Execution of the Circadian Negative Feedback Loop in ...dblab.rutgers.edu/genome_cooperative/publications/Belden_et_al... · Loop in Neurospora Requires the ATP-Dependent Chromatin-Remodeling

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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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-

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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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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,

590 Molecular Cell 25, 587–600, February 23, 2007 ª2007 Elsev

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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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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Molecular Cell


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,


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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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

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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


Molecular Cell


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


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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Molecular Cell


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


Molecular Cell


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


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


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


Molecular Cell


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


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


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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