transcribed and RNA levels are high. In
the second half of the night, highly phosphorylated forms of PER-TIM
repress transcription and RNA levels are low. Advances are produced at
this time. In per-C2 flies, delays are produced at
almost all times, thus only a single situation is shown. These delays
correlate with reduced feedback and relatively high levels of
RNA.
|
|
Although the effect of the PER-C2 protein on temperature
compensation is interesting, particularly given that the deleted sequence falls in the C domain, a region implicated previously in
temperature compensation (Huang et al., 1995b), it may be premature to
conclude that this specific sequence is important for the compensatory property of clocks. In fact, the data presented here run contrary to
the model of Huang et al., which would predict an increase in
intermolecular over intramolecular PER-C2 interactions and hence a
shortening of period with increasing temperature. Other mutations of
per also affect temperature compensation (Huang et al.,
1995a; Sawyer et al., 1997; Hamblen et al., 1998), and at the present
time it is difficult to reconcile all these data with a single
molecular model.
| |
FOOTNOTES |
Received Feb. 25, 1999; revised Nov. 11, 1999; accepted Nov. 18, 1999.
This work was supported by National Institutes of Health Grant NS35703
and American Cancer Society Grant DB-140. A.S. is an assistant
investigator of the Howard Hughes Medical Institute. We thank Michael
Young for useful comments and suggestions, Michael Rosbash for the
anti-PER antibody, Jeffrey Field for critical comments on this
manuscript, and other members of the laboratory for helpful discussions.
Correspondence should be addressed Amita Sehgal at the above address.
E-mail: amita{at}mail.med.upenn.edu.
Dr. Hunter-Ensor's present address: Department of Biology,
Massachusetts Institute of Technology, Cambridge, MA 02139.
| |
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ABSTRACT
INTRODUCTION
