1. Reconstitution of a Functional Core Polycomb Repressive Complex
Nicole J Francis, Andrew J Saurin, Zhaohui Shao, Robert E Kingston 
Molecular Cell 
Volume 8, Issue 3, Pages (September 2001)
DOI: /S (01)

2. Figure 1 The PRC1 Core Complex and PSC Inhibit Chromatin Remodeling
(A) PCC (800 ng) was resolved by 8% SDS-PAGE and stained with colloidal coomassie. Asterisk indicates 70 kDa band which may correspond to HSC70 (see text).
(B) Verification of identity of bands in PCC by Western blot using antibodies to PSC, the Flag epitope (M5, Sigma), Pc, or RING1. The dRING1 signal is a dimer of two closely migrating bands.
(C) PCC and PSC inhibit chromatin remodeling in the plasmid supercoiling assay. PCC (1–2 nM) or PSC (∼4 nM) was added prior to a 20 min preincubation period at 30°C (+ preinc.) or at the same time as hSWI/SNF (− preinc.). N, nicked DNA; L, linear DNA; and SC, supercoiled DNA.
(D) PSC, PH, Pc or dRING1 (∼300 ng each) were resolved by SDS-PAGE and stained with colloidal coomassie
Molecular Cell 2001 8, DOI: ( /S (01) )

3. Figure 2
Efficient Inhibition of Remodeling by PSC and PCC on Nucleosomal Arrays but Not Mononucleosomes
(A) 5S array template used in restriction enzyme assay. Positions of the central nucleosomes (light) are inferred from MNase digestion.
(B) Comparison of inhibition of chromatin remodeling by PRC1, PCC, and PSC in the restriction enzyme accessibility assay using HhaI. First two lanes next to each titration are the negative (no hSWI/SNF, no PcG) and positive controls (+ hSWI/SNF, no PcG), demonstrating that the HhaI site becomes accessible with hSWI/SNF. Increasing concentrations of PcG proteins (as indicated) were added to remodeling reactions containing 1 ng 5S array and 9 ng HeLa polynucleosomes (8 nM nucleosomes) during the preincubation period. The concentration of PRC1 was estimated from Bradford assays, Western blot titrations, and silver stained gels, while that of PCC and PSC is the measured concentration of active DNA binding molecules. rat. to nuc., ratio of PcG proteins to nucleosomes.
(C) Summary of effects of PRC1 and PCC on chromatin remodeling. Data points represent the average of at least three experiments, and error bars are the standard deviations. (D) Mononucleosmes (1 nM) were incubated with increasing concentrations of PSC or PCC followed by addition of hSWI/SNF and PstI
Molecular Cell 2001 8, DOI: ( /S (01) )

4. Figure 2
Efficient Inhibition of Remodeling by PSC and PCC on Nucleosomal Arrays but Not Mononucleosomes
(A) 5S array template used in restriction enzyme assay. Positions of the central nucleosomes (light) are inferred from MNase digestion.
(B) Comparison of inhibition of chromatin remodeling by PRC1, PCC, and PSC in the restriction enzyme accessibility assay using HhaI. First two lanes next to each titration are the negative (no hSWI/SNF, no PcG) and positive controls (+ hSWI/SNF, no PcG), demonstrating that the HhaI site becomes accessible with hSWI/SNF. Increasing concentrations of PcG proteins (as indicated) were added to remodeling reactions containing 1 ng 5S array and 9 ng HeLa polynucleosomes (8 nM nucleosomes) during the preincubation period. The concentration of PRC1 was estimated from Bradford assays, Western blot titrations, and silver stained gels, while that of PCC and PSC is the measured concentration of active DNA binding molecules. rat. to nuc., ratio of PcG proteins to nucleosomes.
(C) Summary of effects of PRC1 and PCC on chromatin remodeling. Data points represent the average of at least three experiments, and error bars are the standard deviations. (D) Mononucleosmes (1 nM) were incubated with increasing concentrations of PSC or PCC followed by addition of hSWI/SNF and PstI
Molecular Cell 2001 8, DOI: ( /S (01) )

5. Figure 3 Inhibition of Chromatin Remodeling by Isolated PSC
(A) Effect of increasing nucleosome concentration on inhibition of remodeling by PSC. The left and middle panels demonstrate that the amounts of HhaI and hSWI/SNF added to reactions are saturating in all conditions since the level of digestion and stimulation of digestion are not altered.
(B) PSC glycerol gradient fractions were resolved by SDS-PAGE and stained with silver after dialysis to remove urea (see Experimental Procedures for details). Asterisk indicates a 70 kDa protein that fractionates away from PSC on the gradient. The high molecular weight material visible as a smear in fractions 5 and 6 is likely to be aggregated PSC.
(C) Restriction enzyme accessibility assay of PSC glycerol gradient fractions. 1, 2, or 4 μl of each fraction was tested in remodeling reactions.
(D) Graph of the midpoint (2 μl) of three titrations similar to the one shown in C; error bars are standard error of the mean. Similar results were obtained from four different glycerol gradients
Molecular Cell 2001 8, DOI: ( /S (01) )

6. Figure 4 PSC and PCC Bind DNA
(A) Filter binding assay for binding of PSC (top panel) and PCC (bottom panel) to DNA. The indicated concentrations of PSC or PCC were titrated into binding reactions containing labeled 155 bp double-stranded DNA at 10–20 pM. The top row of each panel is the top (nitrocellulose) filter and represents protein-DNA complexes, while the bottom row is the bottom (charged nylon) filter and represents unbound DNA.
(B) Data points are the average of binding data from multiple experiments used to determine the KD of PSC and PCC for TPT
Molecular Cell 2001 8, DOI: ( /S (01) )

7. Figure 5
PCC and PSC Block hSWI/SNF-Mediated Changes in Restriction Enzyme Accessibility
(A) PCC and PSC block the hSWI/SNF-induced decrease in SacI digestion. To determine the effects of hSWI/SNF remodeling on SacI digestion, templates were preincubated with PcG proteins followed by addition of hSWI/SNF to some reactions for an additional 30 min; finally, all reactions were digested with 20 U of SacI for 5 min at 30°C. The first lane of each set of three lanes has no PcG protein added; note the decrease in SacI digestion following remodeling by hSWI/SNF.
(B) PSC and PCC inhibit hSWI/SNF effects on access to multiple restriction sites. In each case, the template was incubated with 1.5–2 nM PCC or 3–4 nM PSC for 15 min at 30°C, restriction enzyme was added with or without hSWI/SNF, and incubation was continued for an additional 60 min. The first lane of each set of three lanes has no protein added.
(C) Quantitation of the results from multiple experiments. The left graph represents the average % uncut without hSWI/SNF, and the right graph is the % uncut with hSWI/SNF. Error bars are standard deviations
Molecular Cell 2001 8, DOI: ( /S (01) )

8. Figure 6
PRC1, PCC, and PSC Inhibit Mobilization of Nucleosomes by hSWI/SNF but Do Not Alter Nucleosome Organization
(A) PRC1 inhibits hSWI/SNF-dependent alteration of nucleosome positions, but does not itself alter the pattern of MNase digestion. Left panel shows the effects of PRC1 on MNase digestion while the right panel demonstrates the effect of hSWI/SNF on MNase digestion and the ability of PRC1 to block this effect. Lanes marked M contain markers for nucleosome positions made by partial digestion of unassembled 5S template with EcoR1.
(B) PCC and PSC block hSWI/SNF-induced nucleosome movement, but do not themselves alter the MNase digestion pattern.
(C) Indirect end labeling of MNase digested templates demonstrates that PCC and PSC do not grossly alter nucleosome positions, but that more MNase is required for full digestion of bound templates.
(D) Indirect end labeling of DnaseI-digested templates confirms that PCC and PSC do not alter nucleosome positions; templates incubated with PSC or PCC are more sensitive to digestion by DNase I
Molecular Cell 2001 8, DOI: ( /S (01) )

9. Figure 7 PSC and PCC Exclude hSWI/SNF from Nucleosomal Arrays
(A) ChIP analysis of remodeling reactions.
(B) The ChIP signal represents a ratio of the signal obtained in each lane divided by the signal obtained for 0.15 nM hSWI/SNF; no protein background was subtracted from all lanes, while PSC or PCC + hSWI/SNF lanes were corrected for background due to PSC or PCC alone. This experiment is representative of three experiments
Molecular Cell 2001 8, DOI: ( /S (01) )