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Genetics, Vol. 176, 2099-2108, August 2007, Copyright © 2007
doi:10.1534/genetics.107.075739
,1
* EMBL, Gene Expression Programme, 69117 Heidelberg, Germany and Max-Planck Institute for Developmental Biology, 72076 Tübingen, Germany
1 Corresponding author: EMBL, Gene Expression Programme, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
E-mail: juerg.mueller{at}embl.de
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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To date, 17 different loci in Drosophila are classified as PcG genes because of HOX misexpression phenotypes. Specifically, mutations in Pc, esc, Sex combs on midleg (Scm), Sex combs extra/Ring (Sce/Ring), Posterior sex combs (Psc), Enhancer of zeste [E(z)], Suppressor of zeste 12 (Su(z)12), Polycomb-like (Pcl), super sex combs (sxc), Additional sex combs (Asx), pleiohomeotic (pho), and dSfmbt have all been shown to cause misexpression of HOX genes in Drosophila embryos or larvae (BEACHY et al. 1985; INGHAM 1985; STRUHL and AKAM 1985; STRUHL and WHITE 1985; WEDEEN et al. 1986; JONES and GELBART 1990; MCKEON and BROCK 1991; PATTATUCCI and KAUFMANN 1991; SIMON et al. 1992; FRITSCH et al. 1999; BEUCHLE et al. 2001; BIRVE et al. 2001; KLYMENKO et al. 2006). It is important to note that in the case of some PcG genes, the Drosophila genome contains two loci that encode closely related proteins that function in a redundant fashion. These are the gene pairs polyhomeotic-proximal (ph-p) and polyhomeotic-distal (ph-d), Psc and Suppressor of zeste 2 [Su(z)2], pho and pho-like (phol), esc and esc-like (escl). In the case of esc, ph-p, ph-d, or phol, single mutants are even viable and they show only very mild or no misexpression of HOX genes but, importantly, in double mutant animals that lack the function of both gene pair members, the PcG phenotype is usually much more severe than in either single mutant (DURA et al. 1987; BROWN et al. 2003; WANG et al. 2006).
Biochemical studies on PcG proteins revealed that they exist in distinct multiprotein complexes that contain two or more different PcG proteins. To date, three different PcG protein complexes have been purified from Drosophila: PhoRC, PRC1, and PRC2. PhoRC contains Pho and dSfmbt (KLYMENKO et al. 2006); PRC1 contains Psc, Pc, Ph, Sce/Ring, and Scm (SHAO et al. 1999), and PRC2 contains Esc, E(z), and Su(z)12 (CZERMIN et al. 2002; MÜLLER et al. 2002). These PcG protein complexes possess specific enzymatic and/or chromatin-binding activities by which they modify and interact with the chromatin of HOX and other target genes (reviewed in MÜLLER and KASSIS 2006;; SCHUETTENGRUBER et al. 2007; SCHWARTZ and PIRROTTA 2007). Nevertheless, there are also PcG genes, e.g., Asx or Su(z)2, whose products are not known to be components of any of these complexes. These proteins may exist as individual molecules in the cell, but it is also possible that they are part of other protein complexes that contain additional, as yet unidentified PcG proteins.
Of the Drosophila PcG genes listed above, some genes (e.g., Pc) were identified because heterozygous adults show subtle homeotic phenotypes (e.g., LEWIS 1947). A number of PcG genes were identified in the classic screens for embryonic lethal mutations that cause patterning defects in the embryonic epidermis (NÜSSLEIN-VOLHARD and WIESCHAUS 1980; JÜRGENS 1985). However, only some PcG loci could be identified by this approach because all known PcG genes are expressed in the female germ line and maternally deposited wild-type protein often rescues homozygous mutant embryos to a considerable extent. For example, homozygous E(z), Su(z)12, or dSfmbt mutants survive into the larval stages, and sxc homozygotes even reach the pharate adult stage (INGHAM 1984; JONES and GELBART 1990; BIRVE et al. 2001; KLYMENKO et al. 2006). More than 20 years ago, JÜRGENS (1985) attempted to estimate the total number of PcG genes in Drosophila using the following assay: embryos that are double homozygous for mutations in two different PcG genes typically show strongly enhanced homeotic transformations compared to the single mutants; the phenotype of such embryos is often similar to the null phenotype of the corresponding single mutants lacking both maternal and zygotic gene function. Jürgens used this striking property and generated embryos that were double homozygous for a particular PcG mutation and large chromosomal deficiencies (JÜRGENS 1985). From these tests, Jürgens estimated that the total number of PcG genes in the Drosophila genome would be in the range of 30–40 genes (JÜRGENS 1985). Although this number is frequently cited (e.g., LANDECKER et al. 1994; YAMAMOTO et al. 1997), only 4 new PcG genes have been identified over the past 2 decades. Among those, mutations in Su(z)12 (BIRVE et al. 2001) and dSfmbt (KLYMENKO et al. 2006) show strong classic PcG phenotypes, whereas cramped mutants show mild homeotic transformations in pharate adults (YAMAMOTO et al. 1997). multi sex combs (mxc), finally, encodes the La protein, which is essential for cell viability; however, mild homeotic phenotypes have been observed in hypomorphic mxc adult flies (SANTAMARÍA and RANDSHOLT 1995; SAGET et al. 1998). Together, these observations indicate that several PcG genes have remained unidentified, which prompted us to perform a genomewide systematic genetic screen for novel PcG mutants.
For this screen we made use of the observation that almost all PcG genes are required throughout embryonic and larval development to maintain repression of HOX genes. In particular, clonal analyses had shown that removal of PcG gene function in imaginal discs of developing larvae results in the misexpression of HOX genes (BEUCHLE et al. 2001). Moreover, if animals with such PcG mutant clones are allowed to develop into adults, HOX misexpression manifests itself in characteristic homeotic transformations in the differentiated epidermis (STRUHL 1981; DUNCAN 1982; INGHAM 1984; BUSTURIA and MORATA 1988). In this study, we used this property as the basis for a genetic screen in clones in the adult epidermis.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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EMS mutagenesis and screen:
Isogenic males of the genotypes described above were mutagenized with 25 mM ethyl methanesulphonate (EMS) and mated to females of the appropriate tester strains described above. The F1 progeny was reared at 18°, and F1 adults were screened for appearance of the PcG syndrome. F1 candidate mutants were isolated and back-crossed to the tester strain. The F2 progeny was again reared at 18°, and F2 adults that showed the PcG syndrome were isolated to establish appropriately balanced stocks. The crossing scheme for the screen on chromosome 3 is depicted in Figure 1.
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Complementation analysis:
After mapping of class I and class II mutants to individual chromosome arms, we tested whether the mutations were alleles of already known PcG genes. The following PcG mutations were used for this analysis: Su(z)21.b8, Psce24, PclD5, Asx3 (also known as AsxXT129), E(z)63, Su(z)124, Pc15 (also known as PcXT109), ScmD1, and Sce1. Class I and II mutants that were viable in trans with these mutations were then crossed to each other and complementation groups were established. In all crosses performed in this study, at least 100 F1 animals were scored in the case of noncomplementation.
Mapping of siren1 and siren9:
Meiotic mapping with a ru h th st cu sr e ca chromosome revealed that siren132E16, siren133D19, and siren932A24 are located between the th marker at 72D1 and FRT2A at 79D–F. Subsequent complementation tests were carried out with the following deficiencies in the 72D–79D interval on 3L: W10, Cat, kto2, XS572, ri-79c, ri-XT1, Pc-101, Pc-2q, ED219, ED220, ED4606, ED223, ED224, ED225, ED4782, ED4786, ED4789, ED4799, ED228, ED229, ED4858, ED230, ED231, A13, A16, BSC20, XS705, 25-21, kto22, XS917, BSC2, and XS411.
Both siren132E16 and siren133D19 failed to complement ED228, ED229, XS572, XS705, 25-21, kto22, XS917, BSC2, and XS411, defining a chromosomal interval that contains the CG32221, kto, and Paps genes. Complementation tests showed that siren132E16, siren133D19, and siren133AC9 all fail to complement both kto1 and kto12. We therefore renamed all siren1 alleles as kto32E16, kto33D19, kto33AC9, kto144, kto1251, and kto33AC20, respectively (see Table 1).
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Staining procedures and preparation of embryonic cuticles:
Imaginal discs were stained as described (BEUCHLE et al. 2001) using antibodies against Ubx, Scr, or Kto/Med12. Cuticle preparations were done following standard protocols.
Screen for new calypso alleles:
Males of the same isogenized line that was used for the screen on the second chromosome were mutagenized with 30 mM EMS and crossed to y w; wg/SM6b females. A total of 2500 males from the offspring bearing the mutagenized chromosome over SM6b were tested individually for viability of their mutagenized chromosome over calypso1. One mutant was recovered that failed to complement calypso1 and showed PcG syndrome and Ubx derepression after clone induction in the wing disc. This mutant was named calypso2.
Generation of calypso mutant germ-line clones:
hs-flp slbo-LacZ/w; FRT40 FRT42D y+ calypso2/FRT42D ovoD1 females were heat-shocked at third instar larval or pupal stage and subsequently crossed to w; FRT40 FRT42D y+ calypso2/CyO Ubi-GFP males. calypso2 mutant embryos were distinguished by absence of GFP signal. hs-flp slbo-LacZ; FRT42D ovoD1/CyO line was generated and kindly provided by Hsin-Ho Sung.
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RESULTS AND DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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We then tested each of these candidate PcG mutants for misexpression of the HOX gene Ubx. Specifically, we used the vgBE-Gal4 UAS-flp combination to induce clones of homozygous mutant cells in the wing imaginal disc and stained these discs with an antibody against the Ubx protein. Sixty-two mutants on chromosome 3, 20 mutants on chromosome 2, but none of the mutants on chromosome X showed misexpression of Ubx in the wing disc. Thus, a total of 82 mutants isolated in our screen showed a bona fide PcG phenotype.
The newly identified mutants can be grouped into two classes. Class I mutants show strong and widespread misexpression of Ubx in most clones in the pouch of the wing disc (Figure 3). Misexpression at high levels in the pouch and at lower levels in the notum and hinge region is observed in many of the classic PcG mutants (BEUCHLE et al. 2001). High-level misexpression of Ubx in the wing pouch likely reflects presence of (unidentified) transcription factors or activating signaling pathways that are localized in this area of the disc and promote transcriptional activation of Ubx. In contrast to class I mutants, class II mutants show misexpression of Ubx only in a small subset of clones in the pouch, and the levels of Ubx protein in these mutant clones are typically lower than in class I mutant clones (Figure 3). Interestingly, often only small clusters of cells within a clone show misexpression of Ubx (Figure 3). This suggests that repression is lost in a stochastic manner in the mutant cells within a clone and that the derepressed state is then maintained in a clonal fashion, similar to what is observed in the case of position-effect variegation (EBERT et al. 2006). The basis for this stochastic loss of repression is not known and will require further investigation. Taken together, these Ubx misexpression phenotypes correlated well with the severity of the PcG syndrome in clones in adult flies: class I mutants consistently showed a more extreme PcG syndrome than class II mutants. However, it should also be noted that the less severe PcG phenotype in class II mutants cannot simply be attributed to defects in clone growth; clones of class I and class II mutant cells show comparable growth rates (see Figure 3 for some examples).
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In the following we shall first briefly discuss some general aspects of the screen and shall then focus on the in-depth characterization of two class I and two class II mutants.
Identification of nonredundant PcG genes by screening for loss-of-function phenotypes:
The use of the FLP–FRT system allowed us to screen for PcG mutants on all five major chromosome arms X, 2L, 2R, 3L, and 3R, provided that the mutated genes were located distally to the FRT cassette on these chromosomes. We thus isolated mutations in the known PcG loci Pcl, Asx, Psc, Pc, Su(z)12, E(z), Scm, and Sce but also in previously uncharacterized genes. The inability to isolate alleles in other known PcG genes was either because of simple technical reasons or because of redundancy between two closely related PcG gene products. In particular, we did not isolate mutations in pho because the lack of suitable FRT sites on the fourth chromosome did not allow screening for mutants on this chromosome. Similarly, the sxc gene at 41C on the right arm of chromosome 2 (INGHAM 1984) is located between the centromere and the FRT cassette in 42D, and this precluded the isolation of sxc mutants because no mutant clones could be generated. In the case of the gene pairs esc and esc-like, ph-p and ph-d, Psc and Su(z)2, or pho-like and pho, we did not expect to isolate mutations because the two proteins of each of these gene pairs function in a redundant manner in imaginal discs (DURA et al. 1987; BEUCHLE et al. 2001; BROWN et al. 2003; WANG et al. 2006). Given that Psc null mutant clones show no misexpression of Ubx (BEUCHLE et al. 2001), it was therefore surprising that three class II mutants turned out to be alleles of Psc (Table 1). However, previous studies showed that certain mutant forms of Psc protein integrate into PRC1 in vitro but function in a dominant-negative fashion in vivo (KING et al. 2005). It is possible that our newly identified Psc alleles encode such aberrant forms of Psc protein that might, for example, compete with the redundantly acting Su(z)2 protein for complex formation. Finally, we note that we did not recover any mutant alleles of dSfmbt, even though dSfmbt null mutant clones in imaginal discs show widespread misexpression of HOX genes (KLYMENKO et al. 2006). However, when we tested dSfmbt mutants in our screen assay, i.e., using the vgBE-Gal4 UAS-flp combination for clone induction, we found that the animals fail to hatch from the pupal case. It is therefore possible that we also failed to isolate mutations in uncharacterized PcG genes because of impaired survival.
Taken together, the screening strategy used here allowed us to isolate mutations in most of the nonredundant known PcG loci and, importantly, we also isolated mutations in 10 genes that were previously not known to be required for PcG-mediated repression.
Calypso, a novel Drosophila PcG gene:
One class I mutation on chromosome 2 complemented mutations in any of the known PcG genes on chromosome 2, suggesting that this mutation affects an uncharacterized PcG gene that is strictly required for repression of HOX genes. We named this gene calypso and the identified mutant allele calypso1 (Table 1). calypso1 mutant clones in imaginal discs show widespread misexpression of the HOX genes Ubx and Sex combs reduced (Scr) (Figure 4). To isolate additional calypso alleles, we performed an EMS mutagenesis in which we isolated calypso2 as a mutation that fails to complement the lethality of calypso1 (see MATERIALS AND METHODS). Clones of calypso2 mutant cells in imaginal discs show the same widespread misexpresssion of HOX genes as calypso1 mutant clones (Figure 4), providing further evidence that calypso is a bona fide PcG gene in Drosophila.
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Asx is essential for PcG repression in larvae:
Asx3 (also called AsxXT129) is considered to be a null mutation in Asx (SOTO et al. 1995). We previously reported that clones of Asx3 homozygous cells in imaginal discs show only very mild misexpression of Ubx and grow poorly (BEUCHLE et al. 2001). In the screen reported here, we identified six different Asx alleles as class I mutants that show severe misexpression of Ubx in clones of homozygous cells in the wing disc (Figure 3 and data not shown). None of these new Asx alleles show the growth defects observed in Asx3 mutant clones (Figure 3 and data not shown). One possible explanation for these differences could be that the chromosome carrying the Asx3 allele contains a second-site mutation that impedes cell growth/proliferation and that this phenotype masks the Polycomb phenotype in Asx3 mutant clones. Alternatively, Asx may have a function in cell growth or proliferation in addition to its role in PcG repression, and the Asx alleles that we isolated in our screen may encode proteins that have impaired PcG repressor activity but retain the function needed for cell growth or proliferation. However, the fact that we isolated six independent Asx alleles that cause a strong PcG phenotype with no proliferation defects strongly supports the first explanation. Asx thus appears to be a classic PcG gene that is required for the long-term silencing of HOX genes in embryos and larvae.
Kto/Med12 and Skd/Med13 are required for Ubx repression:
To gain insight into the role of class II mutants in PcG repression, we mapped the siren1 and siren9 mutants by meiotic recombination, deficiency mapping, and complementation tests with candidate mutations. These experiments revealed that siren1 is allelic to kohtalo (kto) and siren9 is allelic to skuld (skd) (see MATERIALS AND METHODS). We therefore renamed the siren1 and siren9 alleles as kto and skd alleles, respectively (Table 1, Figure 3). The finding that we isolated mutations in kto and skd because of their Ubx misexpression phenotype (Figures 2 and 3) was surprising in several respects. kto and skd encode Med12 and Med13, both components of the very same protein complex, the Med12·Med13·Cdk8·CycC submodule of the Mediator complex (LEWIS and REINBERG 2003; BOURBON et al. 2004). The Med12·Med13·Cdk8·CycC subcomplex appears to function as a repressor module within Mediator; biochemical preparations of Mediator that lack it can stimulate transcription in vitro, whereas preparations including it cannot (BORGGREFE et al. 2002; TAATJES et al. 2002; BJÖRKLUND and GUSTAFSSON 2005). The repressive role of Med12 and Med13 is further supported by genetic studies in yeast, flies, and nematodes, where mutations in these Mediator components were identified in a variety of genetic screens for transcriptional repressors (CARLSON 1997; TREISMAN 2001; JANODY et al. 2004; YODA et al. 2005). Interestingly, clonal analyses in the eye/antenna disc showed that Kto/Med12 and Skd/Med13 are required for the repression or downregulation of dachshund (dac), eyeless (ey), and decapentaplegic (dpp) (TREISMAN 2001; JANODY et al. 2004; LONCLE et al. 2007). This is intriguing because these three genes have been identified as target genes to which PcG proteins are bound in tissue culture cells (SCHWARTZ et al. 2006). Puzzlingly, mutations in kto and skd were, however, also identified as dominant suppressors of the extra sex combs phenotype that is observed in Pc heterozygotes and, because of this genetic interaction, these two genes were originally classified as trxG loci (KENNISON and TAMKUN 1988). The notion that mutations in kto and skd suppress traits of the PcG phenotype in a dominant genetic interaction assay but cause misexpression of certain PcG target genes in the recessive condition posed a conundrum. To address this issue, we performed a number of genetic tests. First, we asked whether Ubx misexpression in kto and skd mutant clones (Figure 3) was a specific feature of the kto and skd alleles that we had isolated in our screen or whether it was also observed in clones homozygous for ktoT241 or skdT606, two null alleles that were previously isolated in a different screen (TREISMAN 2001; JANODY et al. 2004). We found that ktoT241 or skdT606 mutant clones in the wing imaginal disc also show misexpression of Ubx (Figure 5), demonstrating that this is a genuine kto and skd loss-of-function phenotype. Moreover, ktoT241 skdT606 double-mutant clones show Ubx misexpression comparable to kto or skd single-mutant clones (Figure 5C), consistent with the observation that other phenotypes caused by the removal of either kto or skd are also not more severe in kto skd double mutants (JANODY et al. 2003). Second, we stained wing discs with clones homozygous for any of our six newly isolated kto alleles with antibodies against Kto/Med12. No Kto/Med12 epitope was detected in clones that were homozygyous for any of these alleles, indicating that none of the six alleles produces a stable full-length Kto/Med12 protein (Figure 5, F and G, and data not shown). No conclusive results were obtained with an antibody against Skd/Med13; this antibody gave uniform staining on wing discs with skdT606 mutant clones, even though the encoded SkdT606 protein is predicted to lack the corresponding epitope (TREISMAN 2001). Finally, we asked whether mutants lacking the CycC or Cdk8 subunits (LONCLE et al. 2007) of the Med12·Med13·Cdk8·CycC mediator submodule would also show PcG phenotypes. We were unable to detect misexpression of Ubx protein in imaginal disc clones that were homozygous for CycCY5 or Cdk8K185, respectively (data not shown). This suggests that Kto/Med12 and Skd/Med13 do not mediate HOX gene repression through the kinase activity of Cdk8. Kto/Med12 and Skd/Med13 may thus have functions that are not linked to their association with CycC and Cdk8, an idea that is supported by the recent finding that several other phenotypes common to kto and skd mutants are not shared by CycC and Cdk8 mutants (LONCLE et al. 2007).
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Concluding remarks:
In this study, we report the results of a genomewide genetic screen for mutations that cause loss of PcG repression in Drosophila. The screening strategy resulted in the isolation of mutant alleles in most of the known nonredundant PcG loci and identified a number of genes that were previously not known to play a role in Polycomb repression. Among these, calypso stands out as a novel PcG gene that is critically required for repression of HOX genes. The observation that calypso is the only new PcG gene with a strong phenotype that we identified, and the fact that we isolated multiple alleles in most of the nonredundant PcG genes, suggest that the majority of PcG genes with strong phenotypes in Drosophila have been identified. Nevertheless, we also isolated multiple alleles for nine different "class II" genes that show milder PcG phenotypes and less widespread misexpression of HOX genes. It is possible that class II genes show milder homeotic phenotypes because their products are not core components of the PcG system or because they are required for repression only at a subset of PcG target genes. However, as in the case of Psc and several other PcG genes, it is also possible that class II mutants show milder phenotypes because their products act redundantly with proteins encoded by other (e.g., class II) genes.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTS AND DISCUSSIONACKNOWLEDGEMENTSLITERATURE CITED |
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