1 Centro de Biología Molecular, Universidad Autónoma de Madrid, CSIC-UAM, Campus de Cantoblanco, Madrid 28049, Spain
2 Department of Human Genetics, 5200 Eccles Institute of Human Genetics, University of Utah, Salt Lake City, UT 84112, USA
*Author for correspondence (e-mail: sakonju{at}genetics.utah.edu)
Accepted March 23, 2001
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SUMMARY |
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Key words: MCP silencer, Abd-B gene, Pleiohomeotic, GAF, PcG, trxG, Drosophila melanogaster
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INTRODUCTION |
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Establishment of the activation and repression domains takes place early in embryonic development and depends on the function of the segmentation genes (Qian et al., 1991, 1993; Zhang et al.,1991). Maintenance throughout development involves transition to a different mechanism and a different set of proteins (Qian et al., 1991, 1993; Zhang et al., 1991; Müller and Bienz, 1991; Pirrotta et al., 1995; Kehle et al., 1998). The trithorax group of genes (trxG) is required for the maintenance of the active transcriptional state of the homeotic genes (Kennison, 1995). In contrast, the genes responsible for the maintenance of the repression, or silencing, are included in the Polycomb group (PcG) of genes. To date, thirteen PcG genes have been molecularly characterized. All the characterized PcG genes, with the exception of pleiohomoetic (pho), encode chromatin-associated proteins with motifs characteristic of chromatin-bound proteins or suggestive of protein-protein interactions (Paro and Harte, 1996). Indeed, some pairs of PcG proteins have been shown to interact in vitro (Peterson et al., 1997; Kyba and Brock, 1998) and, in vivo, they appear to be associated with large protein complexes (Franke et al., 1992; Shao et al., 1999; Tie et al., 2001). Two large PcG protein complexes have been described to be present in Drosophila embryonic extracts. One of these complexes, called PRC1, with an estimated molecular mass of greater than 2 MDa, contains at least four PcG proteins: PC, PH, PSC, and SCM (Shao et al., 1999). The other complex, with a molecular mass of approx. 600 kDa, includes at least two PcG proteins, ESC and E(Z) (Tie et al., 2001). The recent cloning of the pho gene has shown that it encodes a zinc finger protein related to the mammalian transcription factor YY1 and that it recognizes specific DNA sequences, making PHO the only PcG protein so far characterized that has specific DNA binding properties. This finding has led to the proposal that PHO protein binds to DNA and recruits the PcG silencing complexes to specific DNA sequences (Brown et al., 1998).
PcG proteins act on cis-regulatory elements of the homeotic genes to recognize target promoters and maintain the silenced state. These silencing elements are known as Polycomb Response Elements, or PREs (Chan et al., 1994). Several PREs from the bithorax complex (Müller and Bienz, 1991; Busturia and Bienz, 1993; Chiang et al., 1995; Hagstrom et al., 1997) as well as from other genes regulated by the PcG proteins (Fauvarque and Dura, 1993; Kassis, 1994, Gindhart and Kaufman, 1995) have been characterized. They are DNA segments of several hundred base pairs that, when fused to a reporter, are able to silence target promoters in cis (Pirrotta, 1997a; Pirrotta, 1997b; Pirrotta, 1998; Pirrotta, 1999). Moreover, they can mediate pairing-sensitive silencing. Identification of key sequences bound by proteins within the characterized PREs is under way. Recently, specific sequences recognized by PHO have been shown to play an essential role in the silencing activity of three PREs (Brown et al., 1998; Fritsch et al., 1999; Shimell et al., 2000). The same studies showed, however, that the PHO recognition sequences by themselves are not sufficient to serve as a PRE, suggesting the existence of additional protein(s) that bind PREs.
Here we describe a detailed analysis of the MCP element from the iab-5 regulatory region of the Abd-B gene. This fragment corresponds to the genomic region deleted in Mcp mutant alleles, which cause Abd-B to be expressed outside its normal domain (Lewis, 1978; Celniker et al., 1989; Sanchez-Herrero, 1991; Karch et al., 1994). Using reporter gene assays, we have previously shown that the 822 bp MCP behaves like a silencer during proliferation of the imaginal discs and that it is required throughout development (Busturia et al., 1997). Additional properties of the MCP822 fragment are: (1) it silences a reporter gene when placed either upstream or downstream of the associated enhancer (Busturia et al., 1997), (2) it functions in both orientations (Busturia et al., 1997), (3) it behaves as a PRE since PcG functions are required for its silencing activity (Busturia et al., 1997), and (4) it is capable of participating in long-distance trans silencing (Muller et al., 1999). In this paper, we define the MCP silencer as a 138 bp minimal element based upon its ability to maintain silencing during imaginal discs development. Within the minimal MCP element, there are four PHO binding sites and two GAGA factor (GAF) binding sites. We show by mutational analyses that both PHO and GAF binding sites are required for the silencing activity of the MCP element in vivo. We also show that silencing of the minimal element is dependent on the functions of pleiohomeotic and the GAF-encoding gene Trithorax-like.
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MATERIALS AND METHODS |
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Transgenic flies were produced as previously described (Bienz et al., 1988). With all MCP constructs, including MCP822, the expression of the reporter gene in approximately one quarter of the transformant lines is completely silenced throughout the body axis, presumably due to position effects (see Busturia et al., 1997). The total number of transformant lines reported in Figs 1 and 3 excludes these lines. A line was classified as silenced if it showed background X-gal staining in less than 15% of the wing and haltere discs and if the derepressed patches were very small. Those classified as derepressed lines usually showed lacZ expression in nearly 100% of the wing and haltere discs examined. A line with less than complete penetrance of derepression was classified as derepressed if the penetrance was high and the derepressed patches were large. To assay for silencing of the reporter constructs in wing and haltere discs, larvae were grown continuously at 25°C in a humidity controlled chamber in uncrowded vials. X-gal staining of discs were done as previously described (Christen and Bienz, 1994).
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Transgene construction, in vitro mutagenesis and deletion analysis
The PBX-MCP-Ubx promoter-lacZ reporter construct has been described previously (Busturia et al., 1997). This reporter is built upon the ry+ Ubxpp-lacZ transformation vector described by Müller and Bienz (Müller and Bienz, 1991). It carries 3.1 kb of the Ubx promoter sequence upstream of the start site (Saari and Bienz, 1987) and includes the Ubx transcribed sequence to the first 7 amino acids of the ORF, where it is fused to the lacZ sequence. The PBX element is the 5.2 kb PBXO1 fragment, also described previously (Castelli-Gair et al., 1992; Christen and Bienz, 1994; Busturia et al., 1997). In all constructs, the MCP822 element or its mutated or deleted versions are inserted between the PBX and the Ubx promoter fragments (Fig. 1A). The 822 bp MCP element is the SalI-XbaI fragment located about 50 kb downstream of the Abd-B transcriptional unit (Zavortink and Sakonju, 1989), and corresponds to residues 1153 to 1976 of the sequence shown in Fig. 4A in the paper by Karch et al. (Karch et al., 1994). The MCP fragment used in our study lacks the XbaI site at residue 1878 and is also missing two bases present in the Karch sequence. The length of the SalI-XbaI fragment is therefore 822 bp long, even though it is designated in our previous publication as MCP725 (Busturia et al., 1997). MCP fragments in many of our constructs are flanked by 34 bp FRT elements (Qian and Cox, 1995).
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To prepare embryonic nuclear extract, Canton-S embryos were collected overnight (0-20 hours) and processed according to the protocol described by Franke et al. (Franke et al., 1992). Total protein concentration in nuclear extracts was 8.5-9.5 mg/ml. For EMSAs, 1-3 µl of nuclear extract was used in the binding reaction that was identical to that described above for PHO binding assays, except that ZnCl2 was not added.
Effects of PcG mutations and testing genetic interactions
When examining the effect of a single mutation, the mutant chromosome usually came from females and the reporter-carrying chromosome from males. In all experiments, balancer chromosomes were marked with larval markers (GFP, Tb, or Ci-lacZ) so that appropriate genotypes could be identified unambiguously. A chromosome carrying both TrlR85 and the P[MCP822, T8] reporter was used for genetic interaction studies. In these tests, TrlR85 P[T8]/TM6, GFP Tb females were crossed to PcG mutant males (phob/Ci-lacZ or Psch27/CyO, GFP). For each experiment, wing and haltere discs from wild-type siblings were isolated and stained as controls. The numbers reported in Table 1 are compilations from at least two separate experiments for each genotype. The number of wing discs examined ranged from 61 to 590, but for most genotypes 100 to 300 discs were examined.
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RESULTS |
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To test if the PHO binding sites are required for silencing activity in vivo, we mutated the PHO consensus sequences from ATGGC to CGTGC, which eliminates binding as seen above, and tested the mutated silencers in an in vivo reporter assay (Müller and Bienz, 1991; Busturia et al., 1997). This assay utilizes a transgene comprising the PBX enhancer and the proximal Ubx promoter fused to the lacZ reporter gene (Fig. 1A, see Materials and Methods). In embryos, the PBX element activates the Ubx promoter in parasegment (ps) 6 and more posterior segments, while repressing the lacZ expression in ps5 and more anterior segments. However, this repression is not maintained during larval development, leading to derepression of the Ubx promoter in wing discs (ps4 and 5) and the anterior compartment of haltere discs (ps5). In contrast, when the MCP silencer is fused to the PBX-Ubx promoter-lacZ construct, repression in ps5 and more anterior segments is maintained throughout development (Busturia et al., 1997). Thus, this reporter transgene provides a system to assay the ability of silencers to maintain silencing established early in a spatially specific manner.
Figs 1 and 2 show the results of the experiments in which the activity of the mutated MCP fragments were assayed in vivo. Mutating four or five PHO binding sites (4MPHO or 5MPHO constructs) resulted in derepression of the Ubx promoter in the wing discs of almost all transformed lines. Within each derepressed line, penetrance of derepression was nearly 100%, with only a few lines showing a mixture of silenced and derepressed lacZ expression in the wing discs. The degree of derepression, or expressivity, varied from line to line. Some lines showed only small patches of derepression, while in others almost entire wing discs expressed lacZ (e.g. compare Fig. 2B and D). We presume some of this variability results from position effects. However, the presence of weakly derepressed lines suggest that PHO binding site mutations do not remove the entire silencing activity of MCP822. We conclude that PHO binding sites are required for full silencing activity by MCP, but silencing could be mediated by sequences other than PHO binding sites (see below).
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Sequences other than PHO binding sites can independently maintain silencing
The presence of low expressivity lines among transformants carrying constructs with PHO binding sites mutated suggested that sequences other than PHO binding sites may contribute to silencing. To distinguish between residual silencing activity in 5MPHO and contributions from position effects, we compared the silencing activity of a 5MPHO insertion line with that of its derivative line (5MPHO) from which 5MPHO had been excised. If the 5MPHO line contained silencing activity, then it should silence more effectively than its derivative
5MPHO line. Since both are inserted at the same genomic location, the difference can be attributed to the MCP sequences outside of the PHO consensus binding sites. To construct
5MPHO lines, we started with relatively weakly derepressed 5MPHO lines. Since the 5MPHO sequences were flanked with FRTs, we excised them by passaging through male germ cells that expressed FLP recombinase (see Materials and Methods). Fig. 2 shows the results from one of the lines studied. Compared to its parental 5MPHO line (Fig. 2D), the derivative
5MPHO line (Fig. 2E) shows a greater degree of derepression in the wing discs.
5MPHO lines derived from two other 5MPHO lines also showed an increased derepression of the reporter in the wing discs (data not shown). These results indicate that there are sequences distinct from PHO binding sites in MCP that can mediate silencing.
Deletion analysis of the MCP element to localize sequences required for silencing
To identify sequences other than PHO binding sites that contribute to silencing activity of the MCP element, we generated a number of deletion constructs and tested their silencing activity in the reporter assay. The constructs we generated and the results of in vivo functional tests are shown in Fig. 3. Deletions of MCP from the proximal (left) end up to residue 538 (constructs MCP14 and MCP7), did not significantly alter silencing activity. However, deleting MCP to residue 573 (constructs MCP1 and MCP2) eliminated its silencing activity (see Fig. 2C for lacZ expression in the imaginal discs). MCP2 retains the MCP sequence between residues 675 and 822 that is absent from MCP1. Since neither construct retains silencing activity, a key sequence for silencing must be located proximal to the cluster of PHO binding sites. This loss of silencing is not simply due to lack of the PHO site at residue 62 since a number of constructs that lack this PHO site (PHO62, MCP14, MCP7) were able to silence effectively. Rather, comparison of MCP7 and MCP1 indicates that the sequence required for silencing is located between residues 538 and 573. From the distal end, MCP can be deleted to residue 675 without significantly reducing the silencing activity (MCP14, MCP7); however, as expected, deleting to residue 273 (MCP12) eliminated its silencing activity. This deletion analysis indicates that the minimal MCP construct that retains robust silencing activity is the 138 bp (MCP7) fragment that spans from residues 538 to 675.
To test whether the minimal MCP construct is functioning as a PRE, we examined two MCP7 transformant lines, P[T3] and P[T9], in animals heterozygous for PcG mutations. These inserts show background derepression of the lacZ reporter in 5% and 10%, respectively, of the wing discs from wild-type larvae. In contrast, P[T3] and P[T9] reporters in heterozygous Pc3 larvae were derepressed in 85% and 88%, respectively, of the wing discs (Table 1). Similarly, P[T3] and P[T9] reporters in pho1 heterozygous larvae showed lacZ derepression in 64% and 58%, respectively, of the wing discs (Table 1). These results confirm that the minimal MCP138 element contains sequences that recruit PcG silencing complexes.
Assaying DNA binding activities within the minimal MCP silencer element
To identify the essential sequences located outside the PHO cluster region, we synthesized two pairs of oligonucleotides, 37/38 and 39/40, covering the sequences between residues 543 and 607 and tested them in EMSAs using Drosophila embryonic extracts (see Fig. 7 for location of oligos). With oligo 37/38, three shifted bands were observed (Fig. 4, lane 1, arrows). To localize the region of the oligo responsible for the shift, we replaced three subregions of the oligo with runs of T residues (Fig. 4 top) and used them as unlabeled competitors in the bandshift assay. The top shifted band (arrow) is competed away by oligos 37/38B and 37/38C (Fig. 4, lanes 5 and 6). This indicates that the recognition sequence for the protein(s) bound in the top band is in the left third of the oligo. The lower pair of bands (connected arrows) are competed away by oligos 37/38A and 37/38C (Fig. 4, lanes 4 and 6). This indicates that the middle third of the oligo contains the recognition sequence for the protein(s) bound in the lower two bands. Taken together, this analysis suggests that a protein(s) is bound to the left third, and a different protein is bound to the middle third of the oligo, while no protein is bound to the right third. Using a similar logic with overlapping oligos, we determined that a protein binds to a sequence around residues 582-592. Oligo 39/40 band-shifts in EMSA using an embryonic extract that lacks PHO activity, but the overlapping oligo PHO604 does not (data not shown). These analyses suggest that, in addition to PHO proteins, at least three more proteins bind to the 138 bp minimal MCP element. The proposed protein interaction regions are indicated in Fig. 7.
GAGA binding sites are required for MCP silencing activity
Within the two subregions of oligo pair 37/38 exhibiting protein binding activity there are two potential recognition sequences for GAGA factor (GAF), GAGAG and GAGA at residues 548 and 558, respectively (Fig. 7). Most GAF binding sites are composed of longer GA repeats, but as little as three bases (GAG) has been recognized by GAF in vitro (Wilkins and Lis, 1998). To determine if putative GAGA binding sites at residue 548 and 558 are bona fide GAGA binding sites, we tested the ability of in vitro-synthesized GAF to bind to the oligonucleotide 55/56 containing sequence between residues 533 and 572 (Fig. 7). Oligo 55/56 is shifted when incubated with in vitro-synthesized GAF (Fig. 5, lane 2). The two shifted bands (arrows) are likely to correspond to complexes of a full-length GAF and a partial GAF that are synthesized in our in vitro translation system (data not shown). The shifted bands are competed by excess unlabelled competitors with the same sequence as the labeled oligo (lane 6) but not by an unrelated sequence oligo (lane 10). To determine if both the GAGAG548 and GAGA558 sites can bind GAF, we synthesized oligos with mutations in the putative GAF binding sequence at either 548 or 558 (55/56M1 or M2 in Fig. 7), and tested them in the EMSA using in vitro-synthesized GAF. A small amount of labeled oligo 55/56M1, carrying a mutation at GAGAG548, still shifts (Fig. 5, lane 3), suggesting that GAGA558 weakly binds GAF. When GAGA558 is mutated (55/56 M2), mobility shift is robust (Fig. 5, lane 4), suggesting a preferential binding of GAF to the GAGAG548 site. An oligonucleotide with both GAGAG548 and GAGA558 mutated (55/56 M3) does not bind GAF (lane 5). Results consistent with this were obtained in separate experiments in which the mutated oligos were used as unlabelled competitors. The 55/56M1 oligo moderately competes (lane 7) while the 55/56M2 oligo does so strongly (lane 8), but the 55/56M3 oligo with both GAGA sites mutated competes as poorly as the nonspecific competitor (lane 9). These results indicate that both of the potential GAGA binding sites can be recognized by GAF, but that the GAGAG548 site does so much more strongly than the GAGA558 site.
To test the requirement for GAF binding sites in the minimal MCP element (MCP7) in vivo, we mutated both GAGA binding sequences (mutated sequences are shown in Fig. 6) and tested the ability of the mutant construct (MCP7*) to maintain silencing. We find that, unlike the parental MCP7 construct, this mutant construct MCP7* is unable to maintain silencing in most transformed lines (Fig. 3). In these derepressed lines, the lacZ reporter was expressed in nearly the entire wing and haltere discs (Fig. 6). Therefore, in the context of MCP7, GAF binding sites appear to be absolutely required for silencing. Stated another way, this result indicates that PHO binding sites by themselves possess little silencing activity. The same conclusion can be extended to the two other proteins that bind to the MCP7 (Fig. 7). We have not directly tested their participation in silencing, but we can at least conclude that they do not have any silencing function on their own.
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Interactions of Trl and PcG genes
Trl is usually considered to be a member of the trxG genes (Paro and Harte 1996). If GAF is required for silencing, however, Trl mutations might be expected to enhance the phenotypes caused by PcG mutations. Strutt et al. (Strutt et al., 1997) have noted that male flies doubly heterozygous for Pc and Trl mutant alleles show an increased appearance of extra sex combs. In contrast, Kehle et al. (Kehle et al., 1998) did not detect genetic interaction between Pc and Trl mutations. To determine if Pc and Trl genetically interact, we measured the penetrance of homeotic transformations in doubly heterozygous animals (Table 2). In Pc3/TrlR85 double heterozygotes, 45% of males show the extra sex combs phenotype (on either the second, third or both legs). Moreover, 25% of the females show the antenna-to-leg transformation phenotype associated with the PcG mutations. These frequencies are significant increases in penetrance since only 7% of Pc3/+ and no TrlR85/+ males show the extra sex combs phenotype, and 8% of Pc3/+ and no TrlR85/+ females show the antenna-to-leg transformation. Similar results were obtained with another mutant allele of Trl, Trl13C (data not shown). We have also examined genetic interactions between pho and Trl alleles (Table 2). In these experiments, 11% of TrlR85/+; pho1/+ males show the extra sex comb phenotype. This is a modest but still significant interaction as neither pho1/+ nor TrlR85/+ males show extra sex combs. These results indicate Trl and PcG genes interact to silence homeotic genes. It is not clear why Kehle et al. (Kehle et al., 1998) did not observe genetic interactions. We note, however, that Kehle et al. used different alleles from those used by us or by Strutt et al. and measured expressivity, rather than penetrance, of the extra sex comb phenotype.
We have also tested if Trl and PcG genes interact to silence the endogenous Ubx gene and MCP reporter constructs in double heterozygous animals. Compared to that seen for the extra sex comb phenotype, however, genetic interaction between Trl and PcG genes for the Ubx gene or the reporters was not detectable. The endogenous Ubx gene was not derepressed in the third instar wing imaginal discs of Pc3/TrlR85 larvae any more frequently than in the Pc3/+ wing imaginal discs (Table 2). Moreover, there was no difference in the size of UBX-expressing clones in Pc3/TrlR85 and Pc3/+ animals (data not shown). We also examined the effect of Trl and two PcG mutations, Psc and pho, on silencing of the MCP822 P[T8] reporter. The combination of Psch27 and TrlR85 mutations does not induce derepression any more frequently than the combined effect of Psch27 and TrlR85 individual mutations (Table 1). Similarly, the combination of TrlR85 and phob did not show any increase over phob/+ animals. This apparent lack of synergy between Trl and PcG mutations, when looking at the expression of UBX and the reporter genes in the wing discs, may simply mean that these heterozygous conditions do not sensitize animals sufficiently to reveal genetic interactions. It may also reflect the silencing mechanism at this promoter. A possible explanation is presented in the Discussion.
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DISCUSSION |
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Requirement of PHO for maintenance of silencing
We have shown that PHO binding sites within MCP are necessary for maintenance of silencing. We identified five sites within the 822 bp MCP showing homology to the PHO binding consensus sequence (Brown et al., 1998; Mihaly et al., 1998). Four of the five sites bound to in vitro-synthesized PHO protein. Our mutational analysis of these sites indicates that they are required for silencing. However, not all five sites are needed. In the context of the 822 bp MCP sequence, mutated fragments with four functional PHO binding sites were capable of maintaining silencing. That these PHO consensus sites are indeed recognized by PHO in vivo is supported by the effect of pho mutations on the transgene expression: the PBX-MCP-Ubxp-lacZ transgene is partially derepressed in animals heterozygous for pho1 or phob mutations. At present, the molecular mechanism of how PHO contributes to silencing is unknown. PHO exhibits partial sequence similarity to the mammalian transcription factor YY1 and binds to DNA in a sequence-specific manner (Brown et al., 1998). Based on this, it was suggested that PHO acts as a recruiter of the PcG silencing complexes to DNA (Brown et al., 1998). However, it has been shown recently that a LexA-PHO fusion protein, when bound to LexA binding sites, is unable to silence the expression of a reporter gene, while a LexA-Polycomb fusion protein is able to do so in the same system (Poux et al., 2001). Furthermore, PHO is unable to recruit PcG proteins, as observed in immunoprecipitation experiments. These results suggest that PHO cannot by itself recruit silencing complexes. Our finding that MCP constructs with four PHO binding sites (MCP1 and MCP7*) show little silencing activity supports their conclusion that PHO protein cannot recruit silencing complexes by itself. How then does PHO contribute to silencing? As will be discussed more fully below, the contribution of PHO to silencing may require an additional factor. Only in the presence of binding sites for this additional factor can PHOs contribution be revealed. Under this condition, it is still possible that PHO may recruit PcG complexes. The inability of Lex-PHO protein to recruit PcG complexes may also be explained by the absence of a required adaptor protein in the extracts. YY1 has been shown to interact with a newly identified zinc-finger protein called RYBP, which also interacts with the mammalian PcG proteins with ring-finger domains (Garcia et al., 1999). RYBP also interacts with Drosophila PHO protein in vitro (Garcia et al., 1999). There is at least one candidate for a RYBP homolog in the Drosophila genome (our unpublished observation). The Drosophila counterpart of RYBP may also serve as an adaptor protein between PHO and PcG ring-finger domain proteins such as PSC and SU(Z)2.
Silencing function of GAGA binding sites in vivo
We have demonstrated that GAF binding sites are required for efficient silencing by MCP in vivo. We do not know if both sites are required in vivo. In vitro binding of GAF to the GAGAG548 sequence is much stronger than to the GAGA558 sequence (Fig. 5). In addition, the oligonucleotide containing only the GAGA558 sequence does not detectably compete for the nuclear extract factor (presumed GAF) that binds to the oligonucleotide containing GAGAG548 (Fig. 4). Therefore, occupancy of the stronger site may be sufficient to contribute to the maintenance of silencing.
We have not directly demonstrated that it is GAF that binds to these sites in vivo. It remains possible that another GAGA binding protein recognizes these sites and contributes to silencing. However, several lines of evidence suggest the involvement of GAF in silencing. First, PC protein and GAF were found to be co-localized on polytene chromosomes in salivary glands (Strutt et al., 1997). Second, the combination of Pc and Trl mutant alleles enhances the extra sexcomb phenotype and antenna-to-leg transformation caused by Pc mutations (our results, Table 2). Third, Trl mutations can compromise silencing mediated by a Fab-7 PRE fragment (Hagstrom et al., 1997). Finally, the in vitro formation of complexes between PcG proteins and the bxd PRE is dependent on consensus recognition sequences for GAF, and GAF in embryonic extracts co-immunoprecipitates with PC protein (Horard et al., 2000). These results make GAF a good candidate for the protein that binds to the GAGA sites within MCP.
Contribution of GAF and PHO to the maintenance of silencing
How does GAF or perhaps another GAGA binding protein contribute to the silencing by MCP, and what is its relationship to the PHO protein function? We suggest two models to explain their relationship that leads to strong silencing. These models are based on the following observations. First, PHO binding sites by themselves show little silencing activity (MCP1 and MCP7* constructs). Second, GAF or some other protein that binds to MCP can weakly recruit silencing complexes in the absence of PHO binding (5MPHO construct). Third, when present together, GAF and PHO binding sites exhibit robust silencing activity (MCP7 construct). In the first model, GAF and PHO bind to the MCP silencer in a sequential order. One version would be that GAF binding is absolutely required for binding or activity of PHO. GAF may open up chromatin at MCP, allowing binding of PHO. Upon binding, PHO may recruit PcG silencing complexes, although there is still little evidence that this happens. GAF has been shown to induce DNase I hypersensitive sites, or nucleosome-free regions (Lu et al., 1993), and this may create a prerequisite condition for PHO to bind to its recognition sites. There is indeed a DNase hypersensitive region associated with MCP that includes the location of the GAF binding site (Karch et al., 1994).
In a second version of the model, PHO acts as a facilitator of GAF binding by creating some pre-condition, perhaps by bending DNA as YY1 does (Natesan and Gilman 1993). Since PHO binding sites are not absolutely required for MCP silencing activity, GAF presumably can bind weakly to MCP in the absence of PHO. Enhanced binding of GAF leads to increased recruitment of silencing complexes. GAF bound to MCP may recruit PcG silencing complexes by directly interacting with PC or other members of PcG complexes (Horard et al., 2000). Alternatively, GAF could first recruit SIN3 histone deacetylation complexes through its interaction with SAP18 (Espinas et al., 2000), which then might generate a chromatin state favorable for PcG complex binding. Whichever version of the model is correct, the important feature of the model is the sequential recruitment of DNA binding proteins, GAF and PHO, to MCP. Binding of one protein creates a condition favorable to the binding of a second protein, eventually leading to the recruitment of PcG complexes. Note that the requirement of GAF and PHO proteins applies to MCP silencing, but not necessarily to all PREs. Other PREs may use other combinations of proteins. Our model is analogous to Swi5 protein binding to the yeast HO promoter and recruiting the chromatin remodeling complex Swi/Snf. Swi/Snf in turn recruits the histone acetylase complex SAGA, eventually leading to the binding of the transcription factor SBF to the HO promoter (Cosma et al., 1999). In such a sequential recruitment model, compromising one step in the sequence may become rate limitating so that combining two mutations that disable two different steps may not necessarily lead to synergistic effects. This may explain why we did not observe synergistic effects in our assay system when Trl and PcG mutations were combined.
In the second model, GAF and PHO bind to MCP independently of each other. Each protein may induce a unique chromatin modification that, together, can have a positive synergistic effect on the recruitment of PcG silencing complexes. Multi-layered recruitment of silencing complexes may also explain why recruiting LexA-PHO or LexA-GAF fusion protein individually to a LexA binding site failed to assemble PcG silencing complexes (Poux et al., 2000).
A dual role of Trl in regulating homeotic gene expression
Trl is thought to be required for the maintenance of the activation of homeotic genes. Consistent with this, Trl mutations show phenotypes indicative of the loss of activity of homeotic genes and enhance hypomorphic loss-of-function phenotypes (Farkas et al., 1994). Nevertheless, previous studies (Strutt et al., 1997; Hagstrom et al., 1997; Horard et al., 2000) and our work presented here clearly implicate Trl in the repression of homeotic gene transcription. Trl therefore should be classified as belonging to both the trxG and PcG groups. Trl would not be the first gene to be classified as belonging to both positive and negative regulatory groups. LaJeunesse and Shearn (LaJeunesse and Shearn, 1995) found that a mutant allele of Enhancer of zeste (E(z)) caused both derepression and loss of expression of homeotic genes. A recent screen for second site non-complementers of an ash1 mutation, a trxG gene mutation, found that mutations in six PcG genes E(Pc), Psc, Su(z)2, Asx, E(z) and Scm enhanced, rather than suppressed, the ash1 mutation (Gildea et al., 2000). Thus, there appeared to be a group of genes that act in both activation and silencing of homeotic genes. Gildea et al. (Gildea et al., 2000) have called these genes the ETP (enhancer of trithorax and Polycomb mutations) group. Based on genetic and molecular evidence, we suggest that Trl should be included in the ETP group. The mechanism as to how the ETP proteins contribute to both activation and silencing is unclear. Perhaps ETP proteins are core chromatin proteins needed to form both activation and repression complexes, and depending on the cellular transcriptional state, either one or the other is formed.
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ACKNOWLEDGMENTS |
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