To examine the acetylation state of H3 and H4 at frq, we used antisera against diacetylated H3 (K9, K14) and tetra-acetylated H4 in ChIP experiments ( Figures 3A–3D). The C box and PLRE DNA immunoprecipitated in the Δwc-1 and Δwc-1/Δwc-2 deletion strains was usually comparable to what was seen with the VVD antibody, a cytoplasmic protein used as a nonspecific IgG control ( As further controls, we used two different WC-1 antisera in ChIP experiments with Δwc-1, Δwc2, and Δwc-1/Δwc-2 strains ( Figure S1). The formation and/or disassembly of WCC is regulated and appears to occur at the promoter elements, representing an unanticipated mechanism in circadian control of frq. The observation that some WC-1 is always associated with the frq promoter, whereas WC-2 binding is rhythmic, indicates the WCC does not exist solely as a unit. Not surprisingly, we often saw the greatest amount of WC-1 binding after a 15 min light pulse in WT and frq7. We observed only slight changes in WC-1 binding to the C box and PLRE over the circadian cycle, and these were generally less than 2-fold. Shown in Figure 2 is a WC-1 ChIP experiment using samples and conditions identical to those used for WC-2 ( Figure 1). Surprisingly, however, the WC-1 protein is bound continuously to both the C box and PLRE and is clearly detectable over the entire circadian cycle and in light-treated samples. Comparable results showing rhythmic WC-2 association with the C box have been reported elsewhere (Īll models of the circadian feedback loop predict that the WCC acts as a unit and is modified or regulated in the nucleoplasm to affect changes in frq expression, and therefore we expected the results of the WC-1 ChIP experiment to mirror those of WC-2. It is clear from Figures 1B–1E that WC-2 interacted with the C box in a circadian-dependent manner with peaks occurring at the same CT in both WT and frq7 (CT = 20–5) around subjective dawn.
As an added control, oligonucleotide pairs specific to a noncoding region of the Neurospora genome were added to the quantitative PCR reaction to measure nonspecific background. Events that are clock specific occur at comparable subjective circadian times (CTs) in WT (22.5 hr period) and frq7 (29 hr periods), but due to differences in the inherent period lengths, the comparable CT occurs after different numbers of hours in darkness.
ChIP assays were carried out on a strain that is considered wild-type (WT) for circadian-regulated expression (see Experimental Procedures) and the long period mutant frq7, grown both in a 48 hr time course in constant darkness and in response to a 15 min light pulse (LP15) by using nonspecific IgG as a control (data not shown and see Figure S1 in the Supplemental Data available with this article online). To gain a better understanding of the events associated with the expression of frq, we performed in vivo chromatin immunoprecipition (ChIP) experiments with WC-1- and WC-2-specific antibodies and the oligonucleotide pairs shown in Figure 1A. The data suggest that CSW-1 regulates accessibility of promoter DNA, thus generating the sharp transition from the transcriptionally active to the repressed state. One gene, designated clockswitch ( csw-1), is required for clock function its product localizes to the frq promoter, is required for proper frq expression, and has an impact on chromatin structure.
Nuclease accessibility experiments indicate chromatin rearrangement at the frq promoter therefore, 19 genes with homology to ATP-dependent chromatin-remodeling enzymes were deleted and the strains examined for clock phenotypes. Small oscillations in histone acetylation are detected over the circadian cycle with a marked reduction upon light-induced activation. The WC proteins are thought to form an obligate complex however, chromatin immunoprecipitation (ChIP) indicates that WC-2 binds to the frq promoter in a rhythmic fashion, whereas WC-1 is bound continuously. In the Neurospora circadian system, the transcription factors White Collar-1 (WC-1) and White Collar-2 (WC-2) activate expression of frq, whose gene product inhibits its own expression.
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