Figure 5

DNase accessibility assay defines an inducible nucleosome-depleted region on the Nos2 promoter, but DNase accessibility is not altered by LXR activation. Cultured microglia were subjected to in situ chromatin digestion by DNase, followed by quantitative PCR. The shift in C_T values between digested and undigested chromatin reflected nucleosome occupancy. DNase accessibility (% chromatin accessibility) was quantified using the delta delta C_T calculation, normalizing to the Rho promoter. Low % chromatin accessibility indicated high nucleosome occupancy and a high % chromatin accessibility indicated nucleosome depletion. A Schematic representation of genomic DNA regions amplified following DNase digestion relative to transcription start site (right-angle arrow). B The proximal 5′ region flanking the Nos2 gene (b) showed low accessibility in the unstimulated state and significantly higher DNase accessibility following LPS stimulation. The proximal 5′ region flanking the Tnf gene (b) showed constitutive high DNase accessibility. Data are mean + SD. N = 4 independent experiments. *< 0.05, Mann–Whitney. C DNase accessibility is not altered at the Nos2 promoter in the presence of LXR agonist GW3965 (5 μM, GW/LPS). Data for Tnf promoter are also shown. Data are mean + SD. N = 4 independent experiments.