The core of the polycomb repressive complex is compositionally and functionally conserved in flies and humans

Abstract

The Polycomb group (PcG) genes are required to maintain homeotic genes in a silenced state during development in drosophila and mammals and are thought to form several distinct silencing complexes that maintain homeotic gene repression during development. Mutations in the PcG genes result in developmental defects and have been implicated in human cancer. Although some PcG protein domains are conserved between flies and humans, substantial regions of several PcG proteins are divergent and humans contain multiple versions of each PcG gene. To determine the effects of these changes on the composition and function of a PcG complex, we have purified a human Polycomb repressive complex from HeLa cells (hPRC-H) that contains homologues of PcG proteins found in drosophila embryonic PRC1 (dPRC1). hPRC-H was found to have fewer components than dPRC1, retaining the PcG core proteins of dPRC1 but lacking most non-PcG proteins. Preparations of hPRC-H contained either two or three different homologues of most of the core PcG proteins, including a new Ph homologue we have named HPH3. Despite differences in composition, dPRC1 and hPRC-H have similar functions: hPRC-H is able to efficiently block remodeling of nucleosomal arrays through a mechanism that does not block the ability of nucleases to access and cleave the arrays.

FIG. 1.

Purification of the hPRC-H complex. (A) Schematic for fractionation of hPRC-H. (B) class II PcG proteins cofractionate with FLAG-Bmi1 and FLAG-M33. Nuclear extract (NE; 10 μg), heparin peak fraction (Hep. Pk; 4 μg), M2 flowthrough (∼3 μg), and M2 elutions from Bmi1F and M33F lines (50 ng) were visualized with the indicated antibodies. Most of hPRC-H proteins flow through the M2 column because the tagged proteins are expressed at low levels. (C) Composition of the hPRC-H complex. M33F and Bmi1F M2 eluates (25 μg) were separated by SDS-8% PAGE and stained with silver. Proteins identified by MS sequencing that are present in all of the extracts tested are identified. Changes in the intensity of the bands corresponding to HPC2, HPC3, and Bmi1 between the two complexes likely represent a replacement of Pc homologues by epitope-tagged M33 (>) and a change in mobility of epitope-tagged Bmi1 (<). Asterisks mark proteins not consistently observed from preparation to preparation. The values on the left are molecular sizes in kilodaltons.

FIG. 2.

HPH3 is a new PH homologue. (A) Phylogenetic tree of known PH homologues. (B) Domain architecture of HPH proteins. I, homology domain I; Zn, zinc finger; SEP, SEP domain/homology domain II; III, novel homology domain. The values above the sequences refer to percent identity. (C) ClustalW alignment of homology domain III. Sequences identical to HPH3 are highlighted in black. Similar sequences (3 distance units with PAM 62 matrix) are highlighted in gray.

FIG. 3.

Further fractionation of the hPRC-H complex. (A) Immunoblot of 25 μl (∼5%) of each HiTrap-S fraction visualized with the indicated antibody. For SNF2H and YY1, 250 μl was TCA precipitated prior to analysis. (B) Silver staining of HiTrap-S fractions. Fractions (250 μl) were TCA precipitated and separated by SDS-7.5% PAGE.

FIG. 4.

Activity of the hPRC-H complex. (A) Map of the 5S array used in the restriction enzyme and MNase assays. The HhaI site is indicated. (B) Restriction enzyme assay (REA). One nanogram (8 fmol) of nucleosomes was preincubated with increasing amounts of dPRC1 or hPRC-H from M33- and Bmi1-tagged lines. Lanes: 1, no-Swi/Snf control; 2, Swi/Snf (100 ng) only; 3 to 6, ∼0.4, 1.2, 4, and 12 fmol of hPRC-H (M33); 7 to 10, ∼1.2, 3.6, 12, and 36 fmol of hPRC-H (Bmi1). The percentage of template cut by HhaI is indicated under each lane. (C) Quantification of inhibition of remodeling. Same as panel B but with 80 fmol of nucleosomes. The amount (nanograms) of PRC added was calculated by Bradford analysis and comparative silver staining. Molar amounts were determined with an estimate of 500 kDa as the mass of hPRC-H (see Materials and Methods). Half-maximal repression occurs at ratios of hPRC-H to nucleosomes of approximately 1:8. Est. mol, estimated number of moles. (D) Topological assay. Nucleosomal plasmids were preincubated with hPRC-H for 15 min before being challenged with 100 ng of Swi/Snf and 4 U of topoisomerase I. Remodeled templates are visualized as slower-migrating topoisomers. Increasing amounts of hPRC-H increase the inhibition of Swi/Snf remodeling. Lanes: 1, no-Swi/Snf control; 2, Swi/Snf only; 3 to 6, titration of M33F hPRC-H as in panel B. R, relaxed; ∗, linear; S, fully negatively supercoiled.

FIG. 5.

hPRC-H activity is specific to polynucleosomal templates. (A) hPRC-H inhibits Swi/Snf without disrupting nucleosome position. End-labeled 5S arrays were treated as described in the legend to Fig. 4B, except that HhaI was not added. Following incubation of the templates with Swi/Snf, increasing amounts of MNase were added to each reaction mixture. Lanes: 1 to 4, control template; 5 to 8, 25 fmol of Swi/Snf; 9 to 12, same as lanes 5 to 8 with ∼12 fmol of hPRC-H; 13 to 16, with ∼12 fmol of dPRC1. All reaction mixtures are from the same experiment; nucleosome positions align when samples are analyzed side by side (data not shown). (B) hPRC-H does not block Swi/Snf remodeling on mononucleosomes. Internally labeled TPT mononucleosomes (1 ng) (47) were incubated as described in the legend to Fig. 4B with 200 ng of Swi/Snf and PstI. Amounts of hPRC-H (M33) and dPRC1 are ∼1.2, 4, and 12 fmol.