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Previous Article | Next Article 
Molecular and Cellular Biology, September 2002, p. 6070-6078, Vol. 22, No. 17
0270-7306/02/$04.00+0 DOI: 10.1128/MCB.22.17.6070-6078.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Hediye Erdjument-Bromage,3 Zhaohui Shao,1,2,
Paul Tempst,3 and Robert E. Kingston1,2*
Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts 02114,1 Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115,2 Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, New York 100213
Received 12 April 2002/ Returned for modification 10 May 2002/ Accepted 7 June 2002
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ABSTRACT |
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INTRODUCTION |
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The PcG genes were originally identified in drosophila, where homozygous mutant embryos exhibited severe homeotic transformations (reviewed in references 24 and 54). Genetic analysis of these mutants showed extensive derepression of the homeotic genes and suggested that they may play a role in maintenance of silencing. Similarly, targeted deletion of PcG genes in mice results in homeotic transformation of segment identity due to delayed ectopic expression of some Hox genes and is generally lethal (1, 11, 12, 35, 56, 61). The phenotypes of mice lacking single PcG genes are generally milder then those observed in drosophila, at least in part because of the presence of multiple homologues of each drosophila PcG gene in mammals (Table 1). Disruption of both homologues of Psc (Bmi1 and Mel-18), for example, is lethal much earlier in development and causes more extreme Hox gene deregulation than does the disruption of either gene individually (2).
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Gene targeting studies indicate that at least some biological functions of PcG genes, such as regulation of Hox gene expression, have been conserved evolutionarily consistent with conservation of several protein domains between drosophila and mammals. However, many of the PcG genes have also diverged substantially. Furthermore, most of the PcG genes have been duplicated in mammals and different expression patterns and biological functions have been described for different homologues. For example, Bmi1 and Mel-18, the homologues of Psc (9, 63), are less than one-third of the size of Psc (46 and 50 versus 188 kDa) and appear to have opposite effects on cell growth (19, 23, 65). These data suggest that mammalian PcG proteins may interact with different proteins than do drosophila PcG proteins and may have different activities. Comparing composition and functions of complexes purified from human cells to those purified from flies is an important step in investigating these possibilities.
We have recently described the purification and initial functional characterization of the major class II complex from drosophila embryos, drosophila embryonic PRC1 (dPRC1) (45). In parallel with these studies, we have purified and characterized the human counterpart to this complex. A comparison of the components and functions of the class II complexes can reveal core subunits and fundamental activities. We find that only a subset of proteins are conserved between hPRC-H (Polycomb repressive complex from HeLa cells) and dPRC1; most, but not all, of these conserved subunits are PcG genes that emerged from the original developmental screens. Despite the different natures of the complexes, dPRC1 and hPRC-H performed with similar function and efficiency in a variety of protocols that used nucleosomal arrays as template.
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MATERIALS AND METHODS |
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Purification of hPRC-H.
Nuclear extracts were prepared from 100 liters of tagged HeLa cells (cell lines M33F-1 and BMI1F-17) as described previously (53) with 1.5 M KCl for the high-salt extraction buffer. A 50- to 60-mg sample of undialyzed nuclear extract (5 ml) was diluted in BCN buffer (20 mM HEPES, K+ [pH 7.9], 0.2 mM EDTA, 20% glycerol, 0.1% NP-40, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride) containing 300 mM KCl (BCN300) and bound to a 5-ml Hi-Trap heparin column (Amersham-Pharmacia) on an AKTA fast protein liquid chromatography apparatus (Amersham-Pharmacia). Samples were washed with 4 column volumes (CV) of BCN300 and eluted with an 8-CV linear gradient of 300 to 1,000 mM KCl. Fractions from three repeats of this gradient containing the tagged protein (
350 to 550 mM KCl) were pooled and bound to 1 ml of M2 agarose beads (Sigma) overnight. The beads were washed in a column with 20 CV of BCN300 and BCN450, equilibrated with BCN300, and eluted with 1 mg of FLAG peptide (DYKDDDDK) per ml. For analysis on cation-exchange columns, M2 columns were equilibrated with BCN200 and eluted with FLAG/BCN200 directly onto a 1-ml Hi-Trap S column. The S column was transferred to an AKTA fast protein liquid chromatography apparatus exclusively with PEEK tubing (Upchurch). The column was washed with 5 CV of BCN200, and bound proteins were eluted with a 9-CV linear gradient of 200 to 500 mM KCl. Fractions were either immediately resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for immunoblot analysis or precipitated with trichloroacetic acid (TCA) for immunoblot analysis and silver staining. Yields from this column were approximately 50%.
Estimation of hPRC-H concentration. Concentrations of the M2-purified fractions were determined by the Bradford assay (Bio-Rad) and estimated by silver staining compared to bovine serum albumin standards. The molecular mass of the complex was determined by adding up the major bands of the complex as if they were stoichiometric. The molecular mass of 500 kDa agreed with gel filtration chromatography estimates generated with a Superose 6 column (Amersham-Pharmacia) in BCN300. Yields from this column were typically less than 1% (data not shown). The protein molecular size markers used were blue dextran (void), thyroglobulin (670 kDa), catalase (230 kDa), and bovine serum albumin (66 kDa).
MS analysis of hPRC-H. Isolation and mass spectrometry (MS)/MS analysis of the proteins are described in reference 45. The N terminus of the MS-sequenced polypeptide identified as RING1A matched the sequence found in the National Center for Biotechnology Information database under accession number CAC38442. Most of the RING1A sequences in the National Center for Biotechnology Information database (including accession number Q06587) lack 29 amino acids. The additional sequence of RING1A in hPRC-H extends the homology among the known RING1 homologues. To identify HPH3, two peptides, (K/R)MQQPQISVYSGSDR and (K/R)SSLLIEQPVK, were BLAST tested against the full human genome and dbEST. With expressed sequence tags (ESTs) in the database, we generated a contig that contained both peptides in a 2-kb sequence that mapped to chromosome 3. This contig contained the 3.5-kb cDNA CS0DK007YJ17 (Research Genetics). This isolate was sequenced redundantly. Four additional cDNAs tested (American Type Culture Collection) were significantly shorter, and none contained the entirety of the gene. In their cDNA-containing sections, they were identical to the Research Genetics clone.
Immunoblot assays. The antibodies used in the immunoblot assays were M5 anti-FLAG (Sigma), F6 anti-Bmi1 (3), anti-RING1A (46), anti-HPH1 (18) anti-HPC2 (44), anti-SNF2H (7, 26), anti-BRG1 (47), anti-hsp70 (Santa Cruz), anti-RYBP, anti-E2F6 (59), anti-YY1 (Santa Cruz), anti-ENX, anti-EED (50), anti-p325 (26), anti-WCRF (7), anti-CtBP (49), anti-TAFII250 (Santa Cruz), and anti-TBP (Santa Cruz). Proteins were analyzed by standard SDS-7.5 or 8% PAGE, transferred to Immobilon P membranes, and detected with ECL and ECL+ reagents (Amersham-Pharmacia) in accordance with the manufacturer's recommendations.
Activity assays.
The dPRC1, human Swi/Snf, and histones used in activity assays were prepared as previously described (51). Topological assays were carried out as previously described (25), with chromatin assembled with heat-treated Xenopus egg extracts, except that the reaction volume was reduced to 20 µl. The reaction mixtures contained 100 ng of Swi/Snf, 3 ng of chromatinized template, 4 µM ATP, and 2 µM MgCl2 and were electrophoresed on a 1% agarose gel. No change in activity was seen when larger amounts of Swi/Snf were used (data not shown). Assembly and analysis of the 5S array by salt dialysis were done as previously described, and restriction enzyme assays were preformed essentially as previously described (15), with 1 ng of labeled array and 9 ng of HeLa polynucleosomes, as indicated, in 20-µl reaction mixtures. Swi/Snf activity was determined on the basis of the amounts of cutting in reaction mixtures with or without Swi/Snf present (see Fig. 4B, lanes 1 and 2). Micrococcal analysis was performed essentially the same way as the restriction enzyme assay, with 1 ng of end-labeled array. Mononucleosomes were assembled on a TPT-containing DNA sequence (47) by salt dialysis, and reactions were performed under conditions identical to those used for the restriction enzyme assays with 200 ng of Swi/Snf (32).
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RESULTS |
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PcG proteins constitute most of hPRC-H. To identify the components of hPRC-H, we performed MS analyses of the polypeptides isolated from the complex. Purified fractions from M33-tagged cells were separated by SDS-PAGE, and 15 distinct bands were isolated. Peptides from these bands were identified with multidimensional MS, and searches of databases were performed to identify the proteins. Nine of 12 identified bands (excluding the tagged proteins) were identified as PcG genes, including BMI1, RING1A, RING1B, HPH1, HPH2, HPC2, HPC3, and SCMH1 (Fig. 1B). Multiple homologues of the Polycomb protein copurified with the M33-tagged line, suggesting that different Pc homologues may coexist in individual complexes.
One prominent band (135 kDa) was not found in any nonredundant protein database. Two peptide sequences identified from it by matrix-assisted laser desorption ionization-time of flight MS and electrospray ionization-trandem mass spectrometry were used to search the human EST database. On the basis of several contiguous EST sequences, we obtained an apparent full-length cDNA (Research Genetics) containing both MS-sequenced peptides. This cDNA encodes a putative 931-amino-acid protein that is similar to other mammalian homologues of drosophila Ph. We therefore named this novel gene HPH3. HPH3 contains the conserved SEP domain, Zinc finger, and homology region I previously described (3) and has similar degrees of homology to both HPH1 and HPH2 (Fig. 2A). The N-terminal region of HPH3 has significant degrees of similarity to HPH1 (Fig. 2C). This homology extends to murine Ph2 but is not found in the drosophila polyhomeotic genes.
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To confirm that these proteins are robustly associated, we subjected the complex to further fractionation. Purified M33-based complex was applied to a Hi-Trap S column and eluted with a shallow 200 to 500 mM KCl gradient. Proteins in the eluted fractions were followed by silver staining when their characteristic migration allowed clear identification (Fig. 3B) or by Western analysis (Fig. 3A). Each of the class II PcG proteins and HSP70 continued to cofractionate on this column, eluting at 400 mM KCl. None of the bands detectable by silver staining (Fig. 3B) fractionated away from the complex. We concluded that the majority of proteins in hPRC-H are known members of the mammalian PcG gene family.
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A band in hPRC-H preparations that stained lightly with colloidal Coomassie stain and silver, and thus was inferred to be substoichiometric, was identified as SCMH1. Interestingly, the drosophila homologue Scm is found in substoichiometric amounts in dPRC1. Thus, the association of this protein with class II PcG complexes in small amounts is conserved between drosophila and humans, suggesting that a subset of PRC complexes might contain Scm.
Previously, yeast two-hybrid and immunoprecipitation analyses identified several transcriptional repressors that interact with class II PcG proteins in mammals. These include CtBP, RYBP, and two sequence-specific DNA binding factors, YY1 and E2F6, that appear to associate with the class II proteins via RYBP (17, 59). YY1 is homologous to the drosophila PcG protein Pleiohomeotic (Pho) and has been shown to interact with the class I PcG complex proteins (41, 42). YY1 was not detected in bands at the appropriate molecular mass in hPRC-H preparations that contain HPC2 and HSP70. However, fragments of YY1 were detected by MS analysis at an unexpected molecular mass (46 kDa, compared to 68 kDa for intact YY1). Fractions of hPRC-H obtained by Hi-Trap S chromatography were precipitated and tested for the presence of YY1, RYBP, and E2F6. Intact YY1 was detected cofractionating with hPRC-H (Fig. 3A). RYBP and E2F6 were detected with the available antisera in neither the M2 fractions nor the precipitated Hi-Trap fractions. Most of these proteins are not associated with the complex (Fig. 1B and data not shown).
SNF2H, a member of the Swi2/Snf2 family of ATP-dependent chromatin-remodeling factors and a homologue of the drosophila ISWI protein, was also detected in the purified fractions. Comparative Western analysis of SNF2H with RING1A, HPC2, and Bmi1 showed that levels of SNF2H varied significantly from preparation to preparation; even preparations with relatively high levels contained substoichiometric amounts of SNF2H relative to HPH3, as judged by the intensity of colloidal Coomassie staining (data not shown). These levels represent only a small portion of the cellular SNF2H (Fig. 1B). We were unable to detect either p325 or WCRF-180 by immunoblotting, suggesting that hPRC-H does not contain appreciable amounts of the RSF (26) or WCRF (7) complex, both of which contain SNF2H. When TCA-precipitated samples obtained by Hi-Trap S chromatography were analyzed by immunoblotting, the substoichiometric amounts of SNF2H cofractionated with hPRC-H components (Fig. 3A). Together, these data demonstrate that SCM, YY1, and SNF2H can be seen at substoichiometric levels that maintain cofractionation with major hPRC-H components after immunoaffinity and Hi-Trap S fractionation. These proteins are therefore candidates for association with a subset of hPRC-H complexes, and further analysis is required to assess the potential function of these proteins within hPRC-H.
hPRC-H specifically inhibits Swi/Snf remodeling of nucleosomal arrays. The mechanism of PcG repression of transcription in vivo is unknown. However, genetically, it is clear that PcG proteins act antagonistically to trxG-encoded proteins. We previously took advantage of this to develop in vitro assays for dPRC1 by examining its ability to inhibit the activities of the trxG-related human complex Swi/Snf (51). To determine whether this activity was conserved in the mammalian PcG complex despite the changes in the primary sequence and composition of the complex, we investigated whether hPRC-H is also able to inhibit Swi/Snf-dependent remodeling of nucleosomal templates with a variety of assays.
We first examined the ability of hPRC-H to inhibit Swi/Snf-dependent remodeling on linear nucleosomal arrays (Fig. 4A). In vitro and in vivo, the presence of nucleosomes can reduce the accessibility of restriction endonucleases (40). Addition of Swi/Snf to nucleosomal templates can increase restriction enzyme access in an ATP-dependent manner (28). Embryonic dPRC1 is able to block this Swi/Snf-induced increase in restriction enzyme cutting on a polynucleosomal template (15). We tested whether hPRC-H is also able to analogously block Swi/Snf-stimulated restriction enzyme access with a chromatinized template containing two unpositioned nucleosomes flanked on each side by five nucleosomes positioned by repeats of the Xenopus 5S nucleosomal positioning sequence. hPRC-H blocked Swi/Snf-stimulated cutting by HhaI in a manner similar to that of dPRC1. Furthermore, this inhibition was concentration dependent (Fig. 4B). On the basis of calculations of hPRC-H concentration with an estimated size of 500 kDa (see Materials and Methods), hPRC-H tagged on either the Bmi1 or the M33 subunit and dPRC1 have similar specific activities and both can block Swi/Snf activity at concentrations lower than one PRC per nucleosome (Fig. 4C). The inhibition of Swi/Snf-stimulated cutting is strongly reduced when hPRC-H is not preincubated with the template (data not shown).
In addition to increasing the access of restriction enzymes to nucleosomal templates in vitro, Swi/Snf has also been shown to alter the topology of the DNA on closed circular plasmids in the presence of topoisomerase I (25). Previously, dPRC1 was shown to block these topological changes induced by Swi/Snf. We tested the effect of hPRC-H in this assay by preincubating the template with hPRC-H for 15 min and then allowing it to react with Swi/Snf. Increasing amounts of hPRC-H blocked the ability of Swi/Snf to form remodeled products (Fig. 4D, lanes 3 to 6). Taken together, the restriction enzyme assay and topological data suggest that hPRC-H, like dPRC1, can inhibit chromatin remodeling by Swi/Snf.
One explanation for the previous data is that hPRC-H nonspecifically restricts the accessibility of the template to both topoisomerases and restriction enzymes. To evaluate this possibility, we tested the effect of hPRC-H on template digestion by micrococcal nuclease (MNase), which cleaves preferentially in the linker region, creating a defined nucleosomal ladder. Incubation of end-labeled 5S array with Swi/Snf has previously been shown to cause randomization of the normally well-spaced MNase digestion pattern of nucleosomes (21) (Fig. 5A, compare lanes 1 to 4 to lanes 5 to 8, respectively). Preincubation of the template with hPRC-H or dPRC1 (15) prevented Swi/Snf from randomizing the array (lanes 9 to 16). In these reaction mixtures, only a slight decrease in MNase sensitivity was observed, suggesting that hPRC-H blocks Swi/Snf-dependent remodeling but not MNase access.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One major difference between the human and drosophila complexes is the presence of multiple homologues of most of the core proteins in the complex. There are two possible explanations for this result. First, there might be single complexes that contain multiple paralogs of every protein. This possibility is supported by the identification, by both immunoblotting and MS, of HPC2 and HPC3 in fractions purified with tagged M33 (MPC1). Alternatively, there may be multiple highly related complexes that contain single PcG homologues. This possibility is supported by the apparent varied stoichiometry (as judged by Coomassie staining intensity) of the PcG proteins in the complex (Fig. 1) and the significant substoichiometry of SCMH1. The latter hypothesis would help explain a function for some of the specificity seen by the different PcG proteins, such as the binding of CtBP preferentially to HPC2 rather then HPC1 (49). These differences in domains may lead to differential targeting of complexes. Multiple highly related complexes are also seen in other chromatin-modifying factors, such as the Swi/Snf family of complexes (34, 52, 60, 66).
PRC composition is likely to be regulated in both a cell type and developmental manner. Contrary to hPRC-H, dPRC1 associates with stoichiometric amounts of Zeste, most members of the TFIID family, and about a half dozen other non-PcG proteins (45). No members of the TFIID family were found in detectable amounts associated with hPRC-H (data not shown), and mammals have no known Zeste homolog. On the basis of the data in Fig. 4, the role these proteins play in blocking chromatin remodeling appears to be minimal under the conditions that have been tested. This is consistent with the hypothesis that these additional proteins may be involved, instead, in targeting of the complex to specific genes, in stabilizing the association of dPRC1 with template, or in providing additional enzymatic activities. The presence of stoichiometric amounts of non-PcG proteins in the dPRC1 complex may be related to the embryonic state of the extract when there is a transition between establishment of a repressed state on target genes and formation of a structure that can maintain that repressed state. In addition, at early stages of development, repression must be maintained during rapid cycles of replication, which might require a particularly robust mechanism. It remains formally possible that the association of the TAF proteins with dPRC1 occurred during extract preparation and does not reflect an association that is mechanistically significant; however, data from in vivo colocalization studies support the notion that these proteins interact in drosophila embryos (8). While there are already data demonstrating changes in the composition of class II PcG complexes during drosophila and human development, further work on the nature and functional consequences of these changes will be important in explaining the developmental role of these complexes. The identification of core subunits in the PRC family of complexes should allow dissection of the roles of other subunits in changing the capabilities of this family of complexes to maintain a repressed state across the lifetime of an organism.
The importance of the core of the PcG complex in its biochemical activity has recently been demonstrated by studies that have shown that a reconstituted core complex containing four drosophila PcG proteins (Pc, Psc, Ph, and dRING1) has many of the same functions as the complete dPRC1 complex (15). The experiments reported here extend these studies in two important ways. First, we show that a complex exists in HeLa cells that is similar to the reconstituted drosophila core complex in that it is primarily made up of the human homologues of the four core dPRC1 proteins. This suggests that this core complex plays a biologically significant role. Second, the observation (Fig. 4 and 5) that hPRC-H and dPRC1 have similar abilities to inhibit chromatin remodeling shows that, in vitro, this function is conserved despite limited sequence conservation between the human and drosophila PcG proteins. This parallels in vivo experiments in which addition of M33 was able to complement a Pc mutation in the fly (31). The observation that hPRC-H and dPRC1 have similar activities therefore supports the argument that these activities are an important aspect of PcG function.
Given the large differences in composition between the human and drosophila complexes and the additional PcG homologues in humans, it is tempting to speculate on the possible role of the differences. While both organisms use the Polycomb proteins to maintain repression of the Hox genes, mammalian PcG proteins also play key roles in other systems, including the immune system (61). The composition of the complex changes across development as the expression pattern of the paralogs varies. This suggests that hPRC may act more dynamically than its drosophila counterpart. We hypothesize that this variation in composition could lead to changes in targeting of the same biochemical mechanism, leading to distinct biological functions.
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ACKNOWLEDGMENTS |
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This work was supported by grants from the National Science Foundation (to S.S.L.), the National Cancer Institute Cancer Center (to P.T.), and the National Institutes of Health (to R.E.K.).
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FOOTNOTES |
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Present address: ProSeed Capital Holdings CVA, Boston, MA 02109.
Present address: Biogen, Inc., Cambridge, MA 02142.
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REFERENCES |
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