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773 Epigenetic inheritance of expression states in plant development: the role of Polycomb group proteins Claudia Köhler* and Ueli Grossniklaus† Polycomb group (PcG) proteins maintain a repressed state of gene expression over many cell divisions. The recent characterisation of several PcG proteins from plants revealed a remarkable structural and functional conservation of PcG proteins between different kingdoms. In both plants and animals, homeotic genes are among the target genes of PcG complexes, although the structure of these genes is not conserved. However, not all PcG proteins identified in animals are present in plants. Furthermore it becomes clear that PcGmediated repression in plants is more transient compared with the long-lasting effects in animals. This may be related to the absence of PcG proteins thought to be involved in long-term maintenance of PcG repression, suggesting that the mechanisms underlying PcG-mediated repression differ between plants and animals. Addresses Institute of Plant Biology, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland *e-mail: [email protected] † e-mail: [email protected] Current Opinion in Cell Biology 2002, 14:773–779 0955-0674/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S0955-0674(02)00394-0 Abbreviations AG AGAMOUS CLF CURLY LEAF EMF EMBRYONIC FLOWERING ESC Extra sex combs E(Z) Enhancer of zeste FLC FLOWERING LOCUS C FIE FERTILISATION-INDEPENDENT ENDOSPERM FIS FERTILISATION-INDEPENDENT SEED HDAC HISTONE DEACETYLASE PCG Polycomb group TRXG trithorax group MEA MEDEA PRC1 Polycomb repressive complex 1 SU(Z)12 Suppressor of zeste12 VRN2 VERNALISATION2 Introduction Polycomb group (PcG) genes were initially discovered in Drosophila melanogaster through the analysis of mutants exhibiting posterior transformations of body segments. The mutant phenotypes suggested that PcG genes keep homeotic genes in a transcriptionally repressed state. Once the spatial pattern of homeotic gene expression in early embryos is established through the activity of the segmentation genes, PcG proteins maintain this expression pattern throughout the remainder of development [1]. By contrast, proteins of the trithorax group (trxG) are required to maintain homeotic genes in a transcriptionally active state [1]. Many genes of the trxG family were identified as suppressors of mutations in PcG genes. Therefore, it was hypothesised that both classes of genes have opposing functions [2]. However, it was shown that some PcG genes also act as transcriptional activators, suggesting that the initial classification of PcG genes as repressors and trxG genes as activators may be not generally applicable [3]. PcG and trxG genes, which stably maintain gene expression established by other regulators, form a model system to study the cellular memory of transcriptional states. PcG and trxG proteins are generally thought to control higher-order chromatin organisation. Both PcG and trxG proteins form high-molecular-weight complexes, which can bind to specific chromosomal response elements [1,4]. However, no DNA consensus sequence of such elements has yet been defined [5]. It is still unclear how PcG and trxG complexes bound to their target genes can maintain active or repressed states of gene expression through mitosis [4]. To date, the molecular structure of 15 fly PcG proteins has been identified. They belong to diverse structural classes. Homologues of the fly PcG genes have been cloned from many different organisms. In mammals, PcG proteins also regulate expression of homeobox genes, and mutations in PcG and trxG genes cause axial and limb transformations [6]. Furthermore, PcG and trxG genes regulate cell proliferation, and their misexpression is correlated with the development of various cancers [7,8]. In Caenorhabditis elegans, PcG genes are key mediators of transcriptional repression in the germline, and mutations in these genes cause a maternal-effect sterile phenotype [9]. Recent work in plants shows that PcG genes also play important roles at various stages of the plant life cycle [10]. Thus, the exploitation of transcriptional regulation by PcG proteins to control development is remarkably conserved during evolution. In this review, we discuss the structure and function of PcG proteins in plants in comparison to their animal counterparts. Rather than discussing general aspects of chromatin remodelling and modification, we focus on the developmental role of PcG genes during the plant life cycle. CURLY LEAF regulates homeotic gene expression The first PcG gene characterised in Arabidopsis, CURLY LEAF (CLF), is similar to Enhancer of zeste [E(z)] from Drosophila [11] (Figure 1). In addition to some other conserved domains, CLF and E(Z) share the SET domain, a conserved region initially found in three Drosophila proteins, Suppressor of variegation 3-9 [SU(VAR)3-9], E(Z) and TRX [12,13]. The phenotypes of the clf mutant resemble 774 Cell differentiation Figure 1 E(Z) MEA CLF ESC FIE SU(Z)12 FIS2 EMF2 Acidic domain Cysteine-rich domain SET domain 200 amino acids WD repeats 200 amino acids Structural similarities of PcG proteins in different species. The conserved structural elements of the Drosophila proteins E(Z), ESC and SU(Z)12 were compared with structural elements of similar Arabidopsis proteins and are indicated by coloured bars. E(Z), MEA and CLF contain an acidic domain, a cysteine-rich domain and a SET domain [11,15]. ESC and FIE contain seven WD 40 repeats [19]. SU(Z)12, FIS2, EMF2 and VRN2 contain a zinc finger and a conserved region named VEFS box (VRN2, EMF2, FIS2 and SU(Z)12). SU(Z)12, EMF2 and VRN2 also contain a conserved basic domain at the amino terminus [20,31•,34•,36••]. The scale is indicated. Basic domain Zinc finger VEFS box 200 amino acids VRN2 Current Opinion in Cell Biology transgenic plants constitutively expressing the homeotic MADS-box gene AGAMOUS (AG). Flowers of clf plants show partial homeotic transformations of sepals and petals into carpels and stamens, respectively [11]. Expression analysis suggests that CLF is necessary for the maintenance of AG repression during later stages of development, and not for the initial specification of the AG expression domain. Similarly, the E(Z) protein from Drosophila is necessary to maintain the transcriptional repression of homeotic genes in the Antennapedia- and bithorax-complexes (ANT-C and BX-C) [1]. This conservation of PcG function is particularly striking in view of the fact that homeotic genes in plants and animals are structurally unrelated and encode homeobox and MADS-box genes, respectively [14]. This suggests that evolutionary old regulatory complexes have been recruited for similar processes in both plants and animals. In Arabidopsis, CLF seems not to be sufficient for AG repression, as the CLF mRNA is present in all four whorls throughout flower development and overlaps with the AG expression domain in the third and fourth whorls. Therefore, specificity of CLF repression could involve other proteins that form a repressive PcG complex with CLF that is restricted to whorls 1 and 2 [11]. Role of PcG proteins during seed development A genetic screen for gametophytic mutants showing a maternal effect led to the identification of the medea (mea) mutant [15]. Additional alleles of the same gene (FERTILISATION-INDEPENDENT SEED1 [FIS1]), as well as two other loci (FERTILISATION-INDEPENDENT ENDOSPERM [FIE]) and FIS2), were identified in screens for mutants displaying fertilisation-independent seed development [16–21]. All three fis class mutants exhibit a maternaleffect seed abortion phenotype and initiate endosperm formation in the absence of fertilisation with variable penetrance. Seeds derived from female gametophytes carrying a mutation in one of the FIS genes abort, irrespective of whether the paternal allele is mutant or wild type. The embryos in aborting seeds are delayed in their development, and overproliferate to form an abnormally large heart-stage embryo. The endosperm also shows proliferation defects. In addition, the central cell of fis mutants can start dividing without a fertilisation signal. Thus, it was suggested that the primary function of the FIS genes is to regulate cell proliferation [21,22]. The MEA gene encodes another SET-domain protein similar to E(Z). It was shown recently that the SET domain of the SU(VAR)3-9 protein family has histone methyltransferase activity and methylates Lys9 of histone H3 [23]; however, to date neither for E(Z) nor its mammalian homologues, EZH1 and EZH2, has a histone methyltransferase activity been detected [24] (see Update). In Drosophila, the E(Z) protein is found in a 600 kDa protein complex and directly interacts with the WD40 domain containing protein Extra sex combs (ESC) [25]. The FIE protein from Arabidopsis is similar to ESC and was shown to interact with MEA, as was suggested by the common mutant phenotypes of mea and fie [26–28]. E(Z) and ESC are widely conserved and have been shown to interact in Polycombing development in plants Köhler and Grossniklaus 775 Figure 2 CLF FIE AG ? Flowering Gametophyte development ? ? VRN2 FLC Before fertilisation Vernalisation MEA CLF FIE EMF FIE FIS2 ??? AG MEA FIE Germination FIS2 ??? After fertilisation Current Opinion in Cell Biology A speculative view of PcG complexes throughout the Arabidopsis life cycle. In Arabidopsis flowers, a putative complex of CLF, FIE and unknown proteins regulate flower organ development by repressing AG [11,35]. In the female gametophyte, MEA, FIE and FIS2 inhibit a precocious proliferation of the central cell by repressing unknown target genes. It is most likely that the same proteins together regulate embryo and endosperm proliferation during seed development [26,27]. At the seedling stage, CLF, FIE and EMF2 repress the switch from vegetative to generative development. Mutants in all three genes express AG at the seedling stage [11,33,34•]. VRN2, together with other proteins, also controls the onset of flowering by keeping the floral inhibitor FLC repressed after vernalisation [36••]. both mammals and C. elegans [29,30•]. Thus, the interaction between E(Z) and ESC homologues forms the core of an ancient repressor complex deployed in highly diverged species. the mea, fie and fis2 mutants and their overlapping expression patterns suggest that all three proteins are part of one complex regulating common target genes [26,27]. Because neither MEA nor FIE interact with FIS2 in yeast twohybrid studies, it is possible that the interaction is transient, involves other proteins, or that FIS2 is not a component of the MEA–FIE complex at all [27] (see Update). The FIS2 protein is similar to the Drosophila Suppressor of zeste12 (SU[Z]12) protein and is also conserved in vertebrates [31•] (see Update). One characteristic feature of these proteins is a zinc finger similar to fingers found in sequence-specific DNA-binding proteins. However, DNAbinding activity has not been reported for either FIS2 or its homologues. The similarities of the mutant phenotypes of Role of PcG proteins during floral induction The transition from vegetative to reproductive development requires important changes in gene expression, which have 776 Cell differentiation to be tightly regulated. Regulators of flowering time are endogenous factors, such as plant hormones, and environmental factors, such as photoperiod and temperature [32,33]. Mutant analyses in Arabidopsis have defined four major flowering promoting pathways: the photoperiod, the vernalisation (a period of prolonged exposure to cold temperature that accelerates flowering), the autonomous, and the gibberellin pathways. Whereas the first two pathways integrate environmental conditions, the latter two act largely independently of environmental stimuli but depend on the developmental competence of the plant [32,33]. The early flowering mutant embryonic flower2 (emf2) does not produce any rosette leaves but rather initiates small influorescences whose lateral buds produce only flowers but no additional inflorescences. Double mutant analysis demonstrated that the emf2 mutant is epistatic to floweringtime mutants that integrate environmental and endogenous signals [34•], suggesting a major role for EMF2 in the repression of reproductive development. The EMF2 protein shares similarity to the zinc-finger-containing proteins FIS2 and SU(Z)12. The EMF2 mRNA is expressed throughout the life cycle of Arabidopsis without any significant changes during vegetative and reproductive development. Thus, similar to the situation for CLF, specificity of EMF2 repression does not depend on the time of EMF2 transcription but most likely involves other proteins and mechanisms. Likely candidates for interaction partners of EMF2 are CLF and FIE. The clf mutant shows ectopic AG expression, as does the emf2 mutant, and it flowers early, although this phenotype is not as extreme as that of emf2 [11,34•]. Furthermore, EMF2 antisense transgenic plants display curled leaves, a characteristic phenotype of the clf mutant. Finally, a partially complemented fie mutant starts flowering at the seedling stage, similar to emf2 [35]. A putative CLF–FIE–EMF2 complex would be analogous to the MEA–FIE–FIS2 complex, as CLF and EMF are similar to MEA and FIS2, respectively (Figure 2). As there are no other genes with similarity to FIE present in the Arabidopsis genome, it is possible that FIE is a component of several distinct PcG complexes. This is consistent with the observation that fie mutants are only viable as heterozygotes while other fis mutants can be made homozygous, for instance through embryo rescue in culture [18]. VERNALISATION2 (VRN2) is another PcG protein with similarity to FIS2 and SU(Z)12 and was isolated in a screen for mutants that do not respond to cold treatment [36••]. Vernalisation usually occurs at the seedling stage but the transition to flowering happens several weeks later. Thus, the meristem has to remember this stimulus for several mitotic divisions, suggesting an epigenetic mechanism [36••,37]. Vernalisation promotes flowering by downregulating the expression of the MADS-box gene FLOWERING LOCUS C (FLC), which acts as a strong floral repressor by negatively regulating the expression of several flowering promoting genes [38,39]. Several vrn mutants have been isolated. In these mutants, the mRNA levels of FLC are not reduced in response to cold temperature, indicating that they affect regulators of FLC expression [40]. Interestingly, VRN2 function is only revealed in the presence of mutations leading to an upregulation of FLC expression. Thus, the VRN2 protein itself is not required for the initial repression of FLC after cold treatment but rather to keep FLC stably repressed. VRN2 behaves in a functionally similar way to PcG proteins in Drosophila and provides insights into the epigenetic basis of vernalisation. The VRN2 gene is expressed in the absence of vernalisation and the level of VRN2 transcript is not altered in response to vernalisation [36••]. Therefore, VRN2 activity most likely also requires other factors or mechanisms to establish specificity. Specificity of PcG action: how does it come about? The specific expression patterns of PcG-repressed genes in Drosophila contrasts with the widespread expression of PcG proteins [4]. Similar observations were made in plants, as we pointed out for the overlapping expression patterns of the PcG proteins CLF and VRN2 and their target genes AG and FLC, respectively. This implies that highly selective mechanisms target PcG-mediated repression. One mechanism could be that the regulation of specific subunits of the multimeric PcG complexes specifies the onset of repression. Furthermore, it is possible that PcG repression requires silenced genes as templates, allowing the PcG complex to lock a previously established expression pattern in place, whereby active genes are unaffected and silent genes become stably repressed. In this model, the specificity of PcG-mediated repression would be determined by the molecular features of silent chromatin, which allows the establishment or stabilisation of PcG repressive complexes. Molecular differences between transcribed and nontranscribed chromatin could be associated proteins, histone modifications, remodelled nucleosomes and chromatin compaction. Alternatively, the formation of PcG repressive complexes on their respective target genes could be the default state, with features of an actively transcribed gene inhibiting PcG complex assembly [4]. Finally, the control of the subcellular localisation or the binding characteristics by post-translational modifications of PcG proteins could provide an explanation for their specificity. Such modifications could be established by certain endogenous signalling pathways conferring competence to the repressor complexes. Consistent with this hypothesis is the finding that ESC is modified by phosphorylation in Drosophila and that the phosphorylated form preferentially associates with E(Z) [25]. How is the repressive function of PcG complexes achieved? Given the ubiquitous use of histone modifications for gene regulation, it is likely that PcG repression in plants involves histone modifications as well. The correlation between histone acetylation and gene activation, as well as histone deacetylation and gene repression, is well established and has also been observed in plants [41–43]. The Drosophila E(Z)–ESC complex and the corresponding mammalian EZH2–EED complex have been shown to Polycombing development in plants Köhler and Grossniklaus 777 interact with RPD3-like histone deacetylases (HDACs) [44,45••]. RPD3-like HDACs are present in plants and have been shown to be involved in different developmental processes [41,43,46]. Considering the highly conserved interaction between E(Z) and ESC in diverse organisms, it is possible that transcriptional repression mediated by plant PcG complexes also involves histone acetylation and deacetylation. mammals [1]. The identification of the PcG protein CLF as repressor of the MADS-box gene AG indicated a widely conserved function of PcG proteins as repressors of homeotic genes [11]. Another MADS-box gene, FLC, has been shown to be regulated by the PcG protein VRN2 [36••]. Interestingly, homeobox genes of animals and MADS-box genes of plants both function as homeotic genes, but are structurally not related [14]. Differences between PcG complexes of plants and animals In addition to controlling homeotic target genes in development, PcG genes in mammals are also regulators of the cell cycle. Among others, the INK4a locus of mice, which encodes the tumour suppressors and cell cycle inhibitors p16 and p19Arf, is a target gene of the PcG protein BMI-1 [7,8]. In Drosophila and C. elegans, PcG proteins have also been implicated in the control of cell proliferation. Similarly, the phenotype of the mea, fie and fis2 mutants, which produce giant heart-stage embryos and enlarged endosperm, suggests that target genes regulated by the MEA–FIE complex are also involved in controlling cell proliferation. The identification of additional target genes of PcG proteins in plants will be important to elucidate the mechanism of PcG action in plants. Biochemical purification of PcG complexes from Drosophila embryos and mammalian cells demonstrated the existence of at least two types of complexes [25,45••,47]. The first complex is about 600 kDa, contains E(Z) and ESC, and co-purifies with the RPD3 HDAC [45••]. It is thought that the E(Z)–ESC complex establishes a repressed state, before the second class of complexes takes over to maintain this expression state [4,48•]. The second complex, Polycomb repressive complex 1 (PRC1), is a ~3 MDa complex purified from Drosophila embryos [47]. This complex contains the PcG proteins Polyhomeotic, Polycomb, Posterior sex combs and Sex comb on midleg [4]. Although no catalytic activity has been defined for any of the fly PRC1 components, it has been shown that PRC1 blocks chromatin remodelling by the trxG-related SWI/SNF complex in vitro [47]. However, homology searches did not reveal coding sequences with similarity to these proteins in the Arabidopsis genome [49]. In contrast to Drosophila, where PcG-repressed genes remain repressed once this state is established, PcG repression in plants can be relieved in response to either developmental or environmental cues. The repressive function of EMF2, for instance, inhibits or delays the transition to flowering and is most likely relieved in response to certain stimuli that promote flowering [34•]. The same could apply for the action of the MEA–FIE complex. The mea, fie and fis2 mutants start endosperm development in the absence of fertilisation, indicating that one function of this complex is to repress genes before fertilisation and that repression is relieved by a fertilisation-dependent signal [16–21]. Therefore, one could assume that, in contrast to the long-lasting repression mediated by PcG genes in Drosophila, PcG action in plants is more transient. The idea is supported by the absence of PcG proteins similar to those of the PRC1 maintenance complex in plants. Stable repression of genes could require other proteins or mechanisms that evolved independently in plants and animals. An attractive hypothesis is that a more transient transcriptional repression in plants is one consequence of the continuous postembryonic development of their body plan, which is highly flexible and strongly influenced by environmental conditions. Target genes in animals and plants As mentioned before, PcG genes have been defined as regulators of homeotic gene expression in Drosophila and Conclusions The identification of PcG proteins that are involved in several developmental processes in plants has revealed that transcriptional repression using PcG proteins is widely conserved between different kingdoms. Strikingly, among the target genes of PcG complexes in plants and animals are homeotic genes, which are structurally not conserved, and likely also genes that regulate cell proliferation and growth. However, there seem to be differences regarding the stability of PcG-mediated repression between plants and animals. The characterisation of PcG complexes in plants will, therefore, be an essential step for understanding the mechanism(s) underlying the cellular ‘memory’ in plants. Given the recent progress in identifying potential chromatin remodelling factors, additional links between PcG action and developmental decisions will probably be uncovered in plants. It will be important to identify factors regulating PcG protein function at different stages of plant development and to relate their effects to the maintenance of gene expression. Finally, the identification of additional target genes for PcG proteins in plants will be crucial to appreciate PcG action during plant development. Update Recent work has shown that SU(Z)12 is a subunit of the the E(Z)–ESC complex from Drosophila [50••,51••]. Furthermore, it was demonstrated that this complex contains a histone methyltransferase activity that methylates Lys9 and Lys27 of histone H3. The human counterpart to the E(Z)–ESC complex, EZH2–EED, has also been purified and was shown to have histone methyltransferase activity, methylating Lys27 of histone H3 [52••]. 778 Cell differentiation Acknowledgements We apologise to our colleagues whose work we could not cite due to space constraints. C Köhler is an Human Frontier Science Program Fellow, and our work on Polycomb group proteins is supported by the Kanton of Zürich, the Swiss National Science Foundation and a Searle Scholarship to U Grossniklaus. References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Simon JA, Tamkun JW: Programming off and on states in chromatin: mechanisms of Polycomb and trithorax group complexes. 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MES2 and MES6 are similar to E(Z) and ESC, whereas MES3 is a novel protein. MES2 and MES6 were shown to interact with each other and are present together with MES3 in a 255 kDa complex. 31. Birve A, Sengupta AK, Beuchle D, Larsson J, Kennison JA, Rasmuson • Lestander AA, Muller J: Su(z)12, a novel Drosophila Polycomb group gene that is conserved in vertebrates and plants. Development 2001, 128:3371-3379. Mutants in a novel PcG gene, Suppressor of zeste 12 [Su(z)12], exhibit strong homeotic transformations and Su(z)12 function is required throughout Drosophila development to maintain repression of homeotic genes. Furthermore, su(z)12 mutations are strong suppressors of position-effect variegation and SU(Z)12 activity is required for germ cell development. Su(z)12 encodes a PcG protein with similarity to plant FIS2, VRN2 and EMF2, indicating a strong conservation of PcG proteins across kingdoms. 32. Simpson GG, Dean C: Arabidopsis, the Rosetta stone of flowering time? 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