Figure 1.
Concentration dependence of cytosolic extract-mediated chromatin assembly.
A) Varying amounts of yeast cytosolic extract were incubated with core histones and a relaxed, circular plasmid for 1 hour in the presence or absence of rHif1p (as indicated). Following removal of protein, DNA was isolated and resolved by agarose gel electrophoresis and visualized by staining with Sybr Gold. Migration of relaxed (R) and supercoiled (S) forms of the plasmid are indicated. B) Yeast cytoplasmic extract was assayed for chromatin assembly as above with either rHif1p or BSA, as indicated (left panel). The amounts of rHif1p and BSA used in the chromatin assembly assay were resolved by SDS-PAGE and stained with coomassie blue (right panel). For both the left and right panels, all of the lanes were run on the same gel but an irrelevant lane was removed from the figure.
Figure 2.
Fractionation of the chromatin assembly activity by anion exchange chromatography.
A) Cytosolic extract was applied to a DEAE Sepharose column in buffer containing 50 mM NaCl. Proteins that did not bind to the column were collected as the flow-through fraction (FT). Column was then step eluted with buffers containing 100 mM, 250 mM and 500 mM NaCl, as indicated. Aliquots of each fraction were assayed for chromatin assembly activity by incubation with core histones and a relaxed, circular plasmid for 1 hour in the presence or absence of rHif1p (as indicated). Following removal of protein, DNA was isolated and resolved by agarose gel electrophoresis and visualized by staining with Sybr Gold. Migration of relaxed (R) and supercoiled (S) forms of the plasmid are indicated.
Figure 3.
Further fractionation of the chromatin assembly activity.
A) The FT fraction from the DEAE sepharose column was applied to a carboxymethyl Sepharose column in buffer containing 50 mM NaCl. Proteins that did not bind to the column were collected s the flow-through fraction (FT). Column was then step eluted with buffers containing 100 mM, 250 mM, 500 mM NaCl and 1.0 M NaCl as indicated. Aliquots of each fraction were assayed for chromatin assembly activity by incubation with core histones and a relaxed, circular plasmid for 1 hour in the presence or absence of rHif1p (as indicated). Following removal of protein, DNA was isolated and resolved by agarose gel electrophoresis and visualized by staining with Sybr Gold. B) CM sepharose and heparin sepharose columns run in series. The fraction that flows through both columns was assayed for chromatin assembly activity as described above.
Figure 4.
The cytosolic chromatin assembly activity is RNA-dependent.
(A) Partially purified chromatin assembly activity (FT from DEAE, CM and heparin sepharose columns) was treated with RNase or boiled as indicated. Sample was then assayed for in vitro chromatin assembly activity. (B) Partially purified assembly activity was extracted with phenol/chloroform and nucleic acids isolated from the aqueous phase by ethanol precipitation. Recovered nucleic acids were assayed for chromatin assembly activity as described above.
Figure 5.
Purification of an RNA-dependent chromatin assembly activity.
Cytosolic extract was passed through DEAE, CM and heparin sepharose columns as described above. The partially purified fraction was boiled and clarified by centrifugation. The supernatant was resolved by gel filtration chromatography. Indicated fractions were phenol/chloroform extracted and nucleic acids recovered by ethanol precipitation. Isolated nucleic acids were assayed for chromatin assembly activity in the presence and absence of rHif1p (top). Isolated nucleic acids from the indicated fractions were resolved on a polyacrylamide/urea gel and visualized by Sybr Gold staining (bottom).
Figure 6.
RNA-based chromatin assembly factor localizes to a discreet size range.
The material that flowed through the DEAE, carboxymethyl and heparin columns was deproteinized and resolved on a polyacrylamide/urea gel. Segments were cut out of the gel and RNA was eluted (numbered lanes). Recovered RNA was either resolved by polyacrylamide:urea electrophoresis and visualized with Sybr Gold (top) or assayed for chromatin assembly activity with a relaxed circular plasmid, core histones and in the absence or presence of rHif1p, as indicated (bottom). Supercoiled and relaxed plasmid is indicated (S and R, respectively).
Figure 7.
The TPR4 domain of sNASP is important for its activity in RNA-mediated chromatin assembly factors.
Chromatin assembly assays were performed with a constant amount of yeast cytosolic extract as described in the legend to Figure 1 except that aliquots of the reactions were taken at 15′, 30′, 1 hr, 2 hr, 3 hr, 4 hr and 5 hr (left to right on each gel). The gel in the upper left hand corner was performed with extract alone. The gels in each row represent reactions performed with 3 concentrations of the indicated form of NASP (WT = wild type, TRP 4Δ = deletion of TPR4 and 12E/K is E to K mutations in the acidic domain of sNASP). The concentrations of NASP in each reaction are indicated by the coomassie blue stained bands on the left of each row. All of the proteins were run on the same gel.