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

Abstract

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...

Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing

“…As expected, the vvd KO locus was found to be associated with strong DNA methylation (Fig. 4F) as observed previously (Li et al 2015). In addition, the DNA methylation at the vvd KO locus is independent of QDE-2 (Supplemental Fig.…”

“…Second, we showed previously that antisense transcription induces DLDM from an artificial locus and the disiRNA-47 ( frq) locus (Belden et al 2011;Dang et al 2013;Li et al 2015). Here we showed that the mutation of ERI-1-binding sites on qrf RNA severely impaired DLDM at the frq locus (Fig.…”

“…Antisense transcription and ERI-1 binding of RNA are both necessary and sufficient to trigger DLDM Most disiRNA loci are known to produce antisense RNA, and previous studies have shown that convergent transcription leads to DLDM (Lee et al 2010;Belden et al 2011;Dang et al 2013;Li et al 2015). There are 477 genes in Neurospora that are known to produce antisense transcripts (Arthanari et al 2014); therefore, antisense transcription alone is not sufficient to trigger DLDM.…”

“…Transcription of both genes results in transcriptional interference in the form of Pol II stalling and H3K36me3. In addition, H3K9me3 and DNA methylation were also found in the promoter region of frq (Belden et al 2011;Dang et al 2013;Li et al 2015).…”

“…Unlike the previously characterized DNA methylation events in repeat-induced point mutation regions in Neurospora (Galagan and Selker 2004), the disiRNA locus DNA methylation (DLDM) is dependent on transcription and is induced by antisense transcription (Dang et al 2013). Interestingly, frq resides in one of the disiRNA loci (disiRNA-47), and H3K9me3 and DNA methylation in the promoter region of frq require qrf expression (Belden et al 2011;Dang et al 2013;Li et al 2015).…”