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 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.

Keywords: DNA methylation; circadian rhythm; heterochromatin; natural antisense transcripts.

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.

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.

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-1^bd (XB136-6) and qa-2-qrf (XB190-2) with and without QA.

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.

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.

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.

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.

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.