Inhibition of chromatin remodeling by polycomb group protein posterior sex combs is mechanistically distinct from nucleosome binding

Abstract

Polycomb Group (PcG) proteins are essential regulators of development that maintain gene silencing in Drosophila and mammals through alterations of chromatin structure. One key PcG protein, Posterior Sex Combs (PSC), is part of at least two complexes: Polycomb Repressive Complex 1 (PRC1) and dRING-Associated Factors (dRAF). PRC1-class complexes compact chromatin and inhibit chromatin remodeling, while dRAF has E3 ligase activity for ubiquitylation of histone H2A; activities of both complexes can inhibit transcription. The noncovalent effects of PRC1-class complexes on chromatin can be recapitulated by PSC alone, and the region of PSC required for these activities is essential for PSC function in vivo. To understand how PSC interacts with chromatin to exert its repressive effects, we compared the ability of PSC to bind to and inhibit remodeling of various nucleosomal templates and determined which regions of PSC are required for mononucleosome binding and inhibition of chromatin remodeling. We find that PSC binds mononucleosome templates but inhibits their remodeling poorly. Addition of linker DNA to mononucleosomes allows their remodeling to be inhibited, although higher concentrations of PSC are required than for inhibition of multinucleosome templates. The C-terminal region of PSC (amino acids 456−1603) is important for inhibition of chromatin remodeling, and we identified amino acids 456−909 as being sufficient for stable nucleosome binding but not for inhibition of chromatin remodeling. Our data suggest distinct mechanistic steps between nucleosome binding and inhibition of chromatin remodeling.

Figure 1. PSC inhibits remodeling of mononucleosomes poorly

Restriction enzyme accessibility (REA) assays were carried out with PSC at the indicated concentrations and hSwi/Snf. A,B) Representative gels of REAs with 6N (A) and 2N (B) templates. (N refers to nucleosomes, so 6N is a 6-nucleosome template; all of these templates are composed of repeats of the 5S nucleosome positioning sequence.). C) Summary of REAs with 6N and 2N templates. D, E) Representative gel of REA with mononucleosomes assembled on a 157-bp (10-1N) 5S template at 1nM (D) or 5nM (E). Note that PstI was used for digestion in the experiment with 1nM nucleosomes, while HhaI was used with 5nM nucleosomes. Both enzymes have single digestion sites in the template, but produce slightly different digestion patterns (HhaI digests the template into 77 and 80 bp fragments, which are not resolved, while PstI digests it into 99 and 52 bp fragments). Both enzymes were used in these assays and they produce very similar results. D) Summary of REAs with mononucleosome template. Error bars in are SEM.

Figure 2. PSC inhibits mononucleosomes with one long linker or two linkers better than mononucleosomes with one short linker

A-C) Representative REAs on a series of mononucleosome templates. The three templates are all 247-bp long. D) PSC does not inhibit restriction enzyme digestion. Naked DNA at 1nM was incubated with PSC under identical conditions as in the remodeling assays either with or without the restriction enzyme HhaI. E) Summary of REAs on the various mononucleosome templates. Note that even though remodeling of templates with linkers can be inhibited by PSC, higher concentrations are required than for 2N templates (compare with Figure 1C). Error bars are SEM.

Figure 3. PSC binds all mononucleosome templates at low concentrations that do not inhibit chromatin remodeling

A. EMSAs with various mononucleosome substrates at 1nM. Details of quantification of EMSAs are presented in sFigure 5. At low concentrations, PSC binds mononucleosomes (bound (1)), while at higher concentrations it forms a second, slowly migrating species (bound (2)). B. Quantification of fraction bound (bound 1+2) for each template. C. Quantification of slowly migrating species (bound (2)) only. Error bars are SD.

Figure 4. Competition binding assays demonstrate that PSC binds 2N templates preferentially over 1N templates but that binding to both templates is stable

A. Competition assays in which competitor was added simultaneously with labeled substrates, as indicated. B. Competition assays in which competitor was added after a 15-minute pre-incubation with labeled substrate. PSC was used at 1nM and labeled nucleosomes at 0.5nM. Competitor was used up to 10nM.

Figure 5. PSC interacts with itself to likely form dimers

A. FLAG- and HA-tagged PSC were expressed individually or together, and FLAG-PSC immunoprecipitated. When FLAG-PSC and HA-PSC were co-infected (third panel), HA-PSC co-purifies with FLAG-PSC. B. Crosslinking with BM (left) or EDC (right) followed by Western blot analysis. PSC cross-links into high molecular weight species when treated with either cross-linker. C. Glycerol gradient sedimentation of PSC, PSC-dRING, or PCC (PSC+dRING+Pc) followed by Western blot analysis. The peak of FLAG-PSC is in fraction 5, as is the peak for PSC-dRING, while the peak of PCC is in fraction 3. The molecular weight of FLAG-PSC is 171 kDa. Gradients shown were run in 900mM KCl, although similar results were obtained at 300 and 600mM KCl.

Figure 6. Inhibition of chromatin remodeling requires sequences in PSC not required for stable nucleosome binding

A. Schematic diagram of PSC truncations. Note that some of these truncations have been previously characterized [9, 18]. B. Colloidal Blue stained gel of representative preparations of PSC and PSC truncations. Asterisks (*) indicate contaminants that are likely Hsc70 (~70 kDa) and β-tubulin (~55 kDa). C. EMSA and REA of each truncation with a 50-1N-50 mononucleosome (2 linkers). The two shifted species are indicated next to the gels in cases where they form. D. Summary of REAs on two-linker 1N substrate with PSC and the various truncations. E. PSC^456-909 was tested for inhibition of remodeling of a 12N array. Full length PSC, and PSC^456-1603 were included for comparison. F. PSC^456-909 was tested for stable binding to the two-linker 1N substrate, using competition assays as in Figure 4.