Native and recombinant polycomb group complexes establish a selective block to template accessibility to repress transcription in vitro

Abstract

Polycomb group (PcG) proteins are responsible for stable repression of homeotic gene expression during Drosophila melanogaster development. They are thought to stabilize chromatin structure to prevent transcription, though how they do this is unknown. We have established an in vitro system in which the PcG complex PRC1 and a recombinant PRC1 core complex (PCC) containing only PcG proteins are able to repress transcription by both RNA polymerase II and by T7 RNA polymerase. We find that assembly of the template into nucleosomes enhances repression by PRC1 and PCC. The subunit Psc is able to inhibit transcription on its own. PRC1- and PCC-repressed templates remain accessible to Gal4-VP16 binding, and incubation of the template with HeLa nuclear extract before the addition of PCC eliminates PCC repression. These results suggest that PcG proteins do not merely prohibit all transcription machinery from binding the template but instead likely inhibit specific steps in the transcription reaction.

FIG. 1.

(A) 5S array transcription template. The template is made up of repeats of a 5S nucleosome positioning sequence flanking a region of 390 bp. This region contains five Gal4 binding sites, the adenovirus E4 promoter, and a start site for T7 RNA polymerase. The relative locations of primers used to visualize RNA transcription products by primer extension are indicated. (B) Micrococcal nuclease (MNase) analysis of 5S nucleosomal array template. (C) EcoRI digest of 5S array template. Assembled 5S arrays were digested with EcoRI, which cuts between each 5S repeat, reducing the template to 10 196-bp 5S repeat fragments plus one fragment of 431 bp containing the E4 promoter and Gal4 sites. Saturation of assembly is assessed by the proportion of nonnucleosomal mononucleosome-sized fragments. The asterisk indicates the uncut template. (D) Mobility shift analysis. Assembled 5S array templates were compared to naked DNA for a mobility shift indicative of template compaction due to the addition of nucleosomes. (E) Schematic of transcription reaction protocol. RT, room temperature; N.E., nuclear extract. (F) Transcription of the 5S array template is stimulated by hSwi/Snf. Reaction mixtures use 20 ng (∼15 fmol) of 5S nucleosomal array, with and without hSwi/Snf and ATP. hSwi/Snf was added to a molar ratio of approximately one per two nucleosomes. Quantitation is relative to +Gal4-VP16-+Swi/Snf lane.

FIG. 2.

PRC1 and PCC repress transcription on naked and chromatinized templates. PRC1 (A) and PCC (B) were titrated into transcription reaction mixtures by the same protocol as shown in Fig. 1, including a remodeling step with Swi/Snf and ATP. In both cases, 20 ng of chromatinized 5S template or 20 ng of naked 5S template DNA was used. Reaction mixtures contained 10, 20, and 40 fmol of PRC1 (approximately 0.7:1, 1.3:1, and 2.7:1 molar ratio to template, respectively) and 11, 23, and 47 fmol of PCC (approximately 0.7:1, 1.5:1, and 3:1 molar ratio to template, respectively). Exposure time for naked DNA and chromatin reactions was equal for all reactions shown here. Typically, though, transcription on chromatin is less efficient than transcription on naked DNA. Quantitation is relative to −PcG lane for each. (C) PRC1 and PCC were titrated into transcription reaction mixtures on naked DNA templates at higher ratios to template. Reactions contained 7.5, 15, and 30 fmol of PRC1 and 5 ng of naked DNA template (approximately 2:1, 4:1, and 8:1 molar ratio to template, respectively) or 47, 94, and 188 fmol of PCC and 20 ng of naked DNA template (approximately 3:1, 6:1, and 12:1 molar ratio to template, respectively). Quantitation is relative to −PcG lane for each. All experiments were performed at least three times, and results of representative experiments are shown.

FIG. 3.

Analysis of individual PCC subunits in transcription. The subunits that make up PCC were purified individually and added to in vitro transcription reaction mixtures. Titrations for each subunit contained 10, 20, and 40 fmol of total protein and 5 ng of 5S nucleosomal array template (molar ratio of approximately 2.5, 5, and 10 per template, respectively). Also shown is a titration of PCC at these concentrations.

FIG. 4.

PRC1 and PCC repress transcription by T7 RNA polymerase. (A) Schematic of reaction protocol for transcription with T7 RNA polymerase. (B and C) PRC1 (B) and PCC (C) were titrated into T7 transcription reaction mixtures. As with Pol II transcription reactions, 20 ng (∼15 fmol) of assembled 5S array or 20 ng of unassembled 5S array DNA was used in each reaction. Reaction mixtures contained 2.5, 5, 10, and 20 fmol of PRC1 and 17.5, 35, 70, and 140 fmol of PCC. Quantitation is relative to −PcG lane for each.

FIG. 5.

PRC1 efficiently represses templates challenged with HhaI. 5S array templates were cut with HhaI before incubation with PRC1 and then transcribed with T7 RNA polymerase. Templates that were nucleosomal at the HhaI site adjacent to the T7 start site were protected from digestion, while any residual naked templates were digested. Since transcription was initiated more often from templates in which the T7 start site was unprotected, digesting the template with HhaI resulted in a ninefold drop in transcription. Primer extension with the primer used for these reactions gave two products of approximately the expected size. The primer may have recognized two adjacent sequences. Quantitation of transcription products is relative to −PRC1 lane for each set of reactions.

FIG. 6.

PCC inhibits transcription when added before HeLa nuclear extract. The order of addition of reaction components was changed as noted. Reactions used 20 ng of 5S nucleosomal array template. PCC was titrated under each reaction protocol (24, 47.5, and 95 fmol). Quantitation is normalized to −PCC lane for each protocol. The transcription signal was sometimes stronger for the PCC-last protocol (fourfold in this case). This may reflect differences in the time that the template was incubated with HeLa nuclear extract (N.E.) before transcription in this protocol.

FIG. 7.

PRC1 and PCC do not inhibit transcription by blocking the binding of Gal4-VP16. Transcription reaction mixtures containing end-labeled 5S nucleosomal array templates were split, with one portion being analyzed by DNase I footprinting (A) and the other portion being analyzed for transcription (B). Regions that became DNase I hypersensitive upon Gal4 binding are indicated with asterisks, and the region of protection is indicated by the bracket. Reaction mixtures containing PRC1 or PCC used fourfold-lower DNase I concentrations than did the reaction mixtures without PcG complexes (6.25, 12.5, and 25 mU for reaction mixtures with PcG complexes and 25, 50, and 100 mU for reaction mixtures without PcG complexes). Reaction mixtures used 116 fmol of PCC, 150 fmol of PRC1, and 50 ng of 5S array template (3:1 and 4:1 molar ratios, respectively).