The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin

Edited by Steven Henikoff, Fred Hutchinson Cancer Research Center, Seattle, WA, and approved January 21, 2015 (received for review April 2, 2014)
March 23, 2015
112 (14) 4357-4362


The circadian clock is predominantly regulated by transcriptional negative feedback, where the protein(s) arising from the central clock gene(s) inhibit their own expression. In Neurospora and mammals, the clock genes have natural antisense transcripts (NATs): long noncoding RNAs that overlap and are expressed in opposite orientation to the protein coding genes. Previously, we demonstrated that Neurospora frequency NAT is needed for normal DNA methylation. This report demonstrates that proper regulation of the frequency antisense transcript is needed to establish repressive heterochromatin. However, prior to establishing heterochromatin, NAT expression creates a transcriptionally permissive state that helps promote sense transcript expression. Broader implications indicate that NATs, in general, may first promote sense gene expression prior to establishing heterochromatin.


The circadian clock is controlled by a network of interconnected feedback loops that require histone modifications and chromatin remodeling. Long noncoding natural antisense transcripts (NATs) originate from Period in mammals and frequency (frq) in Neurospora. To understand the role of NATs in the clock, we put the frq antisense transcript qrf (frq spelled backwards) under the control of an inducible promoter. Replacing the endogenous qrf promoter altered heterochromatin formation and DNA methylation at frq. In addition, constitutive, low-level induction of qrf caused a dramatic effect on the endogenous rhythm and elevated circadian output. Surprisingly, even though qrf is needed for heterochromatic silencing, induction of qrf initially promoted frq gene expression by creating a more permissible local chromatin environment. The observation that antisense expression can initially promote sense gene expression before silencing via heterochromatin formation at convergent loci is also found when a NAT to hygromycin resistance gene is driven off the endogenous vivid (vvd) promoter in the Δvvd strain. Facultative heterochromatin silencing at frq functions in a parallel pathway to previously characterized VVD-dependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNA-mediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.
In eukaryotes, metazoans, and vertebrates, the circadian rhythm requires timed chromatin remodeling and modifications to ensure the appropriate amplitude, period, and phase of clock gene expression. The need for chromatin regulation arises because the clock is predominantly controlled by a transcriptional negative feedback loop where transcriptional activators drive expression of negative elements that inhibit its own expression (13). In Neurospora crassa, the positive elements are the GATA type transcription factors White Collar-1 (WC-1) and WC-2 that form the White Collar complex (WCC) (4). The WCC drives expression of frequency (frq) that is translated with delays before it associates with FRQ-interacting RNA helicase (FRH) (5, 6). FRQ-FRH inhibits frq expression through a direct interaction with WCC that is mediated by WC-2 (7, 8). FRQ undergoes phase-specific phosphorylation over the course of the day, and this phosphorylation controls regulated transport between the nucleus and cytoplasm, destabilization, and turnover (912). In addition, there appears to be fine-tuned control of chromatin in both the activation and feedback inhibition phases of circadian oscillations (1317).
Recently, we reported that cytosines in the frq promoter are methylated (m5C) and proper regulation of the frq locus is needed for normal DNA methylation (13). The frq locus is composed of three transcripts: the frq gene encoding FRQ protein (6), a natural antisense transcript (NAT) qrf (frq spelled backwards) (18), and a small upstream transcript of unknown function 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 proximal light-regulated element (pLRE) is the predominant cis-acting sequence that controls light-activated expression of frq (19, 20). The antisense light response element (aLRE) is required for light-induced qrf expression (18, 21), whereas the c-box is the major element controlling circadian frq expression (22). We have previously demonstrated that the frq antisense transcript qrf and WCC are needed for normal m5C (13). It was later shown that convergent transcription at frq gives rise to dicer-independent siRNA (disiRNA) and that DNA methylation occurs at disiRNA loci throughout the genome. This work also confirmed the requirement for WCC-mediated expression for m5C at frq (23, 24).
Fig. 1.
Induction of qrf affects DNA methylation at frq. (A) Comparative analysis of disiRNA-sequencing and WC-2 ChIP-sequencing found at the frq loci. (B) Schematic representation of the WT frq locus in Neurospora and the transgenic qa-2-qrf strain used in this study. The qa-2-qrf has the qa-2 promoter in place of the aLRE. (C) Methylation-sensitive Southern blot examining the frq promoter in WT (FGSC2489) and qa-2-qrf (XB141-12) with and without QA. Genomic DNA was digested with the methylation-sensitive restriction enzyme BfuCI (labeled B) or the methyl-insensitive isoschizomer DpnII (labeled D) and probed for a region specific to the pLRE. (D) Same as in C, 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, time in darkness; LL, constant light.
All DNA methylation in Neurospora repeat regions requires the histone H3 Lys 9 (H3K9) methyltransferase (KMT1) DIM-5, heterochromatin protein 1 (HP1), and DNA methyltransferase DIM-2 (2528). The molecular mechanism of DNA methylation proceeds in a stepwise fashion mediated by DCDC (DIM-5/DIM-7/DIM-9–CUL4/DDB1 complex) (25, 28, 29). Both DIM-5 and HP1 are also required for methylation at frq, suggesting that other DCDC components may also be required (30). However, the dependency of qrf expression and the existence of disiRNAs originating from frq suggest a more complicated mechanism because RNAi is not needed for DNA methylation at relics of repeat-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 normal facultative heterochromatin formation with an unanticipated twist. Before heterochromatic silencing, induction of qrf first promotes the expression of frq by generating a more transcriptionally permissive state. High levels of qrf expression that normally occur in response to light ultimately silence frq via heterochromatin formation in a process independent of, but parallel to, VIVD (VVD)-mediated repression. The bimodal activity of NAT expression that first helps promote sense gene expression prior to silencing via heterochromatin also occurs at an artificial sense/antisense reporter system indicating that the dual function may be a general feature of NAT expression.


DNA Methylation at frq Requires Proper Regulation of the qrf.

m5C in the frq promoter requires DIM-2, a functional WCC, and normal qrf expression (13). Promoter DNA methylation also occurs at other convergent transcripts, and these loci all produce disiRNA (24, 32). As a primer for the work presented here, RNA-sequencing data from QDE-2 (Argonaute)–associated RNAs and WC-2 ChIP-sequencing were analyzed at frq and are shown in Fig. 1A (21, 24). To explore the role of the qrf and disiRNAs in directing m5C at frq, we replaced the aLRE with the inducible qa-2 promoter (qa-2-qrf) to control expression by adding quinic acid (QA) to the medium (33) (Fig. 1B). We assayed m5C at frq by performing a methylation-sensitive Southern blot in WT and the qa-2-qrf strain with and without QA under circadian conditions sampling every 4 h (Fig. 1C). Surprisingly, we were unable to detect WT levels of m5C in qa-2-qrf even at high concentrations of QA (10 mM). This unexpected result arose because qrf induction with QA was lower than in WT under light-inducing conditions (Fig. S1 A and B). However, QA induction of qrf was elevated relative to a strain lacking the aLRE that is needed for qrf expression. This strain, called frq10frqccg-2, has the 3′ UTR of frq replaced with the 3′ UTR of clock controlled gene-2 (ccg-2), thus removing the aLRE (18) (below and Fig. S1C). The frq10frqccg-2 strain dramatically reduces the level of qrf expression to the point where it cannot be detected by a Northern blot (18), but qrf can still be detected via strand-specific RT-PCR analysis (Fig. S1C), indicating that it does 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 every condition tested, we observed a reduction in m5C at frq (Fig. 1C and Fig. S2). We then analyzed if the qa-2-qrf strain could suppress the hypermethylation phenotype seen in Δchd1 but found no major change in the hypermethylation phenotype in the double mutant (qa-2-qrf Δchd1), suggesting that CHD1 functions subsequent to qrf (Fig. 1D).

qrf Is Needed for Heterochromatin Formation.

m5C at frq requires both histone H3K9 methylation (H3K9me) and HP1 (30). DIM-5 adds a monomethyl group, dimethyl group, and trimethyl group to H3K9 and is needed for all known DNA methylation in Neurospora (28, 34). The defect in DNA methylation in qa-2-qrf led us to test if qrf is needed for efficient heterochromatin formation. We performed ChIP with an H3K9me3 antibody comparing WT with qa-2-qrf (without QA). Normally, H3K9me3 at frq peaks 30 min after exposure to light-inducing conditions, and we found significantly lower levels of H3K9me3 in qa-2-qrf relative to WT at the pLRE and at the c-box (Fig. 2 A and B). We could still detect some H3K9me3 in qa-2-qrf; however, the qa-2 promoter is leaky, and there is spurious transcription even in the absence of QA.
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 by quantitative PCR assay of DNA isolated with an H3K9me3-specific antibody using oligonucleotides specific to the pLRE. The Δdim-5 and a nonspecific IgG were used as negative controls. (B) Same as in A, except the oligos used detected a region near the c-box. (C) HP1 binding to regions in frq promoter near the pLRE was measured by ChIP using a GFP-specific antibody in strains containing the HP1-GFP fusion protein (XB270-1 and XB265-1). (D) Same as in 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 and then transferred to saturating light and cross-linked at the indicated times before processing.
We next tested if other heterochromatin marks besides m5C and H3K9me3 were altered in qa-2-qrf. We assayed binding of HP1 to the frq promoter via ChIP using an HP1-GFP fusion construct. We detected a lower level of HP1 binding in qa-qrf relative to WT (Fig. 2 C and D). The combination of reduced m5C, H3K9me3, and HP1 binding indicates that facultative heterochromatin formation at frq is dependent on qrf. Moreover, the defect in qrf-dependent heterochromatin is consistent with results showing that long noncoding RNAs are processed into siRNA via the exosome and can induce heterochromatin formation in Schizosaccharomyces pombe (35).

Altered qrf Expression Affects the Circadian Clock.

We next sought to examine how induction of qrf affects clock regulation. The qa-2-qrf strain was crossed to a strain with the frq promoter fused to a codon-optimized luciferase (36) and to a strain harboring the ras-1bd allele to examine circadian output (37). Induction of qrf with 10 mM QA abolished the normal circadian rhythm (Fig. 3A and Fig. S3). In the absence of QA, there was an expected low-amplitude, phase-shifted rhythm. The altered phase is consistent with frq10frqccg-2, which has a small but detectable phase shift relative to WT (18). These data are further support for the idea that facultative heterochromatin is needed to establish the appropriate phase (13, 18). Moreover, replacing the aLRE with the qa-2 promoter appeared to interfere with the normal rhythmic conidia formation (Fig. 3B).
Fig. 3.
Induction of qrf affects the circadian rhythm. (A) frq promoter luciferase traces were obtained for WT (9014-VG3) and qa-2-qrf (XB106-9) with and without 10 mM QA. The relative light units (RLUs) were obtained by analyzing luciferase expression over the entire race tube. Similar results were obtained by performing a sectional analysis (Fig. S3). (B) Standard race tube analysis comparing ras-1bd (XB136-6) and qa-2-qrf (XB190-2) with and without QA.
We next wanted to explore how altering the level of constitutive qrf expression affected the circadian rhythm. We grew the qa-2-qrf strain over a range of QA concentrations and found a low-amplitude rhythm in every condition tested except 10 mM QA (Fig. S4). We then tested whether transient or constitutive induction of qrf was causing the arrhythmic phenotype. We grew the qa-2-qrf strain in normal media and then transiently induced qrf with 10 mM QA for 15 min before transferring the cultures to media 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 was misregulated relative to WT (Fig. S7).

qrf Affects Molecular Oscillations.

To determine why constitutive expression of qrf was causing arrhythmic frq expression, we examined the relative levels of RNA expression originating from the frq locus over circadian time by quantitative RT-PCR analysis using a random hexamer capable of detecting both frq and qrf abundance. We found that the overall transcript levels were elevated in the induced qa-2-qrf strain compared with WT (Fig. 4A). There was also a peak in expression that was delayed 4 h relative to WT. The seemingly obvious explanation is that the increase in expression was due to qrf induction, but data shown below indicate that it is due to an increase in frq expression. To confirm this defect in expression, we examined the rhythm in FRQ protein by immunoblot analysis. We found that induction of qrf had a dramatic effect on the normal protein rhythm typically observed in WT, and we detected a relatively high level of FRQ at every time point examined in qa-2 qrf (Fig. 4B). These results are consistent with the luciferase data and support the observed defect in circadian clock function.
Fig. 4.
qrf affects frq message and FRQ protein levels. (A) Total RNA originating 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 was generated with a random hexamer, so the graph represents both frq and qrf. CT, circadian time. (B) Immunoblot analysis of FRQ in WT (FGSC2489) and qa-2-qrf (XB141-12) with and without QA as indicated.

Expression of qrf Initially Promotes frq Expression.

The elevated level of transcription originating from frq was surprising because our expectation was that qrf induction would cause disiRNA-mediated heterochromatin. Therefore, we tested the individual levels of frq and qrf by strand-specific RT-PCR assay. WT and qa-2-qrf were grown in the dark for 24 h to remove any residual light effects on frq expression, and the growing mycelia were then transferred to fresh medium containing 10 mM QA. The 10 mM QA pulse resulted in roughly fivefold induction of qrf that peaked 15 min after the cultures were transferred to QA medium (Fig. 5A). Surprisingly, induction of qrf had an unanticipated effect on frq expression; instead of disiRNA-mediated transcriptional gene silencing (TGS), there was a dramatic and steady increase in frq expression (Fig. 5B). Therefore, we surmised that induction of qrf might generate a more transcriptionally permissive state that helps promote expression of frq. To explore this idea further, we tested if qrf induction could elevate frq expression in the absence of a functional WCC. We generated a strain that contained both qa-2-qrf and Δwc-2 and tested if induction of qrf elevated frq expression (Fig. 5 C and D). The qa-2-qrf Δwc-2 strain had an increase in frq expression independent of WC-2, and although the absolute levels were less than a strain with a functional copy of wc-2, there was increased expression coinciding with qrf induction. This finding indicates that qrf expression can promote frq expression independent of a functional transcriptional activator.
Fig. 5.
Induction of qrf promotes frq expression. (A) Strand-specific RT-PCR assay 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) Same as in A, except the absolute levels of frq were measured from the same samples. (C) Strand-specific RT-PCR assay measuring absolute levels of qrf in Δwc-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 data are averages of four independent biological replicates, each with two technical replicates. The error bars represent the SEM.

Induction of qrf Creates a More Accessible Chromatin Environment.

To ascertain if qrf induction created a more transcriptionally permissive chromatin state, we measured nucleosome density within frq via ChIP using an antibody that recognizes histone H3. We grew cultures in the dark and then subjected them to a 30-min light pulse, and we found that in WT, the level of histone occupancy decreased at the pLRE and the transcriptional start site in response to light, which is consistent with more accessible light-activated chromatin (Fig. 6 A and B). We then examined the qa-2-qrf strain and found that the nucleosome density was less than WT (Fig. 6 A and B). Collectively, these data point to a dual mode of action where expression of qrf can create a more permissible chromatin environment but is also needed for silencing of the sense transcript via heterochromatin formation.
Fig. 6.
Induction of qrf creates a more permissive chromatin. (A) We analyzed the extent of chromatin compaction by measuring nucleosome density via ChIP with histone H3 antibody. WT and qa-2-qrf were grown in media containing QA in the dark (0) and transferred to light for 30 min (30). The amount of H3 at the pLRE was measured by quantitative PCR assay. (B) Same as in A, except the amount of H3 present at the TSS was determined.

Δvvd as a Model for NAT-Mediated Expression and Silencing.

The notion that NAT expression can first promote sense gene expression by creating a more accessible local chromatin environment and then repress via heterochromatin was entirely unexpected. To examine if bimodal function of NAT expression was a general mechanism, we used the Δvvd strain made by the Neurospora Knockout Consortium (38, 39). In Δvvd, the hygromycin (hph) resistance gene is in the opposite orientation to the normal vvd gene, but the endogenous light-inducible vvd promoter is largely intact and can drive expression of an hph antisense transcript (hphAS). As a result of this design, Δvvd cannot grow on medium containing hygromycin in the light but grows unencumbered in the dark (Fig. 7A). The hph and hphAS transcripts in Δvvd resemble frq and qrf in the frq locus (Fig. 7B). To test if light-induced hphAS can elevate hph expression, we measured both hphAS (Fig. 7C) and hph (Fig. 7D) by strand-specific RT-PCR assay, sampling at 0, 5, 10, 15, 30, and 60 min after transferring to light-inducing conditions. We observed a steady increase in light-induced hphAS driven off the endogenous vvd promoter that peaked at 30 min after transferring cultures to the light (Fig. 7C). We also noted that light-induced hphAS expression caused an increase in the hph gene expression (hph expression is normally constitutive and is driven off the TrpC promoter) (Fig. 7D). This result was not entirely consistent with a silenced hph gene, so we examined hph and hphAS expression up to 4 h in the light. At later time points, there was a slow decline in hphAS and hph transcript levels consistent with TGS (Fig. 7 E and F). To verify that TGS was occurring via heterochromatin formation, we tested whether Δvvd contained m5C via methylation-sensitive Southern blot analysis. There was a significant amount of DNA methylation at the Δvvd locus, indicating heterochromatic gene silencing of hph (Fig. 7G). These data bear striking resemblance to what is observed at frq and indicated that induction of a NAT is capable of first promoting expression of a sense transcript, but is ultimately involved in heterochromatic silencing.
Fig. 7.
Induction of an hphAS promotes and then silences hph. (A) Growth assay comparing WT (FGSC2489) and Δvvd (FGSC11556) in the light and dark on media with and without hygromycin (hyg). Note there is little to no growth of Δvvd on hyg-containing media in the light, but growth is normal if cultures are grown in the dark. (B) Schematic representation of the Δvvd strain. (C) Strand-specific RT-PCR assay in Δvvd examining the absolute level of 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 the normally constitutive TrpC promoter. (E and F) Same as in C and D, except the times were carried out to 240 min in the light. The data are averages of four independent biological replicates, each with two technical replicates. The error bars represent the SEM. (G) DNA methylation Southern blot comparing WT and Δvvd from DNA isolated under light-inducing conditions.

qrf-Mediated Heterochromatic Silencing Functions in Parallel with VVD.

Normally, VVD is involved in down-regulating frq and inhibits WCC-mediated expression by directly interacting with WCC (4044). Therefore, we sought to examine if qrf-induced heterochromatic silencing was dependent on VVD. To examine this possibility, we constructed a strain that contained both Δvvd and qa-2-qrf (qa-2-qrf Δvvd) and compared frq and qrf expression by strand-specific RT-PCR analysis. We compared the qa-2-qrf Δvvd double mutant with WT, qa-2-qrf, and Δvvd with and without QA under light-inducing conditions (Fig. 8) with the hypothesis that if VVD and heterochromatin were independent, we would see no adaption of frq and light-activated expression of frq would remain elevated. In qa-2-qrf exposed to light, frq expression was elevated in medium containing 10 mM QA, but VVD-dependent adaptation still occurred and frq levels dropped between the 15-min and 30-min time points (Fig. 8A). There was also an increase in FRQ protein (Fig. S8 A and B). However, qrf expression was less than WT, which once again demonstrated that qrf expression in qa-2-qrf is less than the amount normally observed in WT under light-inducing conditions (Fig. 8B). In addition, frq expression was elevated in qa-2-qrf relative to Δvvd after 240 min in the light, indicting that high levels of qrf expression are needed for silencing of frq independent of VVD. Moreover, qrf was elevated in Δvvd relative to WT, suggesting that (i) VVD also inhibits qrf expression and (ii) residual adaptation often observed in Δvvd is due to qrf-mediated heterochromatin.
Fig. 8.
VVD and qrf function in separate pathways to attenuate light-mediated frq expression. Message levels of frq (A) and qrf (B) were examined by strand-specific RT-PCR assay in WT (FGSC2489); qa-2-qrf (XB141-12); Δvvd (FGSC11156); and qa-2-qrf, Δvvd (XB222-7) under light-inducing conditions in media containing 0.1% glucose and 10 mM QA. frq (C) and qrf (D) were measured in the same strains under light-inducing conditions in 2.0% glucose without QA.
In the qa-2-qrf Δvvd double mutant, there was no photoadaptation and frq levels remained relatively constant at all times tested out to 240 min in the light. Moreover, there appeared to be a small gradual increase. Interestingly, the levels of frq in the double mutant resembled the qa-2-qrf at the 240-min time point which did not entirely coincide with an additive effect. However, WC-1 is rapidly degraded after light activation (45), and we were unable to detect WC-1 protein in the qa-2-qrf Δvvd double mutant at the 240-min time point. These data indicate that WC-1 protein is consumed faster than it can be replenished and there is no WC-1 available to drive higher levels of expression at later time points (Fig. S8 C and D). Consistent with the notion of a parallel adaptation pathway between VVD and qrf-mediated heterochromatin, there was an increase in frq levels in the double mutant in 2% (wt/vol) glucose (Fig. 8C). Collectively, these results indicate that qrf functions in a pathway alongside VVD and is involved in silencing frq expression in response to light.


In this report, we examined the role of the frq NAT qrf in circadian and light-regulated expression. The results were far more interesting than originally anticipated and appear to document a conserved process where NATs first promote the expression of a sense transcript, by creating a more transcriptionally permissive state, and then repress expression via facultative heterochromatin formation. The inherent notion of this model may seem a bit contradictory, but is perhaps best supported by analysis of hph silencing in Δvvd grown in the light (Fig. 7). In this strain, the hph resistance gene, driven off the constitutive TrpC promoter, was put in opposite orientation to the endogenous vvd gene in the KO. Because only the coding sequence was replaced, the normal vvd light-inducible promoter was left largely intact and there is light-induced expression of an hph antisense transcript. This hphAS is capable of establishing heterochromatin (as evidence by m5C in Δvvd) that prevents growth on medium containing hygromycin when grown in the light. When Δvvd is grown in the dark and then transferred to light, induction of the hph NAT causes a sixfold induction of the TrpC-driven hph peaking at ∼30 min after transfer to light (Fig. 7C). The induction is short lived, and at later times, the levels of both the light-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 the data support a conserved mechanism. First, we recently demonstrated facultative heterochromatin at frq, and a high level of qrf expression is needed for efficient heterochromatin formation. Replacing the endogenous aLRE with the qa-2 promoter caused a defect in m5C, H3K9me3, and HP1 binding (Figs. 1 and 2) largely because qrf expression off the qa-2 promoter was lower than the light-induced WT promoter. Second, induction of qrf in the qa-2-qrf can elevate frq expression, and the increase in frq expression occurs even in the absence of WC-2 (Fig. 5). However, replacing the aLRE with the qa-2 promoter did complicate matters slightly because it is incapable of driving the high level of qrf expression normally observed in WT, and the high level of expression is needed for heterochromatin formation. The inability to induce high levels of expression creates a twofold effect for the qa-2-qrf strain when QA is added to the medium; there is a lack of heterochromatic silencing, and there is NAT-assisted promotion of frq expression. It is the combination of these two factors (lack of heterochromatic silencing and elevated sense gene expression) that causes the clock to become arrhythmic compared with just removing the antisense promoter or with the qa-2-qrf strain grown without QA. In these cases, both strains only display a phase shift.
The model reported here is in contrast to a recent report that suggests qrf inhibits frq by transcriptional interference. In other words, colliding RNA polymerases (PolII) from the sense/antisense pair generate premature termination and abortive transcripts because they run into each other (46). This model, although attractive, seems to contradict normal light-induced frq and qrf expression and does not account for what we observe in Δvvd. Both frq and qrf are expressed at high levels in response to light, 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 qrf in response to light makes transcriptional interference difficult to reconcile. Moreover, Dang et al. (23) have ruled out facultative heterochromatin as a mechanism, even though some of their prior work indicates DNA methylation at convergent transcripts. However, there are a number of similarities between their data and ours, so it remains possible that neither model is mutually 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-dependent heterochromatin are interconnected because VVD inhibits WCC activity at aLRE, driving qrf expression. Thus, there are two mechanisms that mediate light adaptation to attenuate the high level of light-induced frq. One is controlled by a direct interaction between VVD and the WCC to down-regulate frq expression (4244). A second silencing/adaptation process consists of disiRNA-mediated facultative heterochromatin that requires H3K9me3, HP1 binding, and (to a lesser extent) m5C. The molecular mechanism of disiRNA-mediated recruitment of heterochromatin enzymes is still unknown and will likely be the subject of subsequent studies. Of note to many in the mammalian clock community is the notion that the Period 2 locus contains a NAT (16, 47). We have since confirmed that all three Per genes have similar NATs (Fig. S9), suggesting an evolutionarily conserved process of tuning of clock gene expression via heterochromatin. It is interesting to speculate that these Per antisense transcripts are involved in recruiting distinct PER complexes that have the ability to establish facultative heterochromatin. Further support for antisense-guided facultative heterochromatin in clock negative feedback comes from a recent report indicating that the mammalian KMT1, Suppressor of variegation 39 (Suv39H1), is a component of the PER complex (48). Future experiments will provide further insights into these ideas.

Experimental Procedures

A detailed description of the strains used in this report (Table S1), their construction, and media used for growth assays can be found in SI Experimental 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 can likewise be found in SI Experimental Procedures.


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

Supporting Information

Supporting Information (PDF)
Supporting Information


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Information & Authors


Published in

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Proceedings of the National Academy of Sciences
Vol. 112 | No. 14
April 7, 2015
PubMed: 25831497


Submission history

Published online: March 23, 2015
Published in issue: April 7, 2015


  1. circadian rhythm
  2. natural antisense transcripts
  3. heterochromatin
  4. DNA methylation


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


This article is a PNAS Direct Submission.



Na Li
Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901
Tammy M. Joska
Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901
Catherine E. Ruesch
Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901
Samuel J. Coster
Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901
William J. Belden1 [email protected]
Department of Animal Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, NJ 08901


To whom correspondence should be addressed. Email: [email protected].
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; and N.L., T.M.J., and W.J.B. wrote the paper.

Competing Interests

The authors declare no conflict of interest.

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    The frequency natural antisense transcript first promotes, then represses, frequency gene expression via facultative heterochromatin
    Proceedings of the National Academy of Sciences
    • Vol. 112
    • No. 14
    • pp. 4183-E1813







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