Figure 6. SWI1 is required for rhythmic opening of the C box through removal of NucB.

(A) Relative positions of resident nucleosomes, NucB and NucA at the C box (see also reference 31). Arrows indicate primer sets used to detect these regions. (B) Nuclei were isolated by fractionation from samples harvested at indicated hours in the dark and digested with MNase. 20 nanograms of gel-purified mononucleosomal DNA were loaded in each lane. (C) The density of NucB was measured by real-time PCR using a primer set shown in (A, the left primer set) and gel purified mononucleosomal DNA as template (n = 3, error bars represent ± SEM, ***p<0.0005, **p<0.005). NucB occupancy of the C box oscillates between low (CT0 and 21) and high (CT 13-16) in WT as reported by Belden et al. (2007b), whereas NucB is always bound to the C box in Δswi1. NucA and 3.303 (an unregulated control region of genomic DNA distinct from frq) served as controls for equal DNA inputs [31]. (D) Difference in frq promoter chromatin were probed with a limited nuclease digestion assay as described in Experimental Procedures. Cultures were harvested from WT or from Δswi1 cultures at the times in darkness indicated.