(A) Silver stain gel of TAP-purified RSC2, Rpd3S and FACT complexes.
(B) The effect of Rpd3S on RSC-dependent nucleosome remodeling. 2 nM RSC was incubated with 0.3nM 32P-labeled mononucleosome and 0, 13, 39 or 78 nM Rpd3S. The remodeling products were fractionated by native PAGE. A phosphorimage of the gel is shown. See also for the effect of H3K36me3 on Rpd3S in binding and RSC-mediated nucleosome remodeling reactions.
(C) The effect of Rpd3S on RSC dependent nucleosome eviction. Left panel, 6 nM RSC was incubated with 0.3nM mononucleosome and 0, 13, 39 or 78 nM Rpd3S in the presence of 10 ng of pGEM3Z601R acceptor DNA. Bar graph on the right represents quantitation by ImageQuant TL (GE) of the 3 independent experiments. The relative amounts of free DNA generated by eviction were plotted as a bar graph normalized to that generated by 6 nM RSC alone, which was assigned a value of 100. The error bars show +/− standard deviation (SD). The P value is calculated using student’s t-test. See also for the effect of H3K36me3 on Rpd3S in RSC-mediated nucleosome eviction.
(D) Rpd3S-mediated chromatin assembly assay. Left, the reaction contained 18 nM FACT, or 6, 12, 18 nM of Rpd3S respectively, with recombinant Xenopus octamers and a 32P-labeled 601 DNA fragment. A phosphorimage of a native gel is shown. Graph on the right represents quantitation of the amounts of assembled nucleosomes by Rpd3S relative to no Rpd3S control (i.e., octamers alone). The free DNA and assembled nucleosome are indicated. The error bars show +/− standard deviation (SD). The P value is calculated using student’s t-test. For chaperone assays see also for the effect of H3K36me3 on Rpd3S, for the effect of Rpd3S mutants and for the effect of trichostatin.