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 VVDdependent silencing and is needed to establish the appropriate circadian phase. Thus, repression via dicer-independent siRNAmediated facultative heterochromatin is largely independent of, and occurs alongside, other feedback processes.circadian rhythm | natural antisense transcripts | heterochromatin | DNA methylation I n 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 (1-3). 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 (9-12). In addition, there appears to be fine-tuned control of chromatin in both the activation and feedback inhibition phases of circadian oscillations (13)(14)(15)(16)(17).Recently, we reported that cytosines in the frq promoter are methylated (m 5 C) 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 backwar...

The transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. Characterization of the chromodomain helicase DNA-binding enzyme CHD1 revealed DNA methylation in the promoter of the central clock gene frequency (frq) in Neurospora crassa. In this report, we show that the DNA methylation at frq is not only dependent on the DNA methyltransferase DIM-2 but also on the H3K9 methyltransferase DIM-5 and HP1. Histone H3 lysine 9 trimethylation (H3K9me3) occurs at frq and is most prominent 30 min after light-activated expression. Strains lacking dim-5 have an increase in light-induced transcription, and more White Collar-2 is found associated with the frq promoter. Consistent with the notion that DNA methylation assists in establishing the proper circadian phase, loss of H3K9 methylation results in a phase advance suggesting it delays the onset of frq expression. The dim-5 deletion strain displays an increase in circadian-regulated conidia formation on race tubes and there is a synthetic genetic interaction between dim-5 and ras-1bd. These results indicate DIM-5 has a regulatory role in muting circadian output. Overall, the data support a model where facultative heterochromatic at frq serves to establish the appropriate phase, mute the light response, and repress circadian output.

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