 |
INTRODUCTION |
Endogenous circadian clocks control a wide variety of daily
physiological, behavioral, cellular, and biochemical activities in most
eukaryotic and certain prokaryotic organisms. The circadian oscillators
are networks of positive and negative elements that form the core
circadian feedback loops generating the basic circadian rhythmicity (1,
2). The positive elements of the loop activate the transcription of the
negative elements, whereas the negative elements feedback to block
their own activation. In Neurospora, Drosophila, and mammals, the positive elements of the
loops are all heterodimeric protein complexes consisting of
PER-ARNT-SIM (PAS)1
domain-containing transcription factors (1, 2). In each system, the
heterodimeric complex activates the transcription of the negative
elements, and the protein products of the negative elements close the
feedback loop by inhibiting their own transcription through direct
physical interaction with the positive elements (3-9). In these three
systems, the negative elements of the oscillators also activate the
expression of one or two of the positive elements, forming positive
feedback loops that interlock with the negative ones (10-13).
In the Neurospora frq-wc based circadian feedback loops,
WHITE COLLAR-1 (WC-1) and WC-2, the two PAS domain-containing
transcription factors (containing GATA type zinc-finger DNA
binding domains) form heterodimer complexes (14) and act as the
positive components (15, 16). On the other hand, two alternatively
translated FREQUENCY (FRQ) protein forms are the negative elements (7, 15, 17, 18). In constant darkness, the WC-1·WC-2 heterodimeric complex binds to two light-regulated elements (LREs) in the promoter of
frq and activates the transcription of frq (13,
16, 19). In either wc-1 or wc-2 mutants, the
levels of frq RNA and FRQ protein are very low in the dark,
and the circadian clock is not functional under normal conditions (13,
16, 20, 21). In addition, WC-1 is the limiting factor for the formation
of the WC-1/WC-2 complex (13, 18).
After the transcription of frq, two forms of FRQ protein
(large and small FRQ forms) are made (17, 22), and they exist in
homodimeric complexes (15). After their amounts reach a certain level,
they feedback to repress the transcription of frq by
interacting with the WC-1/WC-2 complex (7, 15, 18, 23, 24), thus closing the negative feedback loop. In addition to its role as a
negative element of the loop, FRQ positively regulates the expression of both WC-1 and WC-2 through two different mechanisms, forming positive feedback loops interlocked with the negative feedback loop
(12, 13, 20). Our previous data suggest that the positive feedback
loops are important for the robustness and stability of the clock
(13).
In addition to their essential role in the circadian feedback loop,
WC-1 and WC-2 are also essential components in the light input of the
clock and other light responses in Neurospora (16, 25-27).
In true wc-1 or wc-2 null mutants, most if not
all light responses are abolished, including the light induction of
frq (21, 28). Recently, we (28) and Froehlich et
al. (19) identified WC-1, a flavin dinucleotide (FAD)-containing
protein, as the blue light photoreceptor mediating these light
responses. The result of the in vitro DNA binding assay
suggests that a large WC complex (that is different from the dark
complex) binds to the frq LREs in a
light-dependent manner to mediate light input of the clock
(19). Thus, light irradiation triggers rapid induction of
frq transcription, a process that leads to the resetting of the clock (29). Although the level of WC-1 determines the
concentrations of the WC complex formed in vivo (13), the
WC-2 PAS domain-mediated WC-1/WC-2 complex formation is important for
maintaining the steady state level of WC-1 and its functions in the
circadian clock and light responses (20). Without WC-2 or the formation
of a WC-1/WC-2 complex, the level of WC-1 in the cell is low (20).
These previous studies indicate that WC-1 is a protein with at least
two roles: it is a circadian positive element in the dark and a
photosensing transcriptional activator mediating light responses.
Previously, WC-2 was proposed to function as a scaffold protein that
mediates the FRQ-WC interaction to close the negative feedback loop
(18); however, the involvement of WC-1 in this interaction is unclear.
Although it is clear that WC-2 positively regulates WC-1 by forming the
WC complex, it is not known whether WC-1 regulates the expression of
WC-2. In addition, based on the data described above, there should be
different forms of the WC complexes with distinct functions in the
cell, but no in vivo evidence is available about the nature
of the different WC complexes. Furthermore, we do not know which domain
of WC-1 mediates its interaction with WC-2 and whether different
regions of WC-1 have distinct functional roles. In this study,
experiments were carried out to address these questions. Together, our
results demonstrate that WC-1 is a multifunctional protein with
separable protein domains.
 |
EXPERIMENTAL PROCEDURES |
Strains and Culture Conditions--
The bd, a strain
was used as the wild-type strain in this study. The
wc-1RIP strain was made previously (28), and
sequencing of the wc-1 locus revealed numerous G/C
A/T
point mutations introduced by repeat-induced point mutation (RIP),
resulting in several premature stop codons in the WC-1 open reading
frame. Western blot analysis showed that no WC-1 protein was expressed
in this strain. This wc-1RIP strain was used as the
host strain for various his-3 targeting wc-1 constructs.
The wc-1 mutant (FGSC number 4401, wc-14401) was obtained from the Fungal Genetics
Stock Center. The wc-1 mutant strain,
wc-1ER53, described previously (16), produces a
truncated WC-1 protein. The
wc-1
,qa-wc-1 and
frq
,qa-FRQ strains were described
previously (13).
Liquid culture conditions were the same as those previously described
(7, 15, 22), except a lower glucose concentration was used in the media
for strains used for quinic acid (QA) induction (1 × Vogel's,
0.1% glucose, 0.17% arginine, 0.01 M QA).
Plasmids--
The Myc-WC-1 and Myc-WC-2 constructs were made
previously (20). The Myc-WC-1 construct can rescue the circadian and
light phenotype of the wc-1RIP strain. All deletions
of WC-1 open reading frame were made by using the transformer
site-directed mutagenesis kit (Clontech Laboratories, Inc.), and a Myc-WC-1 plasmid was used as the template. The mutagenic primers used were WC1.LOV (to delete aa 495 to 507), WC1.PASB (to delete aa 647 to 671), WC1.PASC (to delete aa 706 to 720),
WC1.NLS (to delete aa 919 to 926), WC1.Zn (to delete aa 946 to 1049),
WC1.bNLS (to create a stop codon at aa 918), and WC1.NLSa (to create a
stop codon at aa 926). The Myc-WC-1.NruI construct was generated by
digesting the Myc-WC-1 construct with NruI and
AgeI, blunt-ended and self-ligated, resulting in the deletion of aa 766 to 1044 (downstream of the PASC domain). The Myc-WC-1.AgeI construct was generated by cutting the Myc-WC-1 construct
with AgeI, blunt-ended and self-ligated, resulting in the
deletion of the WC-1 open reading frame downstream of the zinc
finger region (from aa 1048).
The qa-wc1 his-3 targeting construct was made
previously (13). To create the qa-wc1.NH construct, the qa-wc1
construct was used as the plasmid template for in vitro
site-directed mutagenesis. The mutagenic primer used was WC1.NH (to
delete aa 103 to 383, upstream of the LOV domain). To create
qa-wc1.BamHI and qa-wc1.MluI constructs, the qa-wc1 construct was cut
by BamHI or MluI, blunt-ended and self-ligated,
to create a truncated WC-1.BamHI (stop at aa 836) or WC1.MluI (stop at
aa 887). All resulting plasmids were targeted by transformation
to the his-3 locus of the host strains as previously
described (30).
Protein and RNA Analyses--
Protein extraction,
quantification, Western blot analysis, and immunoprecipitation assays
were performed as previously described (15, 22). Equal amounts of total
protein (40 µg) were loaded in each protein lane, and after the blots
were developed by chemiluminescence (Amersham Biosciences) they
were stained by Amido Black to verify equal loading of protein.
RNA extraction and Northern blot analysis were performed as described
previously (7). Equal amounts of total RNA (20 µg) were loaded onto
agarose gels for electrophoresis, and the gels were blotted and probed
with an RNA probe specific for frq, albino-3 (al-3), vivid (vvd), or
wc-2 mRNA.
 |
RESULTS |
WC-1 Is Required for the FRQ-WCC Interaction in Vivo--
In the
Neurospora circadian negative feedback loop, the closing of
the loop is achieved by the direct physical interaction between FRQ and
the WC complex (15, 18, 23). To examine whether WC-1 is important for
the formation of the FRQ-WC complex in vivo, two FRQ
expressing wc-1 mutant strains were used:
wc-14401 and
wc-1ER53,qa-FRQ. In the
wc-14401 strain, as described below, the expression
of WC-1 is very low, but the expression of FRQ and WC-2 is near normal
in LL (Fig. 1). In the
wc-1ER53,qa-FRQ strain, the expression of
frq is under the control of the quinic acid-inducible
promoter and can be induced by the addition of QA (Fig. 1). When
the cell lysates of these two strains and a wild-type strain were
subjected to immunoprecipitation with our WC-2 antiserum, although both
FRQ and WC-1 were co-immunoprecipitated with WC-2 in the wild-type
sample, no interaction between FRQ and WC-2 was found in the two mutant
samples. This result demonstrates that the presence of WC-1 is
essential for the FRQ-WC-2 interaction.

View larger version (61K):
[in this window]
[in a new window]
|
Fig. 1.
WC-1 is required for the formation of the
FRQ-WCC complex. Immunoprecipitation assays (IP) using
WC-2 antiserum showing that WC-2 and FRQ do not interact with each
other in vivo in either the wc-14401 or
the wc-1ER53,qa-FRQ strain. Cultures were
grown in LL. To induce FRQ expression in the
wc-1ER53,qa-FRQ strain, 0.01 M QA was added into the medium.
|
|
Because of the low level of WC-1 in wc-2 null strains (20),
we were not able to confirm whether WC-2 is required for the FRQ/WC-1
interaction. However, because of the important role of WC-2 in
maintaining the level of WC-1, both proteins should be important for
the FRQ-WCC interaction in vivo, and FRQ can bind only the WC-1/WC-2 heteromeric complex.
WC-1 Negatively Regulates the Transcription of
wc-2--
Previously, it was shown that the levels of both WC-1 and
WC-2 proteins are positively regulated by FRQ (12, 13, 23). Because of
the low levels of FRQ in wc-1 strains, we expected that the
WC-2 protein level would be low as well. However, we found comparable
amounts of WC-2 in the wc-1RIP strain and in the
wild-type strain (Fig. 2A).
There are two possible explanations for this result. First, the low
level of FRQ in the wc-1RIP strain might be able to
support normal expression of wc-2. Second, WC-1 may
negatively regulate wc-2; therefore, in the
wc-1RIP strain, the negative effect of WC-1 and the
positive effect of FRQ on wc-2 counterbalance each other,
resulting in a normal WC-2 level.

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
The expression level of WC-2 in
frq and wc-1 mutants.
A, Western blot analysis showing that the WC-2 protein level
in the wc-1RIP strain is about the same as in the
wild-type strain (wt). B, Western blot analysis
showing that the levels of WC-1 and WC-2 increased as the level of FRQ
in the frq ,qa-FRQ strain increased.
Liquid cultures were grown in media with different concentrations
(indicated above) of QA and were harvested in LL. C, Western
blot analysis showing that the induction of WC-1 in the
frq ,qa-WC-1 strain slightly
decreased the level of WC-2, whereas the WC-1 induction in the
wc-1ER53,qa-WC-1 strain did not affect
the level of WC-2.
|
|
To test the first possibility, the levels of WC-1 and WC-2 were
monitored in the frq
,qa-FRQ strain
in which the level of FRQ was induced to different levels in the
presence of various concentrations of the QA inducer (Fig.
2B) (13). Therefore, the levels of WC-2 in this strain should inform us about the importance of the FRQ level on the expression of WC-2. When the QA concentration was less than 1 × 10
6 M, FRQ level was low, and the levels of
WC-1 and WC-2 were comparable with those without QA (Fig.
2B) and those in a frq null strain (data not
shown). As the concentration of QA increased, leading to an increase in
FRQ amount, the levels of both WC-1 and WC-2 also increased. At
10
3 M QA, the induction of FRQ appeared to
reach the peak, as did the levels of the WC proteins. This result
suggests that the amount of FRQ determines the level of WC-2. Thus, it
is unlikely that the residual amount of FRQ in the
wc-1RIP strain is able to fully support the
expression of wc-2.
To test the second possibility, we examined the effect of WC-1 on WC-2
in a frq or wc-1 strain in which the expression
of WC-1 is under the control of the QA-inducible promoter
(frq
,qa-wc-1 or
wc-1ER53,qa-wc-1). In the
wc-1ER53,qa-wc-1 strain, the level of WC-2 did not
respond to the change in WC-1 level (Fig. 2C), probably
because the induction of WC-1 in this strain also resulted in an
increase of FRQ, so that the positive effect of FRQ on wc-2
counterbalanced that of the WC-1. However, in the
frq
,qa-wc-1 strain, the WC-2 level
decreased slightly after the induction of WC-1 (Fig. 2C).
This small but reproducible decrease in the WC-2 level is likely due to
the low level of WC-2 in the frq
background;
thus, further reduction of WC-2 by the induction of WC-1 was limited.
In addition, the induction of WC-1 was limited in this strain because
of the absence of FRQ, as FRQ post-transcriptionally regulates WC-1
expression (20).
To further examine the frq-independent role that WC-1 has on
the expression of wc-2, a
frq
,wc-1
double
mutant was made by crossing the frq
strain
with a wc-1
strain. Compared with the low
level of WC-2 in the frq
strain, WC-2 levels
in the double mutants were significantly higher, and their levels were
comparable to those in a wild-type strain (Fig.
3A). To confirm this result,
the qa-wc1 construct was introduced into the double mutant strain. As
expected, the level of WC-2 decreased significantly when WC-1 was
induced in the presence of QA (Fig. 3B). Northern blot
analysis performed using this strain further showed that WC-1
negatively regulated wc-2 at the transcriptional level in a
light-independent manner (Fig. 3C). Together, these results
demonstrate that WC-1 negatively regulates the expression of
wc-2 independent of frq. WC-1 was previously
regarded as a transcriptional activator in the circadian clock and in
light responses, our results here suggested a novel light-independent
repressor function of WC-1.

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 3.
WC-1 negatively regulates the transcription
of wc-2. A, Western blot analysis
showing that in
frq ,wc-1 double
mutants, WC-2 levels were significantly higher than those in the
frq strain. B, Western blot
analysis showing that the induction of WC-1 in a
frq ,wc-1 double
mutant resulted in a decrease of the level of WC-2. Liquid cultures
were grown in LL in media with or without 10 2
M QA for 1 day before harvest. C, Northern blot
analysis reveals that WC-1 down-regulates the transcription of
wc-2. Cultures were grown either in LL or DD (DD20) in media
with or without QA.
|
|
Alternative Protein Initiation of WC-1 and Differential Requirement
of WC-1 for the Light Induction of frq and Other Light-inducible
Genes--
Previously, it was shown that WC-1 is required for the
light induction of frq and other light-inducible genes (16,
25). However, we found one wc-1 mutant strain
(wc-14401) in which no detectable amount of WC-1 was
produced, but it has a near normal amount of FRQ in constant light
(Fig. 4A). In addition, its
FRQ level in the dark was low, a level comparable with that in a
wc-1 null strain (Fig. 4A and data not shown).
Phenotypically, this strain resembles other wc-1 mutant
strains with white mycelia and exhibits arrhythmicity in constant
darkness (data not shown). Northern blot analysis confirmed that the
al-3 gene (a gene required for carotenoid biosynthesis) is
not light-induced in this strain, although frq RNA was
light-induced to a level that was about half of the wild-type induction
level (Fig. 4B).

View larger version (37K):
[in this window]
[in a new window]
|
Fig. 4.
There is alternative protein initiation of
WC-1 in the wc-14401 strain, and
WC-1 differentially regulates the light induction of frq
and other light-inducible genes. A, Western blot
analysis showing the levels of WC-1 and FRQ in both the wild-type
(wt) and wc-14401 mutant strains.
Cultures were grown in LL or DD (DD24). B, Northern blot
analysis showing that in the wc-14401 strain, the
light induction of frq was near normal, whereas the light
induction of al-3 and vvd was mostly abolished.
C, schematic depiction of the domain architecture of the
WC-1 protein along with the mutation in the wc-14401
strain. AD, putative transcription activation domains;
Zn, GATA-type zinc finger DNA-binding domain;
NsiI, the restriction enzyme site used to introduce the 5 c-Myc epitope tag. D, immunoprecipitation assays
(IP) using WC-2 antiserum showing that the truncated
alternatively translated Myc-WC-1 protein co-precipitated with WC-2 in
the wc-1RIP Myc-WC-1.4401 strain. More total cell
extract of the wc-1RIP,Myc-WC-1.4401 strain than
that of the control strain was used in the immunoprecipitation
(reflected by the different amounts of WC-2 in the IP lanes)
to detect the alternatively translated WC-1 protein. The upper
panels were probed with the anti-C-Myc antibody, and the
lower panels were probed with our WC-2 antiserum.
1, wc-1RIP,Myc-WC-1; 2,
wc-1RIP,Myc-WC-1.4401; 3,
wc-1RIP.
|
|
Recently, we and others identified WC-1 as the photoreceptor for
circadian clocks and other light responses (19, 28). In addition, our
analyses of the wc-1RIP strain and other
wc-1 mutant strains have shown that WC-1 is essential for
the light induction of frq (28). To understand the nature of
the wc-1 mutation in this strain, its wc-1 gene was cloned and sequenced. Sequencing of the wc-1 gene of
this strain revealed 1 base pair deletion at nucleotide 184 (C)
of WC-1, causing frameshift and premature protein termination (Fig. 4C). Because this mutant should produce only a small
truncated form of WC-1 (66 aa) and behaves like a wc-1 null
strain, why is frq still light-induced in this strain?
Three possible explanations might explain this result. First, WC-1 may
not be the photoreceptor responsible for the light-activated transcription of frq. However, this possibility cannot fully
explain the existing molecular and biochemical data (19, 28), nor can
it explain the complete abolishment of frq light induction in other wc-1 mutants (16). Second, the 61-aa WC-1
N-terminal region may be capable of sensing light, but there is no
obvious protein motif in this region of WC-1 to suggest such a role.
Third, an undetectable amount of WC-1 may be produced in this strain as
a result of alternative protein initiation using a downstream AUG, and
this truncated WC-1 may be responsible for the light induction of
frq. If this latter possibility is true, it will indicate
that various light-inducible genes may require different amounts of
WC-1 protein. Indeed, a small but detectable amount of light induction
of vvd RNA (31) (less than 1% of the wild-type induction)
was observed in this strain (Fig. 4B), and its conidiation rhythm can still be weakly entrained by light/dark cycles (32), suggesting that there is some residual WC-1 activity in this strain.
Alternative protein initiation can be caused by a "leaky scanning"
mechanism, in which the 40 S ribosomes fail to start translation from
the first AUG and use a downstream AUG instead (33). Upon examining the
wc-1 sequence, we found another AUG at aa 86 with a good
Kozak consensus sequence. To test this hypothesis, the same mutation in
the wc-14401 strain was introduced into a Myc-WC-1
construct (Myc-WC-1.4401), and this construct was transformed into the
wc-1RIP strain. In the Myc-WC-1 construct, WC-1 is
tagged by a 5 c-Myc epitope tag, allowing the detection of the tagged
protein using a monoclonal c-Myc antibody (15, 20). In addition, the
wild-type construct can rescue the circadian and light phenotype of the wc-1 null strain (13). As expected, the transformants
carrying the mutant construct showed a level of FRQ expression in LL
comparable with that in the wild-type strain (data not shown), whereas
no WC-1 protein in the lysate could be detected using the monoclonal c-Myc antibody (Fig. 4D, left). However, by
performing an immunoprecipitation assay with WC-2 antiserum using 5 mg
of the lysate of the Myc-WC-1.4401 strain, a smaller than the wild-type
Myc-WC-1 band was detected (Fig. 4D). The apparent molecular
weight shift indicated that WC-1 translation was reinitiated from a
downstream AUG in the mutant, probably through a leaky scanning
mechanism. We estimated that the amount of Myc-WC-1 in the
Myc-WC-1.4401 strain is less than 1% of the Myc-WC-1 strain. Together,
these data demonstrate that there is alternative protein initiation in
the wc-14401 strain, and the light induction of
frq requires only a very low level of the WC complex.
Therefore, the light induction of frq and other
light-inducible genes was differentially regulated by WC-1, suggesting
that there are different WC-1/WC-2-containing complexes in the cell to
mediate light induction of frq and other genes.
There Are Two Types of WC Complexes and a Portion of WC-1
Self-associates--
Recently, the results of the in vitro
DNA binding assay showed that there are two different WC-1-containing
complexes that bind to the LRE elements of the frq promoter
(28). Whereas the smaller complex that binds to the LRE in the dark is
consistent with it being the WC-1/WC-2 heterodimer, the complex that
binds to the LRE in a light-dependent manner is
considerably larger. Because a similar larger complex was also found
using in vitro translated WC-1 and WC-2 proteins, the WC
proteins may form multimers. Previously, using the in vitro
protein binding assay, it was shown that WC-1 or WC-2 mostly
self-associates instead of forming a WC-1/WC-2 heterodimer (34).
Although it is known that WC-1 and WC-2 form a tight complex in
vivo (14, 28), there is no in vivo evidence to show the
self-association of WC-1 or WC-2.
To examine this, the Myc-WC-1 and the Myc-WC-2 constructs were
transformed into a wild-type strain. Either construct has been shown to
be able to rescue the clock and light phenotypes of either wc-1 or wc-2 mutants (20). As shown in Fig.
5A, Myc-WC-1 was expressed in
the wt,Myc-WC-1 strain at a level that is about 50% of the endogenous
WC-1. To examine the self-association of WC-1, the protein lysate of
this strain was immunoprecipitated using the c-Myc antibody or our WC-1
antiserum. As shown in Fig. 5A, a small amount of WC-1
protein at the position of the endogenous WC-1 co-precipitated with the
Myc-WC-1, and Western blot analysis using the c-Myc antibody showed
that it was not the degradation product of the Myc-WC-1 protein.
Similar results were obtained in four independent experiments.
Comparison of the levels of two different WC-1 forms in the total
extract and the immunoprecipitates showed that about 20-30% of WC-1
self-associated. As expected, both WC-2 and FRQ also
co-immunoprecipitated together with Myc-WC-1. This result indicates
that there are different forms of the WC-1-containing complexes
in the cell. Whereas most of the WC-1 forms a heterodimer with WC-2,
some WC-1 self-interacts to form larger complexes. This interpretation
is consistent with the previous results of the sucrose gradient and
in vitro DNA binding assay (18, 19).

View larger version (43K):
[in this window]
[in a new window]
|
Fig. 5.
A small portion of WC-1 self-associates.
A, immunoprecipitation assays (IP) showing that
Myc-WC-1 co-precipitated with the endogenous WC-1 in the wt,Myc-WC-1
strain. The Western blots were probed with the antiserum indicated at
the left. The wild-type strain was used as the negative
control. B, immunoprecipitation assays showing that there
was no WC-2 self-interaction in vivo. PI, the
wt,Myc-WC-2 extracts were immunoprecipitated with the preimmune
antiserum. The upper panels were probed with the c-Myc
antibody, and the lower panels were probed with the WC-2
antiserum.
|
|
In contrast to WC-1, WC-2 self-interaction was not detected in
vivo. As shown in Fig. 5B, no endogenous WC-2 was found
to co-precipitate with the Myc-WC-2 protein in the wt,Myc-WC-2 strain. This result suggests that the strong self-interaction observed in the
in vitro protein binding assay is very likely an artifact. Because WC-2 is required for maintaining the steady state level of WC-1
through its PAS domain-mediated WC-1/WC-2 interaction (20), the
existence of WC-1 self-association and the absence of WC-2
self-interaction suggest that one WC-2 molecule can form a complex with
more than one WC-1 molecule in the cell.
The WC-1 PASC Domain and Its Immediate C-terminal Region Is
Essential for the WC-1/WC-2 Interaction in
Vivo--
Previously, we showed that the PAS domain of WC-2 is
essential for mediating the WC-1/WC-2 interaction in vivo (20).
However, it is not known which region of WC-1 binds to the WC-2 PAS
domain. Although the PAS domains of both proteins were proposed to
interact with each other based on the in vitro protein
result (34), as we have shown above, the protein-protein interactions
observed in vitro do not necessarily exist in
vivo. Therefore, a series of wc-1 deletion mutants were
created and experiments were carried out to determine which region of
WC-1 mediates the interaction with WC-2 in vivo.
Sequence analysis of WC-1 revealed that it contains two putative
transcription activation domains, three PAS domains (including the LOV
domain), one putative nuclear localization signal (NLS, aa 919 to 926),
and a GATA-type zinc finger DNA-binding domain. A series of internal
and C-terminal deletions of WC-1 were generated. The putative
N-terminal transcription activation domain clearly is dispensable,
because it is missing in the wc-14401 strain (Fig.
4). Fig. 6A is a schematic
diagram of the WC-1 open reading frame and the deletion made in each
mutant. Except for the three constructs at the bottom of the
diagram that were made in the qa-wc-1 plasmid (allowing the expression
of WC-1 in the presence of QA), the rest of the constructs were made in
the Myc-WC-1 plasmid, so that the WC-1 protein can be monitored by the
c-Myc monoclonal antibody. This is important when the level of WC-1 is
low in the mutants. All constructs were transformed into the wc-1RIP strain. Based on the structural predictions
of the three PAS domains, small deletions predicted to abolish the
formation of PAS structures were introduced into each domain.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 6.
The PASC domain of WC-1 is required for the
formation of the WC-1/WC-2 complex. A, schematic
diagrams showing the deletions made in various wc-1 mutants.
B, immunoprecipitation assays (IP) using WC-2
antiserum showing the interaction of WC-1 and WC-2 in various mutants.
The Neurospora protein extracts were either
immunoprecipitated with WC-1 antiserum (right panels) or
analyzed directly with Western blot analyses (left panels).
All constructs indicated were transformed into the his-3
locus of the wc-1RIP strain. PI, the
total extract of the wc-1RIP,Myc-WC-1
strain was immunoprecipitated with the preimmune antiserum. For the
wc-1RIP,qa-WC-1.NH strain, 10 2
M QA was added into the medium.
|
|
To map the WC-1 domain that is essential for the interaction with WC-2,
the lysates of the deletion mutants were subjected to
immunoprecipitation using our WC-2 antiserum. As shown in Fig. 6B, in mutant that contains the deletion of the WC-1
N-terminal region (qa-wc-1.NH) or the disruption of the first two PAS
domains, the formation of the WC-1/WC-2 complex was maintained,
indicating that the entire N-terminal half of the WC-1 protein is not
required for the complex formation. This is consistent with our
previous result showing that the removal of the entire LOV/PAS domain
did not abolish the formation of the WC-1/WC-2 complex (28). However, the disruption of the PASC domain (Myc-WC1.PASC) or the removal of the
C-terminal region downstream of aa 764 (Myc-WC1.NruI) completely abolished WC complex formation. Thus, the PASC domain and its C-terminal region are essential for the formation of the WC complex.
The low level of WC-1 expression in the PASC mutant is consistent with
our previous conclusion that the formation of the WC complex is
important for maintaining the steady state level of WC-1.
Interestingly, although the deletion of the C-terminal of WC-1 in the
Myc-WC1.NruI strain eliminated the WC-1/WC-2 interaction, its level of
WC-1 was significantly higher than that of the PASC mutant. This could
be due to the fact that the removal of the WC-1 C-terminal region
stabilized the protein. Although the LOV and PASB domains are not
required for the formation of the WC complex, their disruptions,
especially the mutation of PASB, did reduce the expression level of
WC-1 significantly. Therefore, it is possible that they also influence
the WC-1/WC-2 interaction.
To further map the region essential for the WC interaction, deletion
mutants downstream of aa 764 were generated. Our results showed that
the region upstream of the putative NLS is required for the
interaction, but the NLS and its C-terminal region are dispensable. As
shown in Fig. 7A, the removal
of the C-terminal region downstream of aa 887 (WC-1.BamHI and
WC-1.MluI) eliminated the WC-1/WC-2 protein interaction; however, in
all mutants that contain the region upstream of the putative NLS (aa
918), the WC-1/WC-2 interaction could still be observed (Fig.
7B). In addition, the deletion of the NLS or its C-terminal
region did not abolish the FRQ-WC interaction. Therefore, the minimal
region that is required for the WC-1/WC-2 interaction appears to be
from the PASC domain to aa 918.

View larger version (68K):
[in this window]
[in a new window]
|
Fig. 7.
The PASC domain and its C-terminal region of
WC-1 are required for the WC-1/WC-2 interaction. A,
immunoprecipitation assays using either WC-1 or WC-2 antiserum showing
that the C-terminal region of the PASC domain is needed for the
WC-1/WC-2 interaction. In the wc-1RIP,qa-WC-1.BamHI
and wc-1RIP,qa-WC-1.MluI strains, 10 2
M QA was added into the liquid medium to induce WC-1
expression. PI, the wild type extract was immunoprecipitated
with the preimmune antiserum. B, immunoprecipitation assays
using WC-2 antiserum showing that the putative NLS and the C-terminal
part of the protein are not required for the WC-1/WC-2 and FRQ-WC
interactions. The constructs indicated above are derived from the
Myc-WC-1 construct, and they were transformed into the his-3
locus of the wc-1RIP strain. PI, the
wc-1RIP,Myc-WC-1.NLS protein extracts were
immunoprecipitated with the preimmune antiserum.
|
|
Because WC-2 is a nuclear protein and its nuclear localization is
independent of WC-1 (14, 20, 35), one possibility for the failure of
the WC-1/WC-2 interactions in some of the mutants is that the mutant
WC-1 proteins failed to enter the nucleus. To exclude this possibility
and to examine the role of the putative NLS signal in the nuclear
localization of WC-1, the localization of WC-1 in the NLS mutants and
other mutants was examined. In these strains, WC-1 proteins were still
found in the nucleus (data not shown). Therefore, the putative NLS
signal and the regions required for the WC-1/WC-2 interaction are not
required for the nuclear localization of WC-1. A putative NLS signal,
located immediately upstream of the zinc finger domain, is also found
in WC-2. It was previously shown that this signal was not required for
the nuclear localization of WC-2 (35). The similarities of these two
putative NLS signals in sequence and location suggest that they may be
part of the domain involved in DNA binding rather than mediating
nuclear import of the proteins, a notion that is supported by the
functional study described below.
The WC-1 NLS and Zinc Finger DNA Domains Are Required for the Dark
Activation of FRQ but Are Not Essential for Light Responses--
To
understand the role of different domains in WC-1 functions, we examined
the light responses and dark expression of FRQ in these mutants. As
expected, in mutants where there was no formation of the WC complex,
the light induction of frq, al-3, and
vvd genes were completely abolished (Fig.
8A), and the expression of FRQ in the dark is very low (Fig. 8B). Although the PASB region
was not required for the WC complex formation, its disruption also abolished both the light and dark function of WC-1, indicating it is a
domain essential for the WC-1 functions. In mutants containing the
deletion of the region upstream of the LOV domain (WC1.NH) or the
C-terminal putative activation domain (Myc-WC1.AgeI), both the light
responses and dark expression of FRQ appeared to be normal (data not
shown). Surprisingly, the light induction of the genes was normal in
mutants containing the deletion of the NLS and the zinc finger
DNA-binding domain (Fig. 8, A and C). However, in
the dark, the levels of FRQ in these strains are very low (Fig. 8,
B and C), a level similar to that of the
wc-1 null strain. These data indicate that although the
putative NLS and the DNA-binding domain are not required for the light
function of WC-1, they are required for the dark activation of
frq and the generation of circadian oscillations in constant
darkness. Therefore, it is likely that the putative NLS and the zinc
finger domain act as one functional motif to activate gene expression in the dark.

View larger version (69K):
[in this window]
[in a new window]
|
Fig. 8.
The putative NLS and the DNA-binding domain
of WC-1 are required for the dark activation of FRQ but not for the
light induction of frq and other genes.
A, Northern blot analysis showing the light induction of
frq, al-3, and vvd mRNA.
DD, cultures were harvested at DD22; LP,
cultures were given a 15-min light pulse at DD22 before harvest.
B, Western blot analysis showing the FRQ expression in LL or
DD22 in various wc-1 mutants. C, Northern and
Western blot analysis showing the expression of frq,
al-3, and FRQ.
|
|
 |
DISCUSSION |
In constant darkness, WC-1 and WC-2, the two PAS-containing
transcription factors, form a complex that binds the
cis-acting elements of the frq promoter and
activates the transcription of frq, leading to the cycling
of frq and the overt rhythmicity (19, 36). Recently, WC-1
has also been identified as the first fungal blue light photoreceptor
that binds FAD as a chromophore and (together with WC-2) binds to the
LREs of the frq promoter in a light-dependent manner (19, 28). Thus, the rapid light-dependent induction of frq transcription mediated by the WC complex is the
mechanism that allows the Neurospora clock to be entrained
by light (16, 29). Therefore, both WC proteins are required for the
clock function in the dark and for the light input of the clock. In addition, the WC complex is required for other light responses in
Neurospora (27).
The physical interaction between the WC complex and FRQ is a crucial
step that closes the Neurospora circadian negative feedback loop (15, 18). Although WC-2 was previously proposed as a scaffold
protein that mediates such an interaction (18), here we show that the
presence of WC-1 is essential for the formation of the FRQ-WC complex
(Fig. 1). In mutants without WC-1, despite the normal expression of
WC-2, no WC-2-FRQ interaction was detected in vivo. Because
WC-1 needs WC-2 to form complexes to maintain its level and because
WC-1 is the limiting factor in WC complexes (13, 18, 20), we think that
WC proteins can interact only with FRQ in vivo as a
WC-1/WC-2 complex. A similar situation may also exist in
Drosophila, in which PER and TIM may only interact with the
dCLK-CYC heterodimer and not with the free CYC protein (37).
Previously, we showed that FRQ positively regulates the expression of
WC-2 and that the PAS domain of WC-2 is required for the formation of
the WC complex and the maintenance of the steady state of WC-1 (13,
20). Here we show that WC-1 negatively regulates the expression of
wc-2 at the level of transcription (Figs. 2 and 3), thus
forming another interacting feedback loop. Interestingly, the
repression of wc-2 by WC-1 is light independent, suggesting
that this function of WC-1 does not require its role in light sensing
(Fig. 3C). Such an interlocked nature of the expression of
the two WC proteins may be important for maintaining an appropriate
ratio of the two proteins in the cell, allowing them to function
properly in the clock and in light responses. The opposite effects of
FRQ and WC-1 on wc-2 may help explain the fact that
WC-2 is not rhythmically expressed (13, 18). Although we
could not exclude the possibility that WC-1 regulates wc-2
indirectly, these data suggest that in addition to being a
transcription activator, WC-1 may function as a transcription repressor
as well.
Consistent with WC-1 being a multifunctional protein, our data also
demonstrate the existence of different forms of the WC complexes and
the significantly different requirements of WC-1 for light induction of
frq and other genes. The differential requirement of WC-1
for light-induced gene expression is highlighted by our result in the
wc-14401 mutant (Fig. 4). In this mutant, resulting
from alternative protein initiation from a downstream AUG, less than
1% of the normal amount of WC-1 was expressed, and it was still
associated with WC-2. However, its light induction of frq
was near normal, whereas the light induction of al-3 and
vvd was mostly abolished. A similar situation was also
observed for a wc-2 mutant. In the
wc-2ER33 mutant (a point mutation in the zinc finger
DNA-binding domain), although its light induction of other genes are
abolished, its light induction of frq was close to normal
(16, 20, 21). Therefore, the light induction of frq, a
step critical in the light resetting of the clock, does not require
fully functional WC proteins, and needs only a very low amount of a WC complex.
Recently, a study by Dragovic et al. (38) showed that in
some wc mutants, conidiation rhythms could still be driven
by light/dark cycles. Although this study suggested the existence of a
wc-independent photoreceptor in Neurospora that
regulates the conidiation process, the interpretation of the results is
complicated by the fact that the
wc-1 strain (generated
by RIP) used in that study may not be a real wc-1 null
strain. In that wc-1 mutant strain, although the light
induction of other genes was eliminated, the light induction of
frq could still be observed, a fact that is in conflict with the results obtained in true wc-1 null strains and other
wc-1 mutants (16, 19, 28). Therefore, based on the results
we presented here, we think that the wc-1 mutant used by
Dragovic et al. (38) is most likely not a real null, and it
could produce a low level of WC-1 (that could not be detected by
regular Western blot analysis) because of alternative protein
initiation from a downstream AUG.
The differential requirement of WC complex in the light induction of
genes and the in vitro DNA binding data (19) suggest the
existence of different WC complexes in the cell, a notion that was
confirmed by the immunoprecipitation assay in a wt,Myc-WC-1 strain
(Fig. 5). Using the extract of this strain, we found that there were at
least two types of WC-1-containing protein complexes in the cell:
~20-30% of WC-1 was found to self-associate to form a large
WC-1/WC-2 complex. The larger WC complex may be the large WC-containing
complex identified by Froehlich et al. (19) using a in
vitro DNA binding assay that binds to the LREs of the
frq promoter in a light-dependent manner and
regulates light responses. In contrast to WC-1, no WC-2 was found to
self-associate in vivo, although strong self-association was
previously observed in vitro (34). Because WC-1 needs WC-2
to form a complex to maintain its steady state level, one WC-2 molecule
should form complexes with more than one WC-1 molecule in the cell. Our
results also confirm the previous results that the majority of WC-1 is
in a WC-1/WC-2 heterodimeric complex (18, 19, 28). The function of the
WC-1/WC-2 heterodimer may be important for the activation of
frq and other genes in the dark (19, 39).
To map the WC-1 domain that mediates its interaction with WC-2 and to
determine the function of various WC-1 domains, we made a series of
wc-1 mutants. Our results indicate that the PASC domain and
its immediate C-terminal region (upstream of the putative NLS) are
required for the formation of the WC-1/WC-2 complex, whereas the rest
of WC-1 is not essential (Figs. 6 and 7). Although the requirement of
the PASC domain for the interaction is consistent with the interaction
mediated by the PAS-PAS interaction of the two WC proteins (20), the
involvement of the PASC C-terminal region indicates that PASC alone is
not sufficient for such an interaction. Consistent with our previous
work of WC-2 (20), these results show that the formation of the WC
complex is essential for their functions in the circadian clock and
light responses and for maintaining the steady state level of WC-1.
However, the existence of the WC-1/WC-2 complex is not sufficient to be
a functional complex, because the PASB domain of WC-1 is required for
both of its dark and light functions whereas it is not required for the
complex formation (Fig. 8, A and B).
One surprising result from the deletion study is that the C-terminal
part of the WC-1 protein (starting from the putative NLS and including
the zinc finger) is not required for the light induction of
frq, al-3, and vvd (Fig. 8).
However, the NLS and DNA-binding domain are required for the expression
of FRQ in the dark. Thus, by deleting this region of WC-1, we separated
the light and dark functions of WC-1. This is in contrast with the mutant lacking the entire LOV domain, in which only the light function
but not the dark function of WC-1 is defective (28). Without the
DNA-binding domain of WC-1, how does WC-1 mediate the light-induced
gene expression? Because WC-1 forms complexes with WC-2, the zinc
finger DNA-binding domain of WC-2 must be responsible for the DNA
binding in these mutants. Interestingly, as described above, in a
wc-2 mutant (ER33) that contains a point mutation in the
DNA-binding domain, the light induction of frq was still
observed, although the light induction of other genes was defective
(16, 20, 21). Together, these data suggest that the light induction of
frq only requires one DNA-binding domain from either of the
WC proteins, but the DNA-binding domain of WC-2 may be essential for
binding to the promoters of other light-inducible genes. On the other
hand, the DNA-binding domains of both WC proteins may be required for
DNA binding and transcription activation of frq in the dark.
In conclusion, our results show that WC-1 is required for the formation
of the FRQ·WC complex and it negatively regulates the expression of
wc-2. In addition, WC-1 differentially regulates the light
induction of frq and other genes, and there are different forms of the WC complexes in the cell, possibly with distinct functions. Together, our data demonstrate that WC-1 is a
multifunctional protein involved in the circadian clock, light
sensing, and transcription repression in Neurospora.