Molecular Cell
Article
Execution of the Circadian Negative FeedbackLoop in Neurospora Requires the ATP-DependentChromatin-Remodeling Enzyme CLOCKSWITCHWilliam J. Belden,1 Jennifer J. Loros,1 and Jay C. Dunlap1,*1Department of Genetics, Dartmouth Medical School, Hanover, NH 03755, USA
*Correspondence: [email protected]
DOI 10.1016/j.molcel.2007.01.010
SUMMARY
In the Neurospora circadian system, the tran-scription factors White Collar-1 (WC-1) andWhite Collar-2 (WC-2) activate expression offrq, whose gene product inhibits its own ex-pression. The WC proteins are thought to forman obligate complex; however, chromatin im-munoprecipitation (ChIP) indicates that WC-2binds to the frq promoter in a rhythmic fashion,whereas WC-1 is bound continuously. Small os-cillations in histone acetylation are detectedover the circadian cycle with a marked reduc-tion upon light-induced activation. Nucleaseaccessibility experiments indicate chromatinrearrangement at the frq promoter; therefore,19 genes with homology to ATP-dependentchromatin-remodeling enzymes were deletedand the strains examined for clock phenotypes.One gene, designated clockswitch (csw-1), isrequired for clock function; its product localizesto the frq promoter, is required for proper frqexpression, and has an impact on chromatinstructure. The data suggest that CSW-1 regu-lates accessibility of promoter DNA, thus gener-ating the sharp transition from the transcrip-tionally active to the repressed state.
INTRODUCTION
Circadian rhythms in eukaryotic organisms are governed
by positive and negative feedback loops resulting in de-
velopmental, behavioral, and other physiological pro-
cesses occurring in a time-of-day-specific manner. In-
volvement of coupled feedback loops is a unifying
principle that underlies our understanding of clock func-
tion and is conserved throughout evolution (Dunlap,
1999; Hirayama and Sassone-Corsi, 2005; Reppert and
Weaver, 2002). In Neurospora crassa, one feedback loop
includes the two transcription factors White Collar-1
(WC-1) and White Collar-2 (WC-2) and the frequency
(FRQ)-FRQ RNA helicase (FRH) complex (Bell-Pedersen
Molecula
et al., 2005; Brunner and Schafmeier, 2006; Dunlap,
2006; Liu and Bell-Pedersen, 2006).
WC-1 and WC-2 are nuclear localized transcription fac-
tors that heterodimerize via Per-Arnt-Sim (PAS) domains
forming the White Collar complex (WCC). Current models
of the Neurospora circadian system assume that the WCC
serves as the positive trans-acting factor responsible for
rhythmic frq expression. The WCC is typical of the
PAS:PAS heterodimeric transcriptional activators charac-
teristic of eukaryotic circadian feedback loops and is re-
sponsible for both light-activated and clock-regulated
transcription of frq (Crosthwaite et al., 1995, 1997). The
elegance of the Neurospora circadian system is high-
lighted by the dual role of WC-1 that serves as both the
photoreceptor (Froehlich et al., 2002; He et al., 2002)
and one of the positive elements involved in frq transcrip-
tion in the dark (Crosthwaite et al., 1997).
FRQ is part of a heteromeric complex that includes FRH
and acts as both a positive and negative regulator to influ-
ence normal clock function (Aronson et al., 1994; Cheng
et al., 2005; Lee et al., 2000; Schafmeier et al., 2006).
The negative feedback loop is believed to occur through
a direct interaction between FRQ, FRH, and the WCC
where FRQ inhibits its own transcription by reducing
WCC activity (Cheng et al., 2001a; Denault et al., 2001;
Froehlich et al., 2003; Schafmeier et al., 2005), presum-
ably by affecting the phosphorylation state of WC-1 and
WC-2 (Schafmeier et al., 2005; He et al., 2006).
Transcriptional regulation at the frq promoter occurs
through binding of the WC proteins to a pair of cis-acting
sequences (see Figure 1) termed Clock box (C box) and
proximal light-regulated element (PLRE) (Froehlich et al.,
2002, 2003). The C box is required for rhythmic expression
of frq and overall clock function in continual darkness,
whereas the PLRE is necessary to establish the proper
phase when entrained by light. Interestingly, both the C
box and PLRE are necessary for high levels of light-in-
duced frq expression. Although much is known about
the regulatory sequences and the components of the cir-
cadian negative feedback loop, little is known about the
actions of the WC proteins at the clock-relevant pro-
moters or the role of histone modifications and chromatin
remodeling. For this reason, and because chromatin
remodeling and the associated modification of histones
is a requisite step in the activation of many genes, we
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frq Regulation Requires clockswitch
Figure 1. WC-2 Protein Binds Rhythmically to the C Box, but Not to the PLRE
(A) A schematic representation of the frq locus in which the shaded box is the coding region and the 50 and 30 UTRs are indicated. The cis-acting
sequences (C box and PLRE, separated by 1001 bp) are also indicated, with +1 marking the major start of transcription. Spliced regions are not
shown, and the cartoon is not to scale (Colot et al., 2005). The arrows represent the primer pairs used for ChIP with the acronyms and product sizes
shown. Single letters above the schematic indicate restriction endonuclease sites: B, BglII; C, ClaI; E, EcoRI; M, MluI; A, AvaI; and F, FspI.
(B) ChIP at the frq promoter done on a clock-WT strain. Quantitative PCR was done in a multiplexed reaction containing either the C box or PLRE-
specific oligonucleotides and the 3.303 oligonucleotide pair. The 3.303 oligonucleotides, specific to an untranscribed region of Neurospora DNA,
were included in each reaction as a measure of nonspecific background binding. Samples were harvested at indicated times after incubation in con-
stant darkness (DD) and in response to a 15 min light pulse (LP15) given after 24 hr in darkness. An equal amount of each lysate was used in reactions
containing a-WC-2 antibody, and DNA was isolated as described in the Experimental Procedures. A 10-fold dilution series of total lysate added in
each reaction is shown at the left. The time cultures were grown in darkness (DD), and the corresponding circadian times (CT) are indicated.
(C) Data from (B) were collected on a phosphoimager, and band intensities were determined by using ImageQuant software. After subtraction of non-
specific background, average values are plotted as a percent of total with error bars representing ±SEM (n = 3–5) Shaded boxes indicate subjective
day and night.
(D and E) Same as in (B) and (C) except that the long period mutant strain frq7 was used in place of WT.
examined the chromatin modifications associated with
frq expression.
Chromatin structure can influence gene expression
by controlling the accessibility of DNA to activators,
repressors, and RNA polymerases (Narlikar et al., 2002).
Posttranslational modifications to histone core proteins,
including acetylation, methylation, phosphorylation, ubiq-
588 Molecular Cell 25, 587–600, February 23, 2007 ª2007 Else
uitination, sumoylation, and ADP-ribosylation, cause dis-
tinct effects on gene regulation and expression by facilitat-
ing changes in chromatin structure (Fischle et al., 2003;
Strahl and Allis, 2000). Histone acetylation, methylation,
and transcription factor binding can serve as a mark for
recruitment of bromo- and chromodomain-containing
ATP-dependent chromatin-remodeling enzymes of the
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frq Regulation Requires clockswitch
Swi2/Snf2 family (de la Serna et al., 2006; Narlikar et al.,
2002). These enzymes, which usually exist as large multi-
subunit complexes, regulate accessibility of DNA through
a number of mechanisms including the translocation of
nucleosomes, the disassembly or assembly of core nucle-
osomes, and exchange of histones with histone variants
(Mellor, 2005; Narlikar et al., 2002). Currently, only a subset
of these helicases is characterized, and there may be
other, yet-to-be determined mechanisms of action with
the unifying theme being regulation of genome structure
to allow accessibility for DNA-specific enzymes.
Experiments in the mammalian circadian system have
revealed that mPer1 and mPer2, which encode central
clock components functionally analogous to FRQ, have
rhythmic AcH3 (K9, K14) modifications and that RNA poly-
merase II (Pol II) binds rhythmically to these promoters
(Etchegaray et al., 2003). Similar results were found at
the Cry locus where the CLOCK-BMAL complex was
found to bind rhythmically. CLOCK itself has histone ace-
tyltransferase activity that is stimulated by binding BMAL
(Doi et al., 2006). Chromatin structure is also remodeled
at the mCry locus in a rhythmic manner, highlighting a pos-
sible role for chromatin modifications and remodeling in
execution of the negative feedback loop (Etchegaray
et al., 2003). Daily oscillations in chromatin modifications
and activator binding are also seen at the mammalian out-
put gene, albumin D element binding protein (Dbp) (Rip-
perger and Schibler, 2006). Finally, the polycomb group
protein, EZH2, localizes to mPer1 and mPer2 promoters
and is involved in circadian-regulated expression in mam-
mals (Etchegaray et al., 2006).
In this report, we show that WC-1 and WC-2 do not bind
to and exit the frq promoter as a unit but instead do so dif-
ferentially as chromatin is remodeled during the transcrip-
tional activation and deactivation of frq. By using targeted
knockouts, we identify a gene clockswitch (csw-1) that
has homology to ATP-dependent DNA chromatin-remod-
eling enzymes and whose deletion leads to disruption of
circadian-regulated banding on race tubes. CSW-1 is re-
quired for normal frq expression and localizes to the frq
promoter. Chromatin structure adjacent to the C box ele-
ment in frq is altered in a circadian manner and appears to
be more accessible to nucleases in the Dcsw-1 strain.
CSW-1 appears to control circadianly regulated frq tran-
scription by creating a more compact chromatin structure
at the C box, suggesting that CSW-1 is required for proper
execution of the transcriptional negative feedback loop.
RESULTS
Differential Binding of the WC Proteins
to the frq Promoter
To gain a better understanding of the events associated
with the expression of frq, we performed in vivo chromatin
immunoprecipition (ChIP) experiments with WC-1- and
WC-2-specific antibodies and the oligonucleotide pairs
shown in Figure 1A. ChIP assays were carried out on
a strain that is considered wild-type (WT) for circadian-
Molecula
regulated expression (see Experimental Procedures) and
the long period mutant frq7, grown both in a 48 hr time
course in constant darkness and in response to a 15 min
light pulse (LP15) by using nonspecific IgG as a control
(data not shown and see Figure S1 in the Supplemental
Data available with this article online). Events that are
clock specific occur at comparable subjective circadian
times (CTs) in WT (22.5 hr period) and frq7 (29 hr periods),
but due to differences in the inherent period lengths, the
comparable CT occurs after different numbers of hours
in darkness. As an added control, oligonucleotide pairs
specific to a noncoding region of the Neurospora genome
were added to the quantitative PCR reaction to measure
nonspecific background. It is clear from Figures 1B–1E
that WC-2 interacted with the C box in a circadian-depen-
dent manner with peaks occurring at the same CT in both
WT and frq7 (CT = 20–5) around subjective dawn. Compa-
rable results showing rhythmic WC-2 association with the
C box have been reported elsewhere (He et al., 2006).
Consistent with results showing that the C box also acts
as a light-response element (Froehlich et al., 2002; He
and Liu, 2005), we found a 10-fold increase in binding after
a 15 min light pulse (LP15). There was no circadian-regu-
lated binding to the PLRE; however, binding of WC-2 at
this element was enhanced greater than 15-fold in re-
sponse to a light pulse (LP15), confirming this site’s role
as the major light element. The differential binding of
WC-2 to these cis-acting sequences confirms the exis-
tence of a complex regulatory mechanism that controls
circadian versus light-regulated transcription at frq.
All models of the circadian feedback loop predict that
the WCC acts as a unit and is modified or regulated in the
nucleoplasm to affect changes in frq expression, and
therefore we expected the results of the WC-1 ChIP ex-
periment to mirror those of WC-2. Surprisingly, however,
the WC-1 protein is bound continuously to both the C
box and PLRE and is clearly detectable over the entire cir-
cadian cycle and in light-treated samples. Shown in Fig-
ure 2 is a WC-1 ChIP experiment using samples and con-
ditions identical to those used for WC-2 (Figure 1). We
observed only slight changes in WC-1 binding to the C
box and PLRE over the circadian cycle, and these were
generally less than 2-fold. Not surprisingly, we often saw
the greatest amount of WC-1 binding after a 15 min light
pulse in WT and frq7. The observation that some WC-1
is always associated with the frq promoter, whereas
WC-2 binding is rhythmic, indicates the WCC does not ex-
ist solely as a unit. The formation and/or disassembly of
WCC is regulated and appears to occur at the promoter
elements, representing an unanticipated mechanism in
circadian control of frq. As further controls, we used two
different WC-1 antisera in ChIP experiments with Dwc-1,
Dwc2, and Dwc-1/Dwc-2 strains (Figure S1). The C box
and PLRE DNA immunoprecipitated in the Dwc-1 and
Dwc-1/Dwc-2 deletion strains was usually comparable
to what was seen with the VVD antibody, a cytoplasmic
protein used as a nonspecific IgG control (Heintzen
et al., 2001).
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frq Regulation Requires clockswitch
Figure 2. WC-1 Is Always Bound to the frq Promoter
ChIP reactions at frq were performed as described in Figure 1 on clock-WT (A and B) and frq7 (C and D) strains by using antibodies specific to WC-1.
Changes in Histone H3 Acetylation Accompany
Chromatin Remodeling
Acetylated histones H3 and H4 are often associated with
transcriptionally active genes, and it was shown that these
modifications are circadianly regulated at the mammalian
Per and Cry loci (Etchegaray et al., 2003). To examine the
acetylation state of H3 and H4 at frq, we used antisera
against diacetylated H3 (K9, K14) and tetra-acetylated
H4 in ChIP experiments (Figures 3A–3D). We found that
the histones are acetylated in constant darkness, but in-
stead of systematic changes, we observed only low-level
fluctuations at the transcriptional start site (TSS) and frq
coding region (FCR) in both WT and frq7. There is a large
reduction in the level of AcH3 (K9, K14) at the TSS in re-
sponse to a 15 min light pulse, and comparable results
were obtained with a tetra-acetylated H4 antibody (data
not shown). The loss of acetylation at the TSS could reflect
differential modifications on the histone tails (i.e., methyl-
ation or phosphorylation) or a disassembly of the nucleo-
somes to allow access for general transcription factors,
similar to what is seen at the yeast PHO5 promoter where
disassembly of nucleosomes is coincident with transcrip-
tional activation (Boeger et al., 2003; Reinke and Horz,
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2003). The slight changes in AcH3 K9, K14 at the TSS
and FCR are consistent with whole-genome analyses in
yeast that indicated the levels of acetylated histones
drop in regions of transcriptionally active genes (Pokholok
et al., 2005; Roh et al., 2004). These results contrast recent
data showing an increase in AcH3 (K14) in the promoters
of light-induced loci (Grimaldi et al., 2006). The discrep-
ancy between results is presumably due to differences
in loci being examined and regions therein.
If the loss of AcH3 (K9, K14) at transcriptionally active
frq is the result of disassembly of nucleosomes, then it
stands to reason that the chromatin structure would be
more susceptible to nucleases. Therefore, we used a mi-
crococcal nuclease (MNaseI) sensitivity assay followed
by indirect end labeling on purified nuclei isolated from
cells grown in the dark or treated with a 15 min light pulse
(Figure 3E). Two major changes in the chromatin structure
were observed in response to light, and these correspond
to the TSS/PLRE (lower arrow) and the C box (upper
arrow) regions. There exists a putative CCAAT box se-
quence near the PLRE/TSS region, so the new MNaseI-
sensitive site seen in LP15 treated nuclei (lower arrow)
may represent a disassembly of nucleosomes to allow
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frq Regulation Requires clockswitch
Figure 3. Loss of Acetylation on Histone H3 Accompanies Chromatin Remodeling
(A–D) ChIP assays at frq in both clock-WT (A and B) and frq7 (C and D) were carried out as described in Figure 1 except that a commercially available
diacetylated (K9, K14) antibody specific to histone H3 (AcH3) (Upstate Biotechnology) was used. Oligonucleotides specific to the major start of tran-
scription (TSS) and the frq coding region (FCR) were used in a multiplexed reaction with the negative control oligos (3.303).
(E) Chromatin structure was visualized by indirect end labeling of partial MNaseI-digested nuclei. Nuclei were isolated from WT Neurospora grown in
the dark for 4 hr (DD) and compared to cells subjected to a 15 min light pulse (LP) after 24 hr in darkness. The upper arrow marks changes at the C box
promoter element, and the lower arrow indicates the presence of a novel MNaseI sites in the light-pulsed sample.
(F) Circadian-regulated chromatin structure was assayed as in (A) except that nuclei were isolated from clock-WT strain grown and harvested after
incubation in the dark for the indicated times (hr). Nuclear isolation and MNaseI reactions were performed in a dark room, and EcoRI was used as the
secondary enzyme for better resolution of the C box region. The arrow indicates a circadianly regulated change in chromatin structure, and the as-
terisks are nonrandom preferential MNaseI cleavage sites.
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frq Regulation Requires clockswitch
access for the general transcription factors. These data
are consistent with an overall change in chromatin struc-
ture when frq becomes transcriptionally active.
Given the light-induced differences in chromatin struc-
ture, it seemed likely that there would also be circadianly
regulated changes, so nuclei isolated from a 32 hr time
course were assayed using conditions designed to re-
solve chromatin structure around the C box. A MNaseI-
sensitive site appears between CT 9 and 17 (DD20–
DD28), a time when frq expression is low, producing
a DNA fragment that migrates between the 1.4 and 1.5
kb markers that is not present at earlier times (Figure 3F,
arrow). The presence of this site may represent a change
in chromatin structure, and because this site is adjacent to
the C box, it seems plausible that a remodeling event oc-
curs and contributes to frq regulation by controlling the
accessibility of the C box to WC-2. This site was difficult
to resolve because of a nonrandom MNaseI site present
in genomic DNA that migrates directly above the site of in-
terest (see also Figure 6A) but is supported by indepen-
dent assessments using ligation-mediated PCR (LMPCR)
(Figure S4) and analysis of mononucleosome occupancy
(Figures 6D and 6E).
Identification of a Circadian-Specific Enzyme with
Homology to Swi2p/Snf2p
The decrease in AcH3 (K9, K14) at the TSS and the
changes in chromatin structure indicated that nucleo-
somes are remodeled at frq and suggest that daily
changes at the frq locus might be an important step in cir-
cadian regulation. To identify the chromatin-remodeling
enzyme(s) responsible for this activity, we systematically
used gene replacement to knock out (Colot et al., 2006)
all 19 genes in Neurospora with homology to yeast Snf2,
Sth2, and other known remodeling enzymes (Borkovich
et al., 2004). Homokaryotic deletion strains were isolated
from germinated ascospores generated by backcrossing
the heterokaryon knockouts to a lab WT (87–74) that con-
tains the bd mutation, which is used to facilitate scoring for
clock phenotypes. The genes deleted are listed in the
Supplemental Data (Table S1). Two of the 19, whose clos-
est homologs are yeast Sth2 and mi-2, appeared asco-
spore lethal, and a third (NCU9106.1), now called clock-
switch (csw-1), is required for normal circadianly
regulated asexual spore development as assayed on
race tubes (Figure 4A). The Dcsw-1 strain had sporadic
conidiation, but circadian regulation of this process was
clearly affected. The Dcsw-1 strain also has a reduced
growth rate and a visible defect in carotenoid biosynthesis
similar to albino mutant strains (data not shown).
The csw-1 locus encodes a 1011 amino acid protein
containing DEXHc and HELICc domains characteristic of
the conserved snf2 domain present in all chromatin-
remodeling enzymes. Its closest homologs are yeast
Fun30 (BLAST 1e�104) and mammalian Etl1 genes about
which little is known. Antibodies generated against the
first 350 amino acids, a region not highly conserved with
other ATP-dependent chromatin-remodeling enzymes,
592 Molecular Cell 25, 587–600, February 23, 2007 ª2007 Elsev
detected a protein with an approximate MW of 115 kDa
not present in Dcsw-1. Examination of cell extracts col-
lected from WT grown in a 48 hr time course indicate
that the CSW-1 protein (Figure 4B) and csw-1 mRNA
(data not shown) are expressed at constitutively low levels
and do not change over the circadian cycle or in response
to light.
CSW-1 Is Required for Circadian-Regulated
frq Expression
The loss of circadian-regulated spore formation on race
tubes suggested that CSW-1 may play a role in regulating
frq expression, so we examined frq mRNA in the Dcsw-1
strain (Figure 4C). Normally, frq mRNA is rhythmically ex-
pressed in the early subjective morning, with the peak oc-
curring between CT 0 and CT 5. In Dcsw-1, the circadian
regulation of frq was quite different compared to WT;
even though both strains started expressing frq at the
same time (8 hr after transfer to darkness), the initial
peak of frq expression in Dcsw-1 consistently occurred
4 hr after WT (CT 9 in Dcsw-1, CT 5 for WT). After the first
peak in frq expression, basal levels of transcription re-
mained higher than normal in Dcsw-1, and there was no
detectable second peak. Examination of FRQ protein ex-
pression was consistent with the northern blots shown in
Figure 4C, indicating an elevated basal level of protein
(Figure 4D). A wide range of phosphorylated forms of
FRQ could be observed after the first round of turnover
and coincides with the apparent loss of circadian rhyth-
micity. The elevated expression of frq and FRQ in Dcsw-
1 during the evening hours (CT12–21) when levels should
be low suggests that CSW-1 is needed to terminate the
positive limb of the circadian feedback loop. We did not
detect any difference in wc-1 or wc-2 transcript levels
due to the loss of CSW-1 (data not shown). These data
suggest that CSW-1 is required for circadianly regulated
frq expression.
CSW-1 Changes Chromatin Accessibility at frq
The phenotypic and molecular defects in the biological
clock indicate that CSW-1 is required to execute the circa-
dian feedback loop, so it seemed reasonable to test if
CSW-1 localized to the frq promoter. A ChIP experiment
using affinity-purified CSW-1 antibody was performed
on WT samples collected over a 48 hr time course. Binding
was difficult to detect, suggesting that CSW-1 is not abun-
dant at the frq promoter; however, there was consistent
and reproducible binding of CSW-1 to both the C box
and PLRE (Figure 5). This binding appeared to be specific
because we did not detect it in the knockout strain
(Figure S2). The difficulties associated with detecting
CSW-1 associated with the C box or PLRE makes sub-
traction of background paramount. For example, we ob-
serve high levels of background for the C box at DD12
and the PLRE at LP15 in Dcsw-1, yet when normalized
to the level of 3.303 band, no significant binding is de-
tected (Figure S2). This contrasts with the identical times
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frq Regulation Requires clockswitch
Figure 4. The csw-1 Gene Is Required for
Normal Circadian-Regulated frq Expres-
sion
(A) Race tube analysis of clock-WT and six indi-
vidual strains deleted for csw-1 indicates loss
of normal rhythmicity in Dcsw-1 strains.
(B) Immunoblot analysis performed on cell
lysates using a CSW-1-specific antibody. Cells
were harvested after growth in the dark for the
indicated times.
(C) Northern blot analysis was done on total
RNA isolated from WT and Dcsw-1 by using
probes specific for frq.
(D) Immunoblot analysis was performed on WT
and compared to Dcsw-1 by using antibodies
specific for FRQ.
in WT when binding is detected, yet there is little or no
3.303 present in these samples.
The binding of CSW-1 appeared periodic with the peaks
occurring between late night (CT 21) and early subjective
day (CT 5) in both WT and frq7, indicating that CSW-1
Molecula
localization is regulated by the clock. There appeared to
be a strong correlation between WC-2 and CSW-1 binding
to the C box, with maximum binding occurring at the same
time. We also detected CSW-1 at the PLRE, although no
transcriptional activation occurs from this element in the
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Figure 5. CSW-1 Shows Rhythmic Association with the C Box
ChIP assays were done as described in Figure 1 by using an affinity-purified antibody to CSW-1.
absence of light. The cumulative data suggest that CSW-1
regulates the accessibility of the frq promoter to WC-2.
Based on homology to other ATP-dependent remodel-
ing enzymes, it seemed reasonable that the gradual
damping and loss of the overt (Figure 4A) and molecular
(Figures 4C and 4D) rhythms in Dcsw-1 was due to
a loss of clock-regulated chromatin remodeling at frq. Pre-
vious data establish the C box as the site of obligate circa-
dian control of frq expression (Froehlich et al., 2003), and
experiments shown here (Figure 3E) indicate that chroma-
tin structure is altered near the C box. To assay if CSW-1
was responsible for catalyzing these changes, a limited
MNaseI digestion on nuclei isolated from a Dcsw-1 strain
was performed and compared to WT by using conditions
described in Figure 3E. The unique MNaseI-sensitive site
that appeared rhythmic in WT and migrated between the
1.4 and 1.5 kb markers in WT seems to always be present
in the Dcsw-1 (Figures 6A and 6B, arrow). This demon-
strates that chromatin structure at frq is more loosely
packed in Dcsw-1 and may indicate that a nucleosome
is remodeled to control accessibility to the C box.
The movement of the individual nucleosome directly ad-
jacent to the C box was next assayed using restriction site
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accessibility experiments with the enzymes AvaI and FspI
(Figure S3). Surprisingly, we saw rhythmic nucleosome re-
modeling in both WT and Dcsw-1; it was clearly apparent
in Dcsw-1 with the peak occurring at CT 5 (DD16). The
only other notable observation from these analyses was
the presence of increased nuclease susceptibility at CT 5
(DD16) in Dcsw-1 over WT, a time when CSW-1 binding
would be at a maximum. The presence of nuclease acces-
sibility rhythms inbothstrains stronglysuggests thatCSW-1
does not act alone in catalyzing the movement of the nucle-
osome adjacent to the C box and that an additional chroma-
tin-remodeling enzyme must also be involved. It also sug-
gests, in light of the MNaseI data (Figures 3F and 6), that
CSW-1 somehow protects the C box from nuclease action.
In an effort to better resolve the CSW-1-dependent
changes in chromatin structure, we performed LMPCR
on WT and Dcsw-1 over a 32 hr time course with specific
focus on the C box element (Figure S4). LMPCR is an as-
say that allows one to properly locate nucleosome posi-
tions and measure changes in movement at the nucleotide
level. We were able to roughly position both nucleosomes,
and densitometric analysis of a hypersensitive site (site 1)
was rhythmic in WT with peaks in accessibility occurring at
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frq Regulation Requires clockswitch
Figure 6. The Accessibility of DNA at the frq Promoter Is Altered in Dcsw-1
(A and B) Chromatin structure was determined by partial MNaseI digestion of purified nuclei from WT (A) and Dcsw-1 (B) as described in Figure 5B.
The arrows indicate changes in the MNaseI digestion profile when WT is compare to Dcsw-1.
(C) Schematic representation of the primers designed to examine nucleosome position in quantitative PCR of mononucleosomes.
(D) Representative experiment from the quantification of mononucleosome positions. The 3.303 primer pair was used as a control for loading.
(E) The amount of Nuc B from WT lysates, normalized to the amount of 3.303 amplified fragment, is reported as the average ± SEM (n = 4–6).
(F) Same as in (E) except that the assay was performed on Dcsw-1.
DD16 and DD32, times when WC-2 binding is at a maxi-
mum. This site also oscillated in Dcsw-1, but the peak in
accessibility was much broader and there was overall
more susceptibility to MNaseI. One of the general obser-
vations from this assay was that sites 1 and 2 were more
Molecula
accessible in Dcsw-1, whereas sites 3 and 4 were more
accessible in WT (compare traces in lower panel). This re-
sult indicates once again that there is an overall change in
accessibility to nucleases in Dcsw-1, and these do not
mirror what we observe in WT. We conclude that, although
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frq Regulation Requires clockswitch
Figure 7. CSW-1 Is Required for Normal Circadianly Regulated WC-2 Association to the C BoxChIP assays were done as described in Figure 1 on Dcsw-1 grown in a 48 hr time course and in response to a 15 light pulse with the WC-2 (A and B)
and WC-1 (C and D) antibodies.
the chromatin structure undergoes cyclical changes in
both strains, it remains in a more open state in Dcsw-1.
To further examine changes in chromatin structure,
quantitative analysis of mononucleosomes surrounding
the C box was performed using primers designed to assay
occupancy of nucleosome A (Nuc A) and nucleosome B
(Nuc B) (see Figure 6C). An additional oligonucleotide
pair spanning the C box was used as a control to measure
if there was complete digestion to mononucleosomes,
and the 3.303 oligo pair was used as a loading control
for normalization. We detected rhythmic levels in the
band corresponding to Nuc B, suggesting the nucleo-
some was remodeled in a circadian manner (Figures 6D
and 6E). At times corresponding to transcriptionally per-
missive states (beginning around DD12 = CT0), there is
a decrease in the levels of Nuc B, and this gradually rises
when transcription is repressed (Figures 6D and 6E). Pre-
sumably, this nucleosome was disassembled, because
when we probed for protection upstream, we found similar
rhythms instead of a corresponding protected region that
should have cycled antiphasic to Nuc B had the nucleo-
some simply been moved (data not shown). When we ex-
amined this same nucleosome in Dcsw-1, we once again
596 Molecular Cell 25, 587–600, February 23, 2007 ª2007 Elsev
detected a fluctuation but found that it did not behave
identically to WT. Nuc B in Dcsw-1 underwent changes
in chromatin structure but did not behave in a typically cir-
cadian manner because its peak occurred later than in WT
before grading to intermediate levels (Figure 6F). It is im-
portant to note that the data derived from examination of
mononucleosome positioning was in excellent agreement
with the LMPCR (Figure S4) and suggests that CSW-1 is
needed to fully close the chromatin structure in order to
facilitate efficient negative feedback inhibition.
Taken together, these data indicate that CSW-1 is local-
ized to frq, is required for normal frq expression, and pro-
vides protection to nucleases but is not solely responsible
for nucleosome movement, supporting the notion that
CSW-1 may play an essential but supportive role as an
ATP-dependent remodeling enzyme. Because of this
and because remodeling enzymes are often necessary
for regulating accessibility of transcription factors, we per-
formed ChIP assays on Dcsw-1 and examined binding of
the WC proteins. WC-2 association with the C box is en-
tirely misregulated in Dcsw-1, with binding occurring
throughout the subjective day and into the night (compare
Figure 7A to Figure 1B). Although there remains a low
ier Inc.
Molecular Cell
frq Regulation Requires clockswitch
amplitude fluctuation in WC-2 binding in Dcsw-1, there is
always a basal level of WC-2 associated with the C box af-
ter the initial change to constant dark conditions, and the
amount of WC-2 bound never decreases to the WT mini-
mum. It is also apparent that the phasing of WC-2 binding
is very different from WT and is consistent with the acces-
sibility experiments determined above. The most notable
effect on WC-2 binding in Dcsw-1 occurs around subjec-
tive dusk from CT 9 to CT 13, a time when frq expression
is normally low and WC-2 is not usually bound in WT,
yet there is substantial binding in the mutant. We were
not able to detect any major differences in WC-1 binding
to the C box, although the relative levels of protein bound
did appear to be appreciably lower in Dcsw-1 (Figure 7C).
The reason for this is unclear but may indicate further mis-
regulation of WCC binding to the frq promoter. Binding of
WC-2 at inappropriate CTs emphasizes the important role
that CSW-1 plays in properly shutting down frq transcrip-
tion and the positive limb, suggesting a mechanism in
which WCC activity is negatively regulated by controlling
accessibility to the C box. Presumably other mechanisms
are also involved, and these include catalytic inactivation
through regulated phosphorylation (He et al., 2006; Schaf-
meier et al., 2005), direct inactivation through association
with FRQ (Denault et al., 2001), or direct inactivation by
protein turnover of transcriptional activators associated
with transcription (Collins and Tansey, 2006; Talora
et al., 1999).
DISCUSSION
We began this analysis from the premise that the circadian
regulation of frq expression, which is required for circa-
dian rhythms in Neurospora, must entail rhythmic binding
of the pertinent transacting factors. These were the White
Collar proteins, WC-1 and WC-2, that have been assumed
to act in an obligate complex (Bell-Pedersen et al., 2005;
Cheng et al., 2001b; Crosthwaite et al., 1997; Froehlich
et al., 2003; Schafmeier et al., 2005). ChIP assays con-
firmed rhythmic association of WC-2, but not of WC-1, in-
dicating a more dynamic regulation that includes complex
formation or disassembly. A trivial explanation would hold
that WC-2 is associated with the C box at all times and
there is rhythmic masking of WC-2 epitopes, presumably
by the FRQ-FRH complex or a yet-to-be-determined com-
ponent. However, we believe this is highly unlikely, be-
cause WC-1, but not WC-2, is present at the PLRE at all
CTs after transition to dark conditions, and if rhythmic
masking was artificially producing the effect at the C
box, then we should see corresponding rhythmic unmask-
ing of WC-2 at the PLRE. Additionally, the polyclonal anti-
sera used for the WC-2 ChIP experiments were directed
against epitopes over the whole protein, and it seems un-
likely that all epitopes would be rhythmically masked.
The exact mechanism of WCC formation needs addi-
tional study, but the data presented here suggest that reg-
ulation of complex disassociation is integral to the regula-
tion of gene expression and is presumably coupled to
Molecula
chromatin remodeling. The differential association of
WC-2 with either the PLRE or C box was dependent on
the regulation conditions (circadian or light activated),
highlighting the existence of an elaborate system regulat-
ing complex formation at a specific element (Froehlich
et al., 2002, 2003). Interestingly, circadian regulation of
WCC binding and disassembly appears to be much
more complicated than initially thought, and this contrasts
its mammalian counterparts. The CLOCK:BMAL1 hetero-
dimer appears much more stable and is bound as a com-
plex to mPer1 over the entire circadian cycle with little to
no change in abundance (Lee et al., 2001), whereas
CLOCK:BMAL1 binds rhythmically to the mCry1 promoter
with peaks in binding occurring coincident with activation
(Etchegaray et al., 2003).
It seemed likely that regulation of WCC assembly at frq
might be controlled at the level of chromatin modifications
or remodeling, but examination of the acetylation state of
core histones revealed only minor changes occurring over
the circadian cycle. However, a large decrease in H3 and
H4 acetylation was observed at the TSS in response to
light that we surmised might be a reduction or reposition
of histones to allow access for the general transcription
factors. Support for this was found by performing nucle-
ase accessibility experiments that show the appearance
of a strong accessible site in a region described as the ma-
jor start of transcription, verifying a chromatin-remodeling
event that is light responsive (Grimaldi et al., 2006). Re-
modeling was also observed at the C box, and not surpris-
ingly, it changed over the circadian cycle, implicating nu-
cleosome translocation as a possible mechanism for
regulating WC-2 binding. This led to a reverse genetic ap-
proach designed to identify the ATP-dependent chroma-
tin-remodeling enzyme(s) responsible for this catalytic ac-
tivity. Phenotypic analysis of deletion strains led to the
discovery of CSW-1, an enzyme that is required for normal
frq expression.
Characterization of Dcsw-1 indicates that CSW-1 nega-
tively regulates either the formation or disassembly of
a transcriptionally active complex composed of WC-1
and WC-2 at the C box and is required to shut down tran-
scription. CSW-1 has strong homology to other Snf2 pro-
teins and appears to be involved in small circadianly reg-
ulated nucleosome changes at the C box ultimately
regulating activator binding. CSW-1 function requires fur-
ther analysis, but the data presented here indicate that it
negatively regulates WCC activity at frq by altering chro-
matin structure. In addition to its role in regulating chroma-
tin structure and WC-2 association with the C box, CSW-1
is required for active transcription of the albino-2 gene in
dark grown cultures (data not shown), suggesting it has
both positive and negative effects on expression depend-
ing on the locus.
The most obvious question that stems from this study is
this: what is the identity of the remaining ATP-dependent
chromatin-remodeling enzyme(s)? The answer to this
question appears to be the Neurospora homolog of Mi-
2. Although Dmi-2 was ascospore lethal, suggesting that
r Cell 25, 587–600, February 23, 2007 ª2007 Elsevier Inc. 597
Molecular Cell
frq Regulation Requires clockswitch
mi-2 is essential, subsequent preliminary data indicate
this is a synthetic-lethal effect when Dmi-2 is combined
with the bd mutation, an output-specific mutation com-
monly used to clarify circadian expression of conidiation
on race tubes. We have since been able to generate a ho-
mokaryon knockout of mi-2 in a WT background, and this
strain also has aberrant circadian-regulated frq expres-
sion (W.J.B. and J.C.D., unpublished data). Analysis of
Dmi-2 is currently ongoing, and elucidation of its role in
conjunction with further analysis of Dcsw-1 should illumi-
nate the role these enzymes play in changing chromatin to
facilitate regulated transcription.
The CSW-1-dependent effects on chromatin structure
are difficult to determine, especially in light of the fact
that Mi-2-catalyzed events are presumably still occurring.
However, it seems evident that CSW-1 is involved in com-
pacting chromatin at the C box consequently controlling
WC-2 association. Proof of CSW-1 remodeling activity
comes from four independent assays: MNaseI digestion
followed by indirect end labeling, restriction site accessi-
bility assays, LMPCR, and quantification of mononucleo-
somes. In all cases, we detected differences between
WT and Dcsw-1, with the general theme being increased
accessibility and inappropriate circadian-regulated
changes and the end result being increased WC-2 associ-
ation. CSW-1 remodeling presumably involves positioning
and stabilizing the nucleosome adjacent to the C box in
a process that may also involve Mi-2. This fits well with
the dynamic nucleosome model whereby multiple remod-
eling enzymes alter chromatin structure in a dynamic fash-
ion in repeated cycles of interconversion, generating per-
missive and nonpermissive states for transcription (Mellor,
2006; Metivier et al., 2003).
It remains possible that CSW-1 functions by a less ca-
nonical role; for example, it could deposit and/or remove
a histone variant in a process similar to Swr1. Swr1 ex-
changes H2A-H2B dimers with H2AZ-H2B dimers gener-
ating flanks around nucleosome-free regions in repressed
promoters that are poised for activation (Mizuguchi et al.,
2004; Raisner et al., 2005; Zhang et al., 2005). It is also
possible that CSW-1 adds or removes heterochromatin
protein-1 (HP1) in a process that simultaneously removes
or blocks WC-2 from the promoter. This would mirror the
regulation of mammalian clock-controlled Dbp where
HP1 is positioned at the E box during the repressive phase
of the circadian cycle (Ripperger and Schibler, 2006).
Lastly, the data are formally consistent with a model in
which the WCC normally enters the C box as a unit to drive
quasisynchronous frq expression and then, associated
with the cycle of rhythmic transcription, WC-2 exits alone
or is turned over there, later followed by loss/displace-
ment of WC-1. In this model, CSW-1 could play a role in
closing down the C box to prevent WCC access; in a
Dcsw-1 strain, unimpeded access would lead to constant
midlevel frq expression no longer coordinated into a cycle
with a peak, and (reflecting steady turnover), reduced
levels of WC-1, both of which were observed. Further ex-
amination of these and other possibilities will undoubtedly
598 Molecular Cell 25, 587–600, February 23, 2007 ª2007 Else
shed light on the role of CSW-1 in circadian-regulated
expression.
Cumulatively, these data indicate that association of
WC-2 occurs in a circadian manner, that assembly or dis-
assembly of WCC occurs at the C box, and that the DNA
helicase CSW-1 is needed to remodel chromatin in a pro-
cess that controls accessibility to transcriptional activator
complex at frq. It is clear that other components, in addi-
tion to those encoded by the standard ‘‘clock genes,’’ are
needed for execution of the circadian cycle, and the ex-
amination of these components may lead analysis of cir-
cadian rhythmicity from the cytoplasm and nucleoplasm
to chromatin.
EXPERIMENTAL PROCEDURES
Strains and Growth Conditions
The clock WT strains, 87–74 (bd, his-3, a) and 328–4 (bd, A), plus the
long period mutant, frq7, all contain the band mutation for visualization
of circadianly regulated conidia formation. Knockouts were generated
in 87–74 by using the split marker strategy with 3 kb of homologous
DNA flanking the hygromycin resistance gene (hph) or by using 1 kb
flanks with the mus-51 stain (Colot et al., 2006). Primary transformants
were confirmed by Southern blot, and strains that contained the
knockout cassette free of ectopic insertions were backcrossed to
328–4. Ascospores were germinated on complete media and geno-
typed. Analysis of rhythms on race tubes (13 Vogel, 0.1% glucose,
0.17% arginine) was done as described (Dunlap and Loros, 2005).
The liquid culture assays were performed in media (2% LCM) con-
taining 13 Vogels and 0.17% arginine with 2% glucose and were
grown at 25�C. Conidia were used to seed mycelial mats in 75 mm petri
dishes, and 0.5 cm plugs were cut from these and used for 50 ml cul-
tures. Mats were harvested by filtration, frozen in liquid nitrogen, and
ground in a mortor and pestle. Time course experiments have been
described previously (Aronson et al., 1994).
Expression of recombinant GST-CSW-1 for antibody production
was done by PCR amplifying full-length csw-1 and inserting it into
pET 41 LIC (Novagen). The vector was cut with EcoRI and then reli-
gated removing the C-terminal portion so that only the N-terminal por-
tion was expressed. The resulting vector pFE2, expressing GST-CSW-
1 amino terminus, was expressed in Rosetta cells grown in TTP media
(2% tryptone, 1.5% yeast extract, 125 mM NaCL, 25 mM Na2HPO4,
15 mM KH2PO4 [pH 7.0]) and induced with 0.5 mM IPTG.
ChIP Assay and Antibodies
The ChIP assays were done as previously described with minor optimi-
zation for use with Neurospora (Komarnitsky et al., 2000; Kuras and
Struhl, 1999). Ground crosslinked cell lysates were resuspended in
10 ml FA lysis buffer (50 mM HEPES-KOH [pH 7.5], 150 mM NaCl,
1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS,
1 mM PMSF) and sonicated eight times for 20 s with a Bronson sonifier
equipped with a microtip and set at 70% amplitude. The mean frag-
ment size was approximately 300–500 bp, and the C box and PLRE
are 1000 bp apart, so very few fragments would contain both sites.
The sonicated cell lysates were cleared of cellular debris using
a 2000 3 g spin. Immunoprecipitated samples were washed four times
with FA lysis buffer by using the same g-force.
The WC-1 and WC-2 antibodies were raised against full-length (WC-
2) or quasi-full-length (WC-1) protein and have been previously de-
scribed (Denault et al., 2001; Lee et al., 2000). Antibodies raised
against diacetylated histone H3 (K9, K14) and tetra-acetylated H4
(K5, K8, K12, K16) were obtained from Upstate Biotech. Polyclonal an-
tibodies were raised against recombinant GST-CSW-1 fusion protein
expressing the N-terminal 350 amino acids of protein. Induced antigen
vier Inc.
Molecular Cell
frq Regulation Requires clockswitch
was purified using glutathione agarose, two rabbits were immunized
with 1 mg/ml at Pacific Immunology Corporation, and sera were col-
lected.
The oligonucleotides used for analysis of DNA-protein interactions
are contained in Table S2. An equal amount of DNA was used in a quan-
titative PCR reaction in the presence of 0.06 mCi/ml [a-32P]dATP by
using the oligonucleotides pairs described in Figure 1, and the samples
were separated on a 10% TBE gel.
MNaseI Assays
Nuclei were isolated in the dark as previously described (Froehlich
et al., 2002; Luo et al., 1998) and then subjected to limited MNaseI di-
gestion following established protocols (Ausubel et al., 1994). Briefly,
equal amounts of nuclei were resuspended in MNaseI buffer A, treated
with 1.0 ml 0.1M CaCl2, and incubated at 37�C for 1.5 min, and then
varying amounts of MNaseI were added and the nuclei incubated for
an additional 1.5 min. The enzyme was inactivated by the addition of
Proteinase K in TENSK buffer, and the nuclei were incubated for a min-
imum of 2 hr to remove the chromatin. The DNA was purified by phenol
chloroform extraction-EtOH precipitation and digested with the indi-
cated restriction endonuclease as the secondary enzyme. DNA frag-
ments were resolved on a 1.5% agarose gel, transferred to positively
charged nitrocellulose, and probed with the indicated frq-specific
probe. LMPCR was performed on isolated nuclei as described (Ausu-
bel et al., 1994; Fragoso et al., 1995). Quantification of mononucleo-
somes was likewise done as previously described (Metivier et al.,
2003).
Northern, Southern, and Western Blot
Standard northern and Southern blots were performed by using digox-
igenin label DNA probes following Roche guidelines. RNA was isolated
from cells using a hot phenol extraction, and 15 or 25 ug/ul of RNA was
fractionated on a 1.3% agarose formaldhyde gel (Aronson et al., 1994).
DNA was isolated using the Puragene Kit following the manufacturers’
protocol. Immunoblot analysis was as described (Garceau et al.,
1997).
Supplemental Data
Supplemental Data include four figures and two tables and can be
found with this article online at http://www.molecule.org/cgi/content/
full/25/4/587/DC1/.
ACKNOWLEDGMENTS
We thank Hildur Colot for help generating the knockout strains. We
also thank Allan Froehlich for his technical help with Neurospora and
for critically reading this manuscript. This work was supported by
grants from the National Institutes of Health to J.C.D. (GM34985 and
GM68087). W.J.B. is funded in part by a Ruth L. Kirschstein Postdoc-
toral Fellowship (GM071223) from the National Institutes of Health.
Received: June 9, 2006
Revised: November 9, 2006
Accepted: January 9, 2007
Published: February 22, 2007
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