The frequency natural antisense transcript firstpromotes, then represses, frequency gene expressionvia facultative heterochromatinNa Li, Tammy M. Joska, Catherine E. Ruesch, Samuel J. Coster, and William J. Belden1

Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901

Edited by Steven Henikoff, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved January 21, 2015 (received for review April 2, 2014)

The circadian clock is controlled by a network of interconnectedfeedback loops that require histone modifications and chromatinremodeling. Long noncoding natural antisense transcripts (NATs)originate from Period in mammals and frequency (frq) in Neuros-pora. To understand the role of NATs in the clock, we put the frqantisense transcript qrf (frq spelled backwards) under the controlof an inducible promoter. Replacing the endogenous qrf promoteraltered heterochromatin formation and DNA methylation at frq. Inaddition, constitutive, low-level induction of qrf caused a dramaticeffect on the endogenous rhythm and elevated circadian output.Surprisingly, even though qrf is needed for heterochromatic silenc-ing, induction of qrf initially promoted frq gene expression bycreating a more permissible local chromatin environment. The ob-servation that antisense expression can initially promote sensegene expression before silencing via heterochromatin formationat convergent loci is also found when a NAT to hygromycin re-sistance gene is driven off the endogenous vivid (vvd) promoter inthe Δvvd strain. Facultative heterochromatin silencing at frq func-tions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriatecircadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of,and occurs alongside, other feedback processes.

circadian rhythm | natural antisense transcripts | heterochromatin |DNA methylation

In eukaryotes, metazoans, and vertebrates, the circadian rhythmrequires timed chromatin remodeling and modifications to

ensure the appropriate amplitude, period, and phase of clockgene expression. The need for chromatin regulation arises be-cause the clock is predominantly controlled by a transcriptionalnegative feedback loop where transcriptional activators driveexpression of negative elements that inhibit its own expression(1–3). In Neurospora crassa, the positive elements are the GATAtype transcription factors White Collar-1 (WC-1) and WC-2 thatform the White Collar complex (WCC) (4). The WCC drivesexpression of frequency (frq) that is translated with delays beforeit associates with FRQ-interacting RNA helicase (FRH) (5, 6).FRQ-FRH inhibits frq expression through a direct interactionwith WCC that is mediated by WC-2 (7, 8). FRQ undergoesphase-specific phosphorylation over the course of the day, andthis phosphorylation controls regulated transport between thenucleus and cytoplasm, destabilization, and turnover (9–12). Inaddition, there appears to be fine-tuned control of chromatin inboth the activation and feedback inhibition phases of circadianoscillations (13–17).Recently, we reported that cytosines in the frq promoter are

methylated (m5C) and proper regulation of the frq locus isneeded for normal DNA methylation (13). The frq locus iscomposed of three transcripts: the frq gene encoding FRQ pro-tein (6), a natural antisense transcript (NAT) qrf (frq spelledbackwards) (18), and a small upstream transcript of unknownfunction that spans the clock box (c-box) promoter element (Fig.1 A and B). The three transcripts are all controlled by the WCC

via binding to three separate cis-acting sequences. The proximallight-regulated element (pLRE) is the predominant cis-actingsequence that controls light-activated expression of frq (19, 20).The antisense light response element (aLRE) is required forlight-induced qrf expression (18, 21), whereas the c-box is themajor element controlling circadian frq expression (22). We havepreviously demonstrated that the frq antisense transcript qrf andWCC are needed for normal m5C (13). It was later shown thatconvergent transcription at frq gives rise to dicer-independentsiRNA (disiRNA) and that DNA methylation occurs at disiRNAloci throughout the genome. This work also confirmed the re-quirement for WCC-mediated expression for m5C at frq (23, 24).All DNA methylation in Neurospora repeat regions requires

the histone H3 Lys 9 (H3K9) methyltransferase (KMT1) DIM-5,heterochromatin protein 1 (HP1), and DNA methyltransferaseDIM-2 (25–28). The molecular mechanism of DNA methylationproceeds in a stepwise fashion mediated by DCDC (DIM-5/DIM-7/DIM-9–CUL4/DDB1 complex) (25, 28, 29). Both DIM-5and HP1 are also required for methylation at frq, suggesting thatother DCDC components may also be required (30). However,the dependency of qrf expression and the existence of disiRNAsoriginating from frq suggest a more complicated mechanismbecause RNAi is not needed for DNA methylation at relics ofrepeat-induced point mutations throughout the genome (31).In this report, we explore the effects of qrf on frq expression.

We found that proper expression of qrf is needed for normalfacultative heterochromatin formation with an unanticipated twist.Before heterochromatic silencing, induction of qrf first promotes

Significance

The circadian clock is predominantly regulated by transcrip-tional negative feedback, where the protein(s) arising from thecentral clock gene(s) inhibit their own expression. In Neuros-pora and mammals, the clock genes have natural antisensetranscripts (NATs): long noncoding RNAs that overlap and areexpressed in opposite orientation to the protein coding genes.Previously, we demonstrated that Neurospora frequency NATis needed for normal DNA methylation. This report demon-strates that proper regulation of the frequency antisensetranscript is needed to establish repressive heterochromatin.However, prior to establishing heterochromatin, NAT expres-sion creates a transcriptionally permissive state that helpspromote sense transcript expression. Broader implications in-dicate that NATs, in general, may first promote sense geneexpression prior to establishing heterochromatin.

Author contributions: N.L., T.M.J., and W.J.B. designed research; N.L., T.M.J., C.E.R., S.J.C.,and W.J.B. performed research; N.L., T.M.J., C.E.R., S.J.C., and W.J.B. analyzed data; andN.L., T.M.J., and W.J.B. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Freely available online through the PNAS open access option.1To whom correspondence should be addressed. Email: belden@aesop.rutgers.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1406130112/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1406130112 PNAS | April 7, 2015 | vol. 112 | no. 14 | 4357–4362

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the expression of frq by generating a more transcriptionallypermissive state. High levels of qrf expression that normally oc-cur in response to light ultimately silence frq via heterochromatinformation in a process independent of, but parallel to, VIVD(VVD)-mediated repression. The bimodal activity of NAT ex-pression that first helps promote sense gene expression prior tosilencing via heterochromatin also occurs at an artificial sense/antisense reporter system indicating that the dual function maybe a general feature of NAT expression.

ResultsDNA Methylation at frq Requires Proper Regulation of the qrf. m5Cin the frq promoter requires DIM-2, a functional WCC, andnormal qrf expression (13). Promoter DNA methylation alsooccurs at other convergent transcripts, and these loci all producedisiRNA (24, 32). As a primer for the work presented here,RNA-sequencing data from QDE-2 (Argonaute)–associatedRNAs and WC-2 ChIP-sequencing were analyzed at frq and areshown in Fig. 1A (21, 24). To explore the role of the qrf and

disiRNAs in directing m5C at frq, we replaced the aLRE withthe inducible qa-2 promoter (qa-2-qrf) to control expression byadding quinic acid (QA) to the medium (33) (Fig. 1B). Weassayed m5C at frq by performing a methylation-sensitive Southernblot in WT and the qa-2-qrf strain with and without QA undercircadian conditions sampling every 4 h (Fig. 1C). Surprisingly,we were unable to detect WT levels of m5C in qa-2-qrf even athigh concentrations of QA (10 mM). This unexpected resultarose because qrf induction with QA was lower than in WTunder light-inducing conditions (Fig. S1 A and B). However, QAinduction of qrf was elevated relative to a strain lacking theaLRE that is needed for qrf expression. This strain, calledfrq10frqccg-2, has the 3′ UTR of frq replaced with the 3′ UTR ofclock controlled gene-2 (ccg-2), thus removing the aLRE (18)(below and Fig. S1C). The frq10frqccg-2 strain dramaticallyreduces the level of qrf expression to the point where it cannot bedetected by a Northern blot (18), but qrf can still be detected viastrand-specific RT-PCR analysis (Fig. S1C), indicating that itdoes not completely abolish qrf expression.The unexpected reduction in m5C in qa-2-qrf with QA

induction led us to examine additional time points. In everycondition tested, we observed a reduction in m5C at frq (Fig. 1Cand Fig. S2). We then analyzed if the qa-2-qrf strain couldsuppress the hypermethylation phenotype seen in Δchd1 butfound no major change in the hypermethylation phenotype inthe double mutant (qa-2-qrf Δchd1), suggesting that CHD1functions subsequent to qrf (Fig. 1D).

qrf Is Needed for Heterochromatin Formation. m5C at frq requiresboth histone H3K9 methylation (H3K9me) and HP1 (30). DIM-5adds a monomethyl group, dimethyl group, and trimethyl group toH3K9 and is needed for all known DNA methylation in Neurospora(28, 34). The defect in DNA methylation in qa-2-qrf led us to testif qrf is needed for efficient heterochromatin formation. Weperformed ChIP with an H3K9me3 antibody comparing WT with

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Fig. 1. Induction of qrf affects DNA methylation at frq. (A) Comparativeanalysis of disiRNA-sequencing and WC-2 ChIP-sequencing found at the frqloci. (B) Schematic representation of the WT frq locus in Neurospora and thetransgenic qa-2-qrf strain used in this study. The qa-2-qrf has the qa-2 pro-moter in place of the aLRE. (C) Methylation-sensitive Southern blot exam-ining the frq promoter in WT (FGSC2489) and qa-2-qrf (XB141-12) with andwithout QA. Genomic DNA was digested with the methylation-sensitive re-striction enzyme BfuCI (labeled B) or the methyl-insensitive isoschizomerDpnII (labeled D) and probed for a region specific to the pLRE. (D) Same as inC, except DNA methylation was examined in a qa-2-qrf Δchd1 (XB223-7)double mutant compared with WT, qa-2-qrf, and Δchd1 (XB131-6). DD, timein darkness; LL, constant light.

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Fig. 2. qrf is needed for normal facultative heterochromatin formation.H3K9me3 and HP1 binding were measured at frq by ChIP in WT (FGSC2489)and qa-2-qrf (XB141-12) strains. (A) Level of H3K9me3 was determined byquantitative PCR assay of DNA isolated with an H3K9me3-specific antibodyusing oligonucleotides specific to the pLRE. The Δdim-5 and a nonspecificIgG were used as negative controls. (B) Same as in A, except the oligos useddetected a region near the c-box. (C) HP1 binding to regions in frq promoternear the pLRE was measured by ChIP using a GFP-specific antibody in strainscontaining the HP1-GFP fusion protein (XB270-1 and XB265-1). (D) Same asin C, except HP1 binding to the transcriptional start site (TSS) was examined.All cultures used in these experiments were grown in the dark for 40 h andthen transferred to saturating light and cross-linked at the indicated timesbefore processing.

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qa-2-qrf (without QA). Normally, H3K9me3 at frq peaks 30 minafter exposure to light-inducing conditions, and we found sig-nificantly lower levels of H3K9me3 in qa-2-qrf relative to WT atthe pLRE and at the c-box (Fig. 2 A and B). We could still detectsome H3K9me3 in qa-2-qrf; however, the qa-2 promoter isleaky, and there is spurious transcription even in the absenceof QA.We next tested if other heterochromatin marks besides m5C

and H3K9me3 were altered in qa-2-qrf. We assayed binding ofHP1 to the frq promoter via ChIP using an HP1-GFP fusionconstruct. We detected a lower level of HP1 binding in qa-qrfrelative to WT (Fig. 2 C and D). The combination of reducedm5C, H3K9me3, and HP1 binding indicates that facultative het-erochromatin formation at frq is dependent on qrf. Moreover,the defect in qrf-dependent heterochromatin is consistent withresults showing that long noncoding RNAs are processed intosiRNA via the exosome and can induce heterochromatin for-mation in Schizosaccharomyces pombe (35).

Altered qrf Expression Affects the Circadian Clock. We next soughtto examine how induction of qrf affects clock regulation. Theqa-2-qrf strain was crossed to a strain with the frq promoter fusedto a codon-optimized luciferase (36) and to a strain harboringthe ras-1bd allele to examine circadian output (37). Induction ofqrf with 10 mM QA abolished the normal circadian rhythm (Fig.3A and Fig. S3). In the absence of QA, there was an expectedlow-amplitude, phase-shifted rhythm. The altered phase is consis-tent with frq10frqccg-2, which has a small but detectable phase shiftrelative to WT (18). These data are further support for the idea thatfacultative heterochromatin is needed to establish the appropriatephase (13, 18). Moreover, replacing the aLRE with the qa-2promoter appeared to interfere with the normal rhythmic con-idia formation (Fig. 3B).We next wanted to explore how altering the level of consti-

tutive qrf expression affected the circadian rhythm. We grew theqa-2-qrf strain over a range of QA concentrations and founda low-amplitude rhythm in every condition tested except 10 mMQA (Fig. S4). We then tested whether transient or constitutiveinduction of qrf was causing the arrhythmic phenotype. We grewthe qa-2-qrf strain in normal media and then transiently inducedqrf with 10 mM QA for 15 min before transferring the cultures tomedia with and without QA. The results indicate that constitutive

induction of qrf disrupts the circadian rhythm (Figs. S5 and S6).We also found that frq-specific disiRNA produced in qa-2-qrf wasmisregulated relative to WT (Fig. S7).

qrf Affects Molecular Oscillations. To determine why constitutiveexpression of qrf was causing arrhythmic frq expression, we ex-amined the relative levels of RNA expression originating fromthe frq locus over circadian time by quantitative RT-PCR anal-ysis using a random hexamer capable of detecting both frq andqrf abundance. We found that the overall transcript levels wereelevated in the induced qa-2-qrf strain compared with WT (Fig.4A). There was also a peak in expression that was delayed 4 hrelative to WT. The seemingly obvious explanation is that theincrease in expression was due to qrf induction, but data shownbelow indicate that it is due to an increase in frq expression. Toconfirm this defect in expression, we examined the rhythm inFRQ protein by immunoblot analysis. We found that inductionof qrf had a dramatic effect on the normal protein rhythm typi-cally observed in WT, and we detected a relatively high level ofFRQ at every time point examined in qa-2 qrf (Fig. 4B). Theseresults are consistent with the luciferase data and support theobserved defect in circadian clock function.

Expression of qrf Initially Promotes frq Expression. The elevatedlevel of transcription originating from frq was surprising becauseour expectation was that qrf induction would cause disiRNA-mediated heterochromatin. Therefore, we tested the individuallevels of frq and qrf by strand-specific RT-PCR assay. WT andqa-2-qrf were grown in the dark for 24 h to remove any residuallight effects on frq expression, and the growing mycelia were thentransferred to fresh medium containing 10 mM QA. The 10 mMQA pulse resulted in roughly fivefold induction of qrf thatpeaked 15 min after the cultures were transferred to QA medium(Fig. 5A). Surprisingly, induction of qrf had an unanticipatedeffect on frq expression; instead of disiRNA-mediated transcrip-tional gene silencing (TGS), there was a dramatic and steadyincrease in frq expression (Fig. 5B). Therefore, we surmised thatinduction of qrf might generate a more transcriptionally per-missive state that helps promote expression of frq. To explorethis idea further, we tested if qrf induction could elevate frq ex-pression in the absence of a functional WCC. We generateda strain that contained both qa-2-qrf and Δwc-2 and tested ifinduction of qrf elevated frq expression (Fig. 5 C and D). Theqa-2-qrf Δwc-2 strain had an increase in frq expression inde-pendent of WC-2, and although the absolute levels were lessthan a strain with a functional copy of wc-2, there was increasedexpression coinciding with qrf induction. This finding indicatesthat qrf expression can promote frq expression independent ofa functional transcriptional activator.

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Fig. 4. qrf affects frq message and FRQ protein levels. (A) Total RNA orig-inating from the frq locus was measured by RT-PCR assay in WT (FGSC2489)compared with qa-2-qrf (XB141-12) grown in the presence of QA. cDNA wasgenerated with a random hexamer, so the graph represents both frq andqrf. CT, circadian time. (B) Immunoblot analysis of FRQ in WT (FGSC2489) andqa-2-qrf (XB141-12) with and without QA as indicated.

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Induction of qrf Creates a More Accessible Chromatin Environment.To ascertain if qrf induction created a more transcriptionallypermissive chromatin state, we measured nucleosome densitywithin frq via ChIP using an antibody that recognizes histone H3.We grew cultures in the dark and then subjected them to a30-min light pulse, and we found that in WT, the level of histoneoccupancy decreased at the pLRE and the transcriptional startsite in response to light, which is consistent with more accessiblelight-activated chromatin (Fig. 6 A and B). We then examinedthe qa-2-qrf strain and found that the nucleosome density wasless than WT (Fig. 6 A and B). Collectively, these data point toa dual mode of action where expression of qrf can create a morepermissible chromatin environment but is also needed for si-lencing of the sense transcript via heterochromatin formation.

Δvvd as a Model for NAT-Mediated Expression and Silencing. Thenotion that NAT expression can first promote sense gene ex-pression by creating a more accessible local chromatin environmentand then repress via heterochromatin was entirely unexpected.To examine if bimodal function of NAT expression was a generalmechanism, we used the Δvvd strain made by the NeurosporaKnockout Consortium (38, 39). In Δvvd, the hygromycin (hph)resistance gene is in the opposite orientation to the normal vvdgene, but the endogenous light-inducible vvd promoter is largelyintact and can drive expression of an hph antisense transcript(hphAS). As a result of this design, Δvvd cannot grow on mediumcontaining hygromycin in the light but grows unencumbered inthe dark (Fig. 7A). The hph and hphAS transcripts in Δvvd re-semble frq and qrf in the frq locus (Fig. 7B). To test if light-induced hphAS can elevate hph expression, we measured bothhphAS (Fig. 7C) and hph (Fig. 7D) by strand-specific RT-PCRassay, sampling at 0, 5, 10, 15, 30, and 60 min after transferring tolight-inducing conditions. We observed a steady increase in light-induced hphAS driven off the endogenous vvd promoter thatpeaked at 30 min after transferring cultures to the light (Fig. 7C).We also noted that light-induced hphAS expression caused anincrease in the hph gene expression (hph expression is normallyconstitutive and is driven off the TrpC promoter) (Fig. 7D). Thisresult was not entirely consistent with a silenced hph gene, so weexamined hph and hphAS expression up to 4 h in the light. Atlater time points, there was a slow decline in hphAS and hph

transcript levels consistent with TGS (Fig. 7 E and F). To verifythat TGS was occurring via heterochromatin formation, wetested whether Δvvd contained m5C via methylation-sensitiveSouthern blot analysis. There was a significant amount of DNAmethylation at the Δvvd locus, indicating heterochromaticgene silencing of hph (Fig. 7G). These data bear striking re-semblance to what is observed at frq and indicated that inductionof a NAT is capable of first promoting expression of a sense tran-script, but is ultimately involved in heterochromatic silencing.

qrf-Mediated Heterochromatic Silencing Functions in Parallel withVVD. Normally, VVD is involved in down-regulating frq andinhibits WCC-mediated expression by directly interacting withWCC (40–44). Therefore, we sought to examine if qrf-inducedheterochromatic silencing was dependent on VVD. To examinethis possibility, we constructed a strain that contained both Δvvdand qa-2-qrf (qa-2-qrf Δvvd) and compared frq and qrf expressionby strand-specific RT-PCR analysis. We compared the qa-2-qrfΔvvd double mutant with WT, qa-2-qrf, and Δvvd with andwithout QA under light-inducing conditions (Fig. 8) with thehypothesis that if VVD and heterochromatin were independent,we would see no adaption of frq and light-activated expression offrq would remain elevated. In qa-2-qrf exposed to light, frq ex-pression was elevated in medium containing 10 mM QA, butVVD-dependent adaptation still occurred and frq levels droppedbetween the 15-min and 30-min time points (Fig. 8A). There wasalso an increase in FRQ protein (Fig. S8 A and B). However, qrfexpression was less than WT, which once again demonstratedthat qrf expression in qa-2-qrf is less than the amount normallyobserved in WT under light-inducing conditions (Fig. 8B). Inaddition, frq expression was elevated in qa-2-qrf relative to Δvvdafter 240 min in the light, indicting that high levels of qrf ex-pression are needed for silencing of frq independent of VVD.Moreover, qrf was elevated in Δvvd relative to WT, suggestingthat (i) VVD also inhibits qrf expression and (ii) residual adaptationoften observed in Δvvd is due to qrf-mediated heterochromatin.In the qa-2-qrf Δvvd double mutant, there was no photo-

adaptation and frq levels remained relatively constant at all timestested out to 240 min in the light. Moreover, there appeared tobe a small gradual increase. Interestingly, the levels of frq in thedouble mutant resembled the qa-2-qrf at the 240-min time pointwhich did not entirely coincide with an additive effect. However,WC-1 is rapidly degraded after light activation (45), and we wereunable to detect WC-1 protein in the qa-2-qrf Δvvd double mu-tant at the 240-min time point. These data indicate that WC-1protein is consumed faster than it can be replenished and there isno WC-1 available to drive higher levels of expression at latertime points (Fig. S8 C and D). Consistent with the notion ofa parallel adaptation pathway between VVD and qrf-mediatedheterochromatin, there was an increase in frq levels in the doublemutant in 2% (wt/vol) glucose (Fig. 8C). Collectively, these

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Fig. 5. Induction of qrf promotes frq expression. (A) Strand-specific RT-PCRassay was used to measure absolute levels of qrf in WT (FGSC2489) and qa-2-qrf(XB141-12) in response to a 10 mM QA pulse in constant darkness. (B) Sameas in A, except the absolute levels of frq were measured from the samesamples. (C) Strand-specific RT-PCR assay measuring absolute levels of qrf inΔwc-2 (FGSC11124) and Δwc-2, qa-2-qrf (XB229-1) in response to a QA pulse.(D) Same as in C, except the absolute levels of frq were examined. The dataare averages of four independent biological replicates, each with twotechnical replicates. The error bars represent the SEM.

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results indicate that qrf functions in a pathway alongside VVDand is involved in silencing frq expression in response to light.

DiscussionIn this report, we examined the role of the frq NAT qrf in cir-cadian and light-regulated expression. The results were far moreinteresting than originally anticipated and appear to documenta conserved process where NATs first promote the expression ofa sense transcript, by creating a more transcriptionally permissivestate, and then repress expression via facultative heterochro-matin formation. The inherent notion of this model may seema bit contradictory, but is perhaps best supported by analysis ofhph silencing in Δvvd grown in the light (Fig. 7). In this strain, thehph resistance gene, driven off the constitutive TrpC promoter,was put in opposite orientation to the endogenous vvd gene inthe KO. Because only the coding sequence was replaced, thenormal vvd light-inducible promoter was left largely intact andthere is light-induced expression of an hph antisense transcript.This hphAS is capable of establishing heterochromatin (asevidence by m5C in Δvvd) that prevents growth on medium

containing hygromycin when grown in the light. When Δvvd isgrown in the dark and then transferred to light, induction of thehph NAT causes a sixfold induction of the TrpC-driven hphpeaking at ∼30 min after transfer to light (Fig. 7C). The in-duction is short lived, and at later times, the levels of both thelight-driven hphAS and trpC-driven hph decrease.Although, Δvvd represents an artificial system, the same basic

genetic structure of a sense/antisense pair exists at frq and thedata support a conserved mechanism. First, we recently dem-onstrated facultative heterochromatin at frq, and a high level ofqrf expression is needed for efficient heterochromatin formation.Replacing the endogenous aLRE with the qa-2 promoter causeda defect in m5C, H3K9me3, and HP1 binding (Figs. 1 and 2)largely because qrf expression off the qa-2 promoter was lowerthan the light-induced WT promoter. Second, induction of qrf inthe qa-2-qrf can elevate frq expression, and the increase in frqexpression occurs even in the absence of WC-2 (Fig. 5). How-ever, replacing the aLRE with the qa-2 promoter did complicatematters slightly because it is incapable of driving the high level ofqrf expression normally observed in WT, and the high level ofexpression is needed for heterochromatin formation. The in-ability to induce high levels of expression creates a twofold effectfor the qa-2-qrf strain when QA is added to the medium; there isa lack of heterochromatic silencing, and there is NAT-assistedpromotion of frq expression. It is the combination of these twofactors (lack of heterochromatic silencing and elevated sensegene expression) that causes the clock to become arrhythmiccompared with just removing the antisense promoter or with theqa-2-qrf strain grown without QA. In these cases, both strainsonly display a phase shift.The model reported here is in contrast to a recent report that

suggests qrf inhibits frq by transcriptional interference. In otherwords, colliding RNA polymerases (PolII) from the sense/anti-sense pair generate premature termination and abortive tran-scripts because they run into each other (46). This model,although attractive, seems to contradict normal light-induced frqand qrf expression and does not account for what we observe inΔvvd. Both frq and qrf are expressed at high levels in response tolight, and if the PolII enzymes are interfering with each other,one might expect no expression of mature transcripts in light-pulsed samples. The simultaneous strong induction of frq and qrfin response to light makes transcriptional interference difficult toreconcile. Moreover, Dang et al. (23) have ruled out facultative

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Fig. 7. Induction of an hphAS promotes and then silences hph. (A) Growthassay comparing WT (FGSC2489) and Δvvd (FGSC11556) in the light and darkon media with and without hygromycin (hyg). Note there is little to nogrowth of Δvvd on hyg-containing media in the light, but growth is normalif cultures are grown in the dark. (B) Schematic representation of the Δvvdstrain. (C) Strand-specific RT-PCR assay in Δvvd examining the absolute levelof the hphAS after transfer to light sampling at the indicated times.(D) Same as in C, except we examined the hph sense transcript driven off thenormally constitutive TrpC promoter. (E and F) Same as in C and D, exceptthe times were carried out to 240 min in the light. The data are averages offour independent biological replicates, each with two technical replicates.The error bars represent the SEM. (G) DNA methylation Southern blotcomparing WT and Δvvd from DNA isolated under light-inducing conditions.

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heterochromatin as a mechanism, even though some of theirprior work indicates DNA methylation at convergent tran-scripts. However, there are a number of similarities betweentheir data and ours, so it remains possible that neither model ismutually exclusive.The qrf-mediated silencing reported herein appears to be

separate and parallel to VVD-mediated adaptation; however,at the same time, VVD-mediated adaptation and qrf-dependentheterochromatin are interconnected because VVD inhibits WCCactivity at aLRE, driving qrf expression. Thus, there are twomechanisms that mediate light adaptation to attenuate the highlevel of light-induced frq. One is controlled by a direct interactionbetween VVD and the WCC to down-regulate frq expression (42–44). A second silencing/adaptation process consists of disiRNA-mediated facultative heterochromatin that requires H3K9me3, HP1binding, and (to a lesser extent) m5C. The molecular mechanism ofdisiRNA-mediated recruitment of heterochromatin enzymes is stillunknown and will likely be the subject of subsequent studies. Ofnote to many in the mammalian clock community is the notionthat the Period 2 locus contains a NAT (16, 47). We have sinceconfirmed that all three Per genes have similar NATs (Fig. S9),

suggesting an evolutionarily conserved process of tuning of clockgene expression via heterochromatin. It is interesting to speculatethat these Per antisense transcripts are involved in recruiting distinctPER complexes that have the ability to establish facultativeheterochromatin. Further support for antisense-guided facultativeheterochromatin in clock negative feedback comes from a recentreport indicating that the mammalian KMT1, Suppressor of var-iegation 39 (Suv39H1), is a component of the PER complex (48).Future experiments will provide further insights into these ideas.

Experimental ProceduresA detailed description of the strains used in this report (Table S1), theirconstruction, and media used for growth assays can be found in SI Experi-mental Procedures. In addition, the protocols used for the DNA methylation-sensitive Southern blots, RT-PCR assay, ChIP assay (including antibodies),Western blots, and disiRNA extraction and disiRNA Northern blots canlikewise be found in SI Experimental Procedures.

ACKNOWLEDGMENTS. We thank Dr. Eric Selker for providing the HP1-GFPused in the ChIP experiments. We also thank Dr. Wendie Cohick for comments.This work was supported by NIH Grant R01GM101378 (to W.J.B.).

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