Polycomb repressive complex 1 (PRC1) disassembles RNA polymerase II preinitiation complexes

Abstract

Despite the important role of Polycomb in genome-wide silencing, little is known of the specific biochemical mechanism by which it inactivates transcription. Here we address how recombinant Polycomb repressive complex 1 (PRC1) inhibits activated RNA polymerase II preinitiation complex (PIC) assembly using immobilized H3K27-methylated chromatin templates in vitro. Recombinant PRC1 inhibited transcription, but had little effect on binding of the activator as reported previously. In contrast, Mediator and the general transcription factors were blocked during assembly or dissociated from preassembled PICs. Importantly, among the PIC components, Tata Binding Protein (TBP) was the most resistant to eviction by PRC1. Immobilized template experiments using purified PRC1, transcription factor II D (TFIID), and Mediator indicate that PRC1 blocks the recruitment of Mediator, but not TFIID. We conclude that PRC1 functions to block or dissociate PICs by interfering with Mediator, but leaves TBP and perhaps TFIID intact, highlighting a specific mechanism for PRC1 transcriptional silencing. Analysis of published genome-wide datasets from mouse embryonic stem cells revealed that the Ring1b subunit of PRC1 and TBP co-enrich at developmental genes. Further, genes enriched for Ring1b and TBP are expressed at significantly lower levels than those enriched for Mediator, TBP, and Ring1b. Collectively, the data are consistent with a model in which PRC1 and TFIID could co-occupy genes poised for activation during development.

FIGURE 1.

Binding of PRC1 to H3K27me3 chromatin in vitro. A, schematic of the MLA synthesis at histone H3 lysine 27. Below the schematic is a Western blot showing the specific detection of H3K27me3 MLA by antibody against HK27me3. Unmod., unmodified. B, schematic of the immobilized chromatin template and chromatin assembly. A Coomassie Blue-stained gel of the purified recombinant Xenopus laevis histones used is shown on the left. The extent of chromatin assembly upon the addition of increasing amounts of histone octamers was monitored by EMSA; equivalent amounts of chromatin were utilized in all experiments. The positions of the free DNA and tri-nucleosome (Tri Nuc) are indicated with arrows. RT primer was used in primer extension to measure RNA during in vitro transcription. C, Coomassie Blue-stained gel of purified PRC1 showing the CBX2, BMI1, and Ring1b subunits. 2 μg of PRC1 expressed in and purified from the Bac-to-Bac baculovirus system (Invitrogen) is shown. D, immobilized template analysis of PRC1 binding to naive (unmodified) and H3K27me3 chromatin. 125 fmol of immobilized chromatin was incubated with increasing amounts of PRC1 from 62.5 to 250 nm. After washing, the bound proteins were eluted and subjected to immunoblotting. E, statistical analysis of data. Three replicates were quantitated using the LI-COR imaging package and graphed. The values were normalized to the highest point of naive chromatin used, which was set at 100%. Error bars indicate S.D.

FIGURE 2.

PRC1 blocks transcription and PIC assembly. A, PRC1 blocks in vitro transcription. The flowchart indicates the order of protein addition. GAL4-VP16 was prebound to the template for 20 min, and 250 nm PRC1 was added. After 30 min, HeLa nuclear extract was added in the presence of nucleotides. After 30 min, the mRNA products were isolated and measured by primer extension. B, PRC1 blocks PIC assembly. An immobilized template assay using HeLa nuclear extract, in the presence and absence of activator and PRC1, was performed. An immunoblot of the isolated complexes comparing binding of select components of the PIC is shown. C, immunoblot signal was quantified using the LI-COR imaging software and graphed, and the statistical significance between mock- and PRC1-treated templates was calculated using a two-tailed Student's t test (n = 3). p values were determined to be *, 0.025, **, 0.03, ***, 0.015, ****, 0.022, and *****, 0.001 for the indicated signals quantified. Signals were normalized to that of activator-stimulated recruitment in lane 2 (100%). Error bars indicate S.D.

FIGURE 3.

PRC1 dissociates preassembled PICs. A, PRC1 silences in vitro transcription from preassembled PICs. The flowchart indicates the order of binding. GAL4-VP16 was prebound to the template for 20 min, after which HeLa nuclear extract was added to allow PIC assembly. After 30 min, PICs were isolated, and PRC1 was added in the presence of nucleotides. After 30 min, the mRNA products were isolated and measured by primer extension. B, PRC1 disassembles a PIC in an immobilized template assay. The flowchart indicates the order of protein addition. An immunoblot comparing binding of select components of the PIC in the presence of increasing amounts of PRC1 is shown. C, immunoblot signal was quantified using the LI-COR imaging software and graphed, and the statistical significance between recruitment of proteins to mock- and PRC1-treated templates was calculated using a two tailed Student's t test (n = 3). The p values determined were *, 0.001, **, 0.002, ***, 0.020, ****, 0.003, and *****, 0.001 for the indicated signals measured. Signals were normalized to that of activator-stimulated recruitment in lane 2 (100%). Error bars indicate S.D.

FIGURE 4.

TFIID binds the immobilized template in the presence of PRC1. A and B, purified TFIID, PRC1, and GAL4-VP16 were incubated with 125 fmol of G5E4T template in immobilized template assays. Assays were performed to investigate both dissociation (A) and blocking (B) of TFIID binding. C and D, a quantitation of three replicate experiments for both dissociation (C) and blocking (D) assays is shown. The immunoblot signals were quantified using the LI-COR system, and values were normalized to the amount of protein bound when added alone. Error bars indicate S.D.

FIGURE 5.

TFIID but not Mediator binding is resistant to PRC1. Purified TFIID, Mediator, PRC1, and GAL4-VP16 were incubated with 125 fmol of template in an immobilized template assay. Following prebinding of the activator, PRC1 was bound to the template. TFIID or Mediator was then incubated with the template either alone or in combination. The Western blot signals for representative subunits of TFIID (TAF1, TAF2, TAF4, TBP), Mediator (MED23, MED6), and PRC1 (BMI1, CBX2) are shown.

FIGURE 6.

PRC1 and TBP enrichment overlaps in vivo. A, the distributions of TBP and Ring1b from −5 kb to +5 kb, centered at the transcriptional start site (TSS) for all annotated genes in mouse ES cells, are shown as heat maps of −log (p value (p-val)). Binding peaks were sorted into differential binding clusters (K1–3) comparing TBP and Ring1b across all genes in the genome. B, RNA-Seq expression analysis was used to determine the mean expression for genes in clusters K1–3. A box and whisker plot of the expression for each cluster is shown for reads per kilobase of exon per million (RPKM) fragments mapped. Asterisks indicate that the change in expression is significant between the indicated clusters as measured using a Kolmogorov-Smirnov test. Error bars indicate S.D. C, a Venn diagram comparing the enrichment of TBP alone, TBP and Ring1b, or Ring1b alone to all genes in mouse ES cells is shown.

FIGURE 7.

Genome-wide analysis of Ring1b, TBP, and Mediator in mouse ES cells. A, the distributions of TBP, Ring1b, and MED1 enrichment −5 kb to +5 kb, centered at the transcriptional start site (TSS) for all annotated genes in mouse ES cells, are shown as heat maps of −log (p value). Peaks were called and sorted into differential clusters (L1–4) based on the enrichments of TBP, Ring1b, and MED1 across all genes in the genome. Only genomic regions enriched for TBP are shown. B, mean expression analysis of genes in clusters L1–4 is shown. A box and whisker plot of the expression for each cluster indicates the mean expression for each cluster in reads per kilobase of exon per million (RPKM). The dashed line represents a 2-fold change in expression from the overall mean. The p values resultant from Kolmogorov-Smirnov test comparing clusters L1–4 are shown. Error bars indicate S.D. C–F, GO term analysis of clusters L1–4. The top six GO terms for each expression cluster are shown, and the −log of the p value of confidence for each term is graphed. G–J, the binding profile for a representative gene from each cluster is shown. Values plotted are −log (p value).