Diverse functions of Polycomb group proteins during plant development

Seminars in Cell & Developmental Biology 14 (2003) 77–84

Diverse functions of Polycomb group proteins during plant development José C. Reyes a , Ueli Grossniklaus b,∗ a

Instituto de Bioquimica Vegetal y Fotosintesis, Centro de Investigaciones Isla de la Cartuja, Av. Americo Vespucio s/n, E-41092 Sevilla, Spain b Institute of Plant Biology, University of Zürich, and Zürich-Basel Plant Science Center, Zollikerstrasse 107, CH-8008 Zürich, Switzerland

Abstract Polycomb group (PcG) proteins play essential roles in animal and plant life cycles by controlling the expression of important developmental regulators. These structurally heterogeneous proteins form multimeric protein complexes that control higher order chromatin structure and, thereby, the expression state of their target genes. Once established, PcG proteins maintain silent gene expression states over many cell divisions providing a molecular basis for a cellular ‘memory.’ PcG proteins are best known for their role in the control of homeotic genes in Drosophila and mammals. In addition, they play important roles in the control of cell proliferation in vertebrate and invertebrate systems. Recent studies in plants have shown that PcG proteins regulate diverse developmental processes and, as in animals, they affect both homeotic gene expression and cell proliferation. Thus, the function of PcG proteins has been widely conserved between the plant and animal kingdoms. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Cellular memory; Chromatin structure; Genomic imprinting; Plant development; Polycomb group proteins

1. Introduction Over the last few years, the prominent role of chromatin structure and organization in the control of eukaryotic gene expression has become apparent. The dynamic nature of chromatin and the machinery that modifies its components play a central role in gene regulation (reviewed in [1–3]). Recent studies have identified several multiprotein complexes that regulate the structure of chromatin (reviewed in [4]). In many systems, such complexes ensure the stable inheritance of gene expression states over many cell divisions. Thus, they are part of a cellular ‘memory’ which allows the stable propagation of ‘on’ or ‘off’ states of gene expression despite the disruption of transcription during mitosis. This cellular ‘memory’ implies the involvement of an epigenetic mark, i.e. information not present in the DNA sequence itself, which labels particular genes as being in an active or inactive state. Some well-studied, evolutionarily conserved chromatin components involved in this cellular memory are the proteins of the Polycomb and trithorax groups (PcG and trxG), which are involved in the transcriptional control of many developmental regulators (reviewed in [5,6]). They are best ∗ Corresponding

author. Tel.: +41-1-634-82-40; fax: +41-1-634-82-04. E-mail address: [email protected] (U. Grossniklaus).

known for their role in regulating homeotic (HOX) genes in Drosophila where they are responsible for the maintenance of stable expression states during development. The spatial expression domains of the HOX genes are initially set by the segmentation genes. Once these transcriptional regulators decay, the PcG and trxG proteins maintain their expression for the remainder of development. The PcG and trxG genes were initially defined genetically through their regulatory interactions with the HOX genes: PcG proteins are involved in maintaining their inactive state whereas trxG proteins maintain their active state. However, this classification may be oversimplified as some PcG proteins behave as activators in certain contexts [7,8]. The PcG and trxG contain structurally diverse proteins that are part of several multiprotein complexes. They do not only regulate HOX gene expression but are more general chromatin regulators that control diverse target genes. Many PcG and trxG proteins are involved in the regulation of growth and cell proliferation and were found to play a major role in human cancer (reviewed in [9]). Over the last few years, it was found that PcG proteins have evolutionarily conserved functions in plants, worms and mammals. In this review we focus on the role of PcG proteins in plant development where they are involved in the regulation of both cell proliferation and homeotic gene expression. Based on recent studies from other systems, we discuss how plant

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PcG complexes may regulate plant developmental processes at the biochemical level. 2. The plant life cycle Plant PcG proteins were found to control aspects of seed development, floral induction and floral organogenesis. These developmental processes occur at various stages of the plant life cycle, which alternates between a diploid sporophyte (spore-producing organism) and a haploid gametophyte (gamete-producing organism). Unlike in animals, where the meiotic products differentiate directly into gametes, the meiotic products of plants (spores) undergo several mitotic divisions to form multicellular gametophytes (reviewed in [10,11]). In seed-free plants, the gametophytes are free-living organisms that often represent the dominant generation of the life cycle. In flowering plants, they consist of only a small number of cells and develop within the sexual organs of the flower, the anthers (male gametophytes) and ovules (female gametophytes). Double fertilization initiates seed development and reconstitutes the sporophyte. It involves two pairs of gametes: one of the sperm cells delivered by the pollen tube fertilizes the egg producing the zygote, whereas the second sperm fertilizes the central cell resulting in the formation of the endosperm. Enclosed by the seed coat the coordinated development of embryo and endosperm, which is thought to play a nutritive role during seed development and/or germination, leads to the formation of the mature seed. After germination, the apical meristems of the seedling produce the root and shoot system, respectively. During vegetative growth, the shoot apical meristem produces rosette leaves before it switches to reproductive growth (floral induction). Floral induction is under the control of both environmental signals and the developmental competence of the plant (reviewed in [12]). Upon floral induction, the shoot apical meristem produces inflorescence and eventually flowers. Differentiation of specific cells within the reproductive organs of the flower and meiosis finally conclude the sporophytic phase of the plant life cycle. 3. Maternal control of seed development by PcG proteins The medea (mea) maternal effect mutant was recovered in a screen for gametophytically acting genes in Arabidopis thaliana. In a mea heterozygote, half of the seeds abort irrespective of the nature (MEAp or meap ) and dosage of the paternal allele [13]. These seeds are derived from a mea mutant female gametophyte (embryo genotypes meam /MEAp and meam /meap ; referred to as mea embryos hereafter) and show a developmental delay. They eventually abort at the late heart stage when sibling seeds (embryo genotypes MEAm /MEAp and MEAm /meap ) have completed morphogenesis and undergo seed desiccation. Rescue of

mea embryos in culture allows the recovery of homozygous mea/mea plants, which display no obvious mutant phenotypes except for 100% seed abortion suggesting that MEA is specifically required for seed development. The mea embryos show a delayed progression through embryogenesis with a dramatic over-proliferation phenotype [13]. The endosperm also shows proliferation defects, with more nuclei in the chalazal cyst but less in the peripheral region, and defects in endosperm polarity [13–15]. In addition to this seed abortion phenotype, female gametophytes lacking MEA activity initiate endosperm proliferation in the absence of fertilization at a low frequency [14,16,17]. This second phenotype, whose relationship to seed abortion remains unclear, was the basis for other independent screens that lead to the isolation of the fertilization-independent seed (fis1, fis2, fis3) and fertilization-independent endosperm (fie) mutants [14,18–20]. Together, the known mutants of the fis class define three loci, mea (fis1), fis2, and fie (fis3) that share the maternal effect seed abortion and fertilization-independent endosperm phenotypes. Molecular cloning of MEA showed that it encodes a SET domain protein with high similarity to the Drosophila PcG protein Enhancer of zeste [E(Z)] (Fig. 1A) [13]. The molecular nature of MEA suggested an involvement in the control of gene regulation through modulating higher order chromatin structure. Furthermore, the involvement of PcG proteins in controlling cell proliferation in invertebrates, vertebrates and plants suggested an evolutionary conserved function of PcG proteins [13,21–23]. Based on the similarities of the phenotypes between mea and the fis mutants it was suggested that the MEA, FIS2 and FIE proteins may be part of a multiprotein PcG complex [16]. This hypothesis gained support by the cloning of FIE, which encodes a WD-40 repeat protein with high similarity to the PcG protein Extra sex combs (ESC) of Drosophila (Fig. 1B) [24]. In Drosophila and mammals, the E(Z) and ESC proteins or their mammalian counterparts, respectively, were shown to interact directly and to be part of a mutliprotein complex of about 600 kDa [25–27]. This direct interaction is also conserved between the plant proteins MEA and FIE in yeast two-hybrid assays [28–30]. Furthermore, this interaction was confirmed in vitro and the expression domains of the two genes were found to partially overlap during female gametophyte and seed development consistent with the interaction being of functional relevance in plants [29]. Recently, we could show that the MEA and FIE proteins elute in high molecular weight fractions of about 600 kDa in gel filtration experiments providing additional evidence for a structural and functional conservation of the FIE/MEA [ESC/E(Z)] complex between plants and animals (Köhler and Grossniklaus, unpublished data). Initially, the cloning of the FIS2 gene was less revealing. It encodes a protein containing many repeats of 17 and 22 amino acids, respectively, and a Zn-finger domain with similarity to transcriptional regulators [17]. With the cloning of the Drosophila Suppressor of zeste 12 [Su(z)12] PcG gene,

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Fig. 1. Schematic domain organization of Arabidopsis Polycomb proteins. (A) Domain organization of Drosophila E(Z) and its Arabidopsis counterparts. This family is characterized by a C-terminal SET domain indicated in red. The cysteine-rich CXC domain, which is characteristic for the E(Z) subclass of SET domain proteins, is shown in green. A putative SANT domain (present in SWI3, ADA2, N-CoR, and TFIIIB) is indicated in yellow. The acidic EZD1 domain is shown in purple and a domain characterized by five cysteine residues (called EZD2 or C5 ) is shown in blue. (B) Domain organization of Drosophila ESC and Arabidopsis FIE proteins. Seven WD-40 domains are depicted in green. (C) Domain organization of Drosophila SU(Z)12 and related Arabidopsis proteins. The four proteins are characterized by the presence of a C-terminal conserved domain of 150 amino acids called VEFS (depicted in black) and a C2 H2 -type zinc finger (striped red). In addition, FIS2 contains a number of repeats of 17 and 21 amino acids (shown in white), which are absent in the other members of the family. Protein lengths in amino acids are shown on the right.

its putative function became more apparent [31]. FIS2 and SU(Z)12 share a C-terminal domain of approximately 150 amino acids of unknown function (Fig. 1C). Based on the similarity of the mea, fie and fis2 phenotypes, it is likely that FIS2 is part of the FIE/MEA complex.

4. Transcriptional regulation of the FIS genes by genomic imprinting The cloning of the FIS genes yielded insights into the molecular mechanism of their function and suggested that they control gene expression by regulating chromatin structure. The FIE/MEA complex is likely to maintain stable expression states of as yet unknown target genes over many cell divisions during seed development. The nature of the maternal effect, however, remained unclear. In principle, three alternative hypotheses could account for the gametophytic maternal effect embryo lethal phenotype observed in the three fis class mutants: (i) haplo-insufficiency in the triploid endosperm (genotype, e.g. meam /meam /MEAp ), (ii) absence of a gene product required for seed development that is deposited prior to fertilization in the egg and/or central cell(s), or (iii) parent-of-origin-dependent activity of the gene after fertilization (genomic imprinting) [10].

The mea locus was found to be regulated by genomic imprinting, that is only transcripts derived from the maternally inherited MEA allele could be detected after fertilization [28,32,33]. A demonstration that a locus is regulated by genomic imprinting requires (i) that maternal and paternal alleles show differential expression levels (this can also occur for gene products deposited in the gametes prior to fertilization) and (ii) that the locus is actively transcribed after fertilization. Both requirements were shown to be true for the mea locus in the early endosperm by looking directly at nascent MEA transcripts before and after fertilization [32]. This finding was confirmed using allele-specific RT-PCR and showed that at these early stages, the paternal MEA allele was neither expressed in the embryo nor the endosperm [32]. Subsequently, it was found that for a large number of loci no paternal activity can be detected during the first few divisions after fertilization in either the embryo or the endosperm [34]. This finding suggested that, in general, paternal activation occurs only after a few cell divisions, although there are exceptional loci with earlier activation [35,36]. This raised the question whether MEA was regulated in a way similar to paternally inherited alleles of these other genes or whether the paternal MEA allele was silent for a longer period during seed development. At a late stage of seed development,

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MEA was found to be differentially expressed in the endosperm but not the embryo [33]. This suggests that, at least in the endosperm, the regulation by genomic imprinting at the mea locus persists for a longer period than at paternal alleles of other genes studied that become active at 2 or 3 days after pollination. The fact that reactivation of a silent paternal MEA allele can rescue seed abortion at a stage later than the general paternal activation provides further support for this interpretation [32]. However, in some accessions the silent state of the paternal MEA allele in the endosperm does not appear to be stable at late stages and breaks down in a fraction of seeds [28]. Recent quantitative allele-specific RT-PCR studies have shown that throughout the morphogenetic phase of seed development the expression levels of maternal and paternal MEA alleles are drastically different. At later stages expression of the paternal allele may be detectable although at a reduced level compared to transcripts derived from the maternal allele (Page, Gagliardini and Grossniklaus, unpublished data). Taken together these data suggest that paternally inherited MEA alleles are in a silent state for a much longer period than paternally inherited alleles of an average gene. To distinguish between the two behaviors of paternally inherited genes, we referred to the wide-spread, general phenomenon as genome-wide and to the more persistent phenomenon as gene-specific genomic imprinting [37]. For the two other FIS genes, FIS2 and FIE, the nature of the maternal effect is less clear as studies investigating genomic imprinting are based largely on reporter gene constructs. Paternally inherited alleles of both FIS2 and FIE are not expressed at early stages of seed development after fertilization [28,30]. The expression profile of FIE appears to be similar to that of paternally inherited alleles of other genes and does not seem to be under a specific regulation in this respect. Therefore, it is likely that FIE is required at an earlier stage than MEA resulting in maternal effect seed abortion even though it is biparentally expressed later during seed development. A difference between MEA and FIE has also been observed with respect to other characteristics [30]. For FIS2 it is unclear whether the silent state of the paternal allele lasts much longer than that of paternally inherited alleles of other genes because even the maternal allele is transiently expressed and not active at late stages of seed development. Interestingly, many imprinted genes in mammals show effects on fetal growth with striking similarities to the mea seed phenotype suggesting similar evolutionary pressures that led to the evolution of genomic imprinting in seed plants and mammals. Evolutionary aspects of genomic imprinting and its regulation have been the topic of several publications [13,28,32,38,39] and recent reviews [37,40–42] and will not be discussed any further. A final point we want to make, however, is that the FIS genes participate in epigenetic gene regulation at two distinct levels. First, the FIS genes are regulated by genomic imprinting, be it gene-specific as at the mea locus or not, such that only maternal product

is provided at crucial stages of seed development. Second, the FIS proteins are part of a PcG complex that maintains stable expression states during development. This opens the possibility for extensive maternal control over seed development through a two-step mechanism. This maternal control involves the transcriptional regulation through genomic imprinting of crucial chromatin regulators, which in turn maintain expression states of their target genes over many cell divisions. It is likely that genes controlling cell proliferation will be among the target genes of the FIE/MEA complex. Some target genes may even be under allele-specific control. For instance, it could be envisioned that the FIE/MEA complex associates with certain silent paternal alleles after fertilization and maintains them in a repressed state throughout seed development whereas the maternally inherited alleles, which may be active prior to fertilization, are not targeted by the complex. In this way, the FIE/MEA complex would itself be involved in the control of gene expression by genomic imprinting. Indeed, it was recently found that the mammalian homologs of E(Z) and ESC are associated with imprinted X-inactivation in mouse trophoblast cells [43,44].

5. PcG-dependent control of reproductive development The first characterization of a PcG gene in plants showed that similarly to their Drosophila counterparts some Arabidopsis PcG proteins control homeotic gene expression [21]. In plants, homeotic genes specify the identity of the four whorls of flowers. AGAMOUS (AG) is a C-class homeotic gene normally expressed in whorls 3 and 4 and is required for correct development of stamens and carpels. In the curly leaf (clf) mutant, the AG gene is ectopically expressed in leaves and other plant organs from early developmental stages onwards [21]. As a consequence, clf plants show small and curled leaves, early flowering and partial homeotic transformations similar to those observed in plants where AG is ectopically expressed under the viral 35S promoter. Therefore, CLF acts as a stable repressor of AG expression in those organs where AG should not be expressed. Like MEDEA, CLF encodes a SET domain protein with high similarity to the Drosophila E(Z) protein. While CLF seems to control AG and probably also AP3 expression, other Arabidopsis PcG proteins have a more general role in regulating reproductive development. For instance, in an Arabidopsis line where a FIE–GFP protein is expressed during seed formation but not on later stages of development, effects on floral induction were observed [45]. This transgenic line showed viable seed formation in a homozygous fie background, suggesting a rescue of the seed abortion phenotype. However, the transgenic plants displayed a strong early flowering phenotype characterized by the presence of flower-like structures and organs at the seedling stage. These data suggest that FIE represses the transition to flowering early during vegetative development when the plant normally produces rosette leaves but no

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inflorescences or flowers. Such seedlings lacking FIE activity express several homeotic genes such as AP1, LFY, AG, AP3 and PI. The phenotype of these transgenic lines has striking similarity to that reported for the embryonic flower mutants (emf1 and emf2) [46]. The emf mutants do not produce any rosette leaves. Rather, emf seedlings give rise to inflorescences whose lateral buds produce only flowers but not additional inflorescences. The emf2 plants also show ectopic expression of the AG gene. Like FIS2, EMF2 is another member of the small family of Arabidopsis proteins with similarity to SU(Z)12 from Drosophila [47]. Another Arabidopsis protein with similarity to SU(Z)12 that was recently identified is VERNALIZATION2 (VRN2) [48]. Vernalization refers to an extended period of exposure to low temperatures that accelerates flowering. As vernalization occurs at the seedling stage but affects floral induction, which often occurs many months later, it was hypothesized to involve an epigenetic memory mechanism. Vernalization induces the down-regulation of the FLOWERING LOCUS C (FLC) transcript. FLC encodes a MADS domain transcription factor and is a key regulator of the vernalization response that represses the transition to flowering. The repression of FLC is epigenetically maintained when the temperature returns to normal levels, thereby stimulating the transition to flowering. Several vrn mutants were isolated that are unable to reduce FLC mRNA levels in response to cold temperature. In the vrn2 mutant, FLC is normally repressed in response to vernalization but upon a shift to higher temperatures, the FLC mRNA level is not stably repressed and returns to higher levels conferring a vernalization-insensitive phenotype. Therefore, the role of VRN2 is to stably maintain repression of FLC after vernalization, consistent with its suggested function in a PcG multiprotein complex. In support for this hypothesis, the first intron of FLC is more sensitive to DNAse treatments in a vrn2 mutant suggesting that VRN2 alters the chromatin structure of the FLC locus [48].

6. How do PcG proteins repress transcription? In Drosophila, genetic and biochemical data have demonstrated that PcG proteins are associated with two different multimeric complexes. The Polycomb repressive complex 1 (PRC1 complex) contains Polycomb, Polyhomeotic, Posterior sex combs, and other factors including a number of TAFII factors (for review see [6]). This complex is able to inhibit ATP-dependent chromatin remodeling activity by the SWI/SNF complex in vitro [49]. Putative open reading frames encoding the PcG proteins present in the PRC1 complex are not found in the Arabidopsis genome, suggesting that a similar complex is not present in Arabidopsis. A second Drosophila PcG complex contains the ESC and E(Z) proteins together with the histone deacetylase RPD3, strongly indicating a role for histone deacetylation in PcG-mediated repression [50]. As described above, an

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interaction between FIE and MEA has been demonstrated, supporting the existence of a ESC/E(Z)-like complex in Arabidopsis [28–30]. Recent data indicate the presence of Drosophila (Kingston and Simon, personal communication) and human (Reinberg, personal communication) SU(Z)12 proteins in the respective ESC/E(Z) complexes (see ‘Note added in proof’). Intents to demonstrate interaction of FIS2 with either FIE or MEA by yeast two-hybrid assays has been unsuccessful. However, the fact that loss of function fis2 mutants display the same phenotype as fie and mea suggests that they act together. It is possible that FIS2 requires additional components to interact with the FIE/MEA complex. Whether histone deacetylases are present in the FIE/MEA complex is presently unknown. Analogous to the FIE/MEA complex, which controls seed development, the existence of a similar PcG complex formed by FIE, CLF and EMF2 or VRN2, which controls reproductive development, can be hypothesized (Fig. 2). However, the phenotypic data discussed above suggest a more complex picture. While fie, clf and emf2 mutants show ectopic expression of AG, the FIE and EMF2 proteins seem to control several additional homeotic genes that are not deregulated in the clf background. Furthermore, vrn2 mutants show a normal regulation of floral homeotic genes. These data suggest the existence of several PcG complexes controlling reproductive development (Fig. 2). In addition to MEA and CLF a third Arabidopsis E(Z)-like gene (EZA1) has been found in the Arabidopsis genome. The function of EZA1 is unknown at the moment. The Drosophila ESC/E(Z) complex also contains an RbAp protein (also referred to as p55 or MSI1 protein), a WD-40 repeat histone binding protein that often co-purifies with histone deacetylase complexes [50]. Five RbAp48-like proteins (AtMSI1-5) have been identified in the Arabidopsis genome, whose functions are currently being elucidated. The mechanism by which the ESC/E(Z) complex represses transcription and marks epigenetically silenced loci is still unclear. E(Z)-like proteins contain a SET domain, which confers histone methyltransferase activity in other chromatin proteins such as Su(var)3-9 [51] (see ‘Note added in proof’). Methylation of histone H3 at lysine 9 has been demonstrated to be involved in gene silencing, heterochromatin formation and acts as a signal for other epigenetic marks such as DNA methylation (reviewed in [52]). Lysine 9 of histone H3 can also be acetylated. Therefore, an attractive possibility is that ESC/E(Z)-like complexes repress transcription by combining deacetylation and methylation at lysine 9 of histone H3 or other histone lysine residues. Histone methylation can then trigger silencing by recruiting other factors that promote heterochromatinization of the locus, i.e. a more compact and less accessible chromatin structure. Contrasting this hypothesis, Orlando and co-workers [53] have demonstrated that binding of Drosophila PcG proteins to repressed promoters does not exclude general transcription factors. They propose that PcG complexes maintain repression by inhibiting general

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Fig. 2. Schematic representation of the Drosophila ESC/E(Z) complex and putative Arabidopsis ESC/E(Z)-like complexes. Extensive genetic and biochemical evidence indicates the presence of a 600 kDa complex in Drosophila which contains ESC, E(Z), the RbAp protein p55 and RPD3, a histone deacetylase. In Arabidopsis, the existence of different ESC/E(Z)-like complexes is proposed. Interacting proteins that were shown to be part of high molecular weight complexes and/or participate in physical interactions are fully colored. Proteins participating in hypothetical complexes for which no experimental evidence regarding direct interactions is available, are striped.

transcription factor-mediated activation of transcription. In this context it is important to remember that plants seem to lack the PRC1 complex and, therefore, differences in PcG-mediated silencing are expected between plants and animals. None of the proteins that have been reported to form the ESC/E(Z)-like complexes have specific DNA-binding capability. How then does the ESC/E(Z)-like complex recognize and bind-specific target genes? Analysis of Drosophila PcG-dependent cis-regulatory elements, called Polycomb response elements (PREs), demonstrated the presence of DNA-binding sites for transcription factors such as GAGA and Zeste which have been shown to interact with PcG complexes [54]. The gap gene product Hunchback has also been implicated in establishing PcG repression. However, a physical interaction between this transcription factor and PcG proteins has not been demonstrated [55]. These data suggest that specific DNA-binding proteins recruit PcG complexes to PREs. Interactions between DNA-binding proteins and PcG proteins have not yet been reported in plants. Tissue-specific expression of the AG gene is controlled by a regulatory element placed in the second intron of the gene. Positive and negative transcription factors such as LEAFY and AP2 bind this element to control AG expression. Reporter gene fusions demonstrated that this region is required for CLF-mediated repression suggesting the existence of a PRE-like element in this region [56] but it has not been further characterized to date.

7. Conclusion In Drosophia, PcG proteins maintain expression states that are established during embryonic development and

thereby fix developmental decisions. PcG repression is established when segmental differentiation starts. Once established, Drosophila PcG repression is maintained throughout the entire live cycle of the fly. A somewhat similar situation may occur during seed formation where PcG function is essential for normal development and mutations in the FIS genes result in seed abortion. It is likely that the FIE/MEA complex regulates a particular set of target genes whose repression (or possibly activation) is established during seed development and maintained for the remainder of this phase of the life cycle. In contrast, a novel role of plant PcG seems to be the repression of developmental pathways that would be active by default in the absence of PcG proteins (e.g. endosperm proliferation or reproductive development). Many plant PcG proteins may be active from an early stage of development onwards. As a consequence, PcG-mediated repression has to be released at some point during normal development in response to certain stimuli that allow progression through the life cycle (e.g. fertilization or induction of flowering). This fundamental difference implies that plant PcG-mediated repression may be the default state, which has to be overcome by some unknown mechanism. Furthermore, the default activity of PcG complexes and their inactivation may explain why PRC1 is apparently not present in plants. It has been proposed that ESC/E(Z), which transiently interacts with PRC1 in Drosophila, plays a major role in establishing PcG-mediated repression but is not present at later stages of development [57]. In contrast, PRC1 is present at later stages and has a long-lasting effect on gene expression, an effect that would not be required during plant development as PcG complexes are inactivated upon specific stimuli. New light will be shed onto such differences and similarities in the function of PcG complexes in animal and plants through

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functional characterization of additional components of PcG complexes, their biochemical activities and regulation.

8. Note added in proof It was recently demonstrated that Drosophila and human ESC/E(Z) complexes have histone methyltransferase activity [58–60]. Both the human and the Drosophila complexes are able to methylate lysine 27 of histone H3, while the Drosophila complex also methylates lysine 9 of histone H3. The presence of SU(Z)12 in the ESC/E(Z) complex was also demonstrated. The existence of histone H3 methylated at lysine 27 in Alfalfa has been known for over 10 years [61] suggesting a similar connection between PcG-mediated regulation and histone H3 modification in plants.

Acknowledgements We apologize to our colleagues whose work on PcG proteins was not covered in this review and thank D. Reinberg, R. Kingston, and J. Simon for providing information prior publication. Research in this area is supported by Grant PB98-0688 from the Spanish DGESIC to J.C.R., as well as Grant 31-64061.00 of the Swiss National Science Foundation, Grant BBW 00.0313 of the Bundesamt für Bildung und Wissenschaft as part of the APOTOOL Project (QLG2-2000-00603) in Framework V of the European Union and Searle Scholarship to U.G.

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