Polycomb-group (Pc-G) Proteins Control Seed Development in Arabidopsis thaliana L.

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1 Journal of Integrative Plant Biology 2007, 49 (1): Invited Review. Polycomb-group (Pc-G) Proteins Control Seed Development in Arabidopsis thaliana L. Xiao-Xue Wang and Li-Geng Ma * (National Institute of Biological Sciences, Beijing , China) Abstract Polycomb-group (Pc-G) proteins repress their target gene expression by assemble complexes in Drosophila and mammals. Three groups of Pc-G genes, controlling seed development, flower development and vernalization response, have been identified in Arabidopsis (Arabidopsis thaliana L.). MEDEA (MEA), FER- TILIZATION INDEPENDENT SEED2 (FIS2), and FERTILIZATION INDEPENDENT ENDOSPERM (FIE) are Pc-G genes in Arabidopsis. Their functions in seed development have been extensively explored. The advanced findings of molecular mechanism on how MEA, FIS2 and FIE control seed development in Arabidopsis are reviewed in this paper. Key words: Arabidopsis; FIS1/MEA; FIS2; FIS3/FIE; Pc-G genes. Wang XX, Ma LG (2007). Polycomb-group (Pc-G) proteins control seed development in Arabidopsis thaliana L. J Integr Plant Biol 49(1), Available online at Polycomb-group (Pc-G) genes are highly conserved regulatory factors that are responsible for the maintenance of silent states of genes. Pc-G genes were initially characterized in Drosophila mutants that failed to maintain the transcriptional repression of homeobox genes in the HOX cluster (Francis and Kingston 2001). Pc-G proteins assemble into two distinct complexes to exert their respective functions by modifying chromatin structure. The first types of Pc-G complexes described in Drosophila, called the E(z)/ESC complex or the Polycomb Repressive Complex 2 (PRC2), contains four PcG proteins: Enhancer of zeste [E(z)], extra sex combs (ESC), suppressor of Zeste 12 (Su(z)12) and the histone binding protein NURF-55. E(z) methylates lysine 27 of histone H3 (H3K27) (Francis et al. 2001; Czermin et al. 2002; Cao and Zhang 2004), creating an epigenetic mark that leads to the recruitment of the second type of Pc-G complex, named PRC1, via binding of the Received 19 Jul Accepted 13 Oct Publication of this paper is supported by the National Natural Science Foundation of China ( ) and Science Publication Foundation of the Chinese Academy of Sciences. * Author for correspondence. Tel: +86 (0) ; Fax: +86 (0) ; <maligeng@nibs.ac.cn> Institute of Botany, the Chinese Academy of Sciences doi: /j x chromodomain of one of its components, the Polycomb (PC) protein. PC is a core component of PRC1, together with Polyhomeotic (PH), Posterior sex combs (PSC), and dring (Shao et al. 1999). PRC1 can mediate silencing of target genes, by interfering with SWI/SNF chromatin remodeling machinery, blocking transcriptional initiation, or recruiting additional silencing activity (Shao et al. 1999; Francis and Kingston 2001; Müller et al. 2002; Czermin et al. 2002; King et al. 2002; Dellino et al. 2004; Lavigne et al. 2004; Plath et al. 2004). The plant Pc-G genes were identified genetically from screens for mutations affecting seed formation, flower development and vernalization response. Several Pc-G proteins have been identified as developmental regulators in plants. Experimental data indicates the presence of PRC2, ESC-E(z)-like complexes in plants, no evidence for the existence of PRC1, however, has been reported. To date, three PRC2 complexes have been well characterized. The MEA-FIE complex has specific functions during gametophyte and early seed development, including suppression of seed development in the absence of fertilization and repression of PHERES1 (PHE1) (Ohad et al. 1996; Chaudhury et al. 1997; Grossniklaus et al. 1998; Kiyosue et al. 1999; Ohad et al. 1999; Köhler et al. 2003). A second PRC2 complex, the CURLY LEAF (CLF) complex, most likely represses transcription of floral homeotic genes, such as the MADS-box gene AGAMOUS (AG). The CLF complex probably consists of MSI1, the E(z)

2 Pc-G in Seed Development 53 homolog CLF, FIE, and the Su(z)12 homolog EMBRYONIC FLOWER2 (EMF2) (Goodrich et al. 1997; Kinoshita et al. 2001; Yoshida et al. 2001; Hennig et al. 2003; Chanvivattana et al. 2004; Katz et al. 2004; Schönrock et al. 2006). The CLF complex represses the expression of MADS-box gene AGL19. AGL19 is a potent floral activator. AGL19 chromatin is strongly enriched in trimethylation of Lys 27 on histone H3 (H3K27me3), which is mediated by the Pc-G proteins CLF and MSI1 in the absence of cold. Prolonged cold relieves AGL19 from the CLF complex repression. Elevated AGL19 levels activate LFY and AP1 and eventually cause flowering. The third potential PRC2- like complex is the VERNALIZATION (VRN) complex. The existence of the VRN complex was hypothesized because the Su (z)12 homolog VRN2 is required for maintaining repression of the MADS-box gene FLOWERING LOCUS C (FLC) after vernalization and for vernalization-induced H3 methylation at the FLC locus (Chandler et al. 1996; Gendall et al. 2001; Bastow et al. 2004; Chanvivattana et al. 2004; Sung and Amasino 2004). In this review, we will focus on the recent advances on the molecular repression mechanism of the three Pc-G proteins, MEA/FIS1, FIS2 and FIE/FIS3, in Arabidopsis seed development. mea, fis2 and fie are Mutations That Allow Endosperm Development Without Fertilization The two products of fertilization, which are the embryo and endosperm, display distinct patterns of development. Fertilization of the egg cell by a sperm cell gives rise to a diploid embryo (Goldberg et al. 1994). Fertilization of the central cell by the second sperm cell generates the triploid endosperm. Distinguishing to embryogenesis, fertilized triploid central cell nucleus, also termed primary endosperm nucleus, undergoes a series of mitotic divisions to produce a syncytium of nuclei that surround the embryo and fill the expanding central cell (Mansfield and Briarty 1990a,b; Webb and Gunning 1991; Berger 1999; Brown et al. 1999). The endosperm nurtures the developing embryo and is ultimately absorbed by embryo development. To understand how fertilization initiates reproductive development, three mutants, fis1/mea, fis2 and fis3/fie, have been isolated (Ohad et al. 1996; Chaudhury et al. 1997; Grossniklaus et al. 1998; Kiyosue et al. 1999; Ohad et al. 1999). The fis-class mutations (fis1/mea, fis2 and fis3/fie) allow for the replication of the central cell nucleus without fertilization. In the fis mutants, a number of steps in seed development occur without pollination, including the autonomous development of diploid endosperm, a low frequency development of globular, embryo-like structures, and the partial development of ovules into seeds, indistinguishable from developing sexual seeds in size and external morphology. Most fis-class seeds do not develop beyond the endosperm cellularization stage before atrophying. These results indicate that a substantial activation of genes involved in seed development is induced in plants carrying the mutated alleles at the FIS loci. All of the three genes have been isolated (Ohad et al. 1996; Chaudhury et al. 1997; Grossniklaus et al. 1998; Kiyosue et al. 1999; Ohad et al. 1999). It is known that FIS1/MEA and FIS3/FIE are Arabidopsis homologs of the Drosophila Pc-G genes E(z) and ESC, respectively (Grossniklaus et al. 1998; Kiyosue et al. 1999; Ohad et al. 1999). FIS2 is a homolog of the recently identified Drosophila Pc-G gene Su (z)12 (Luo et al. 1999; Birve et al. 2001). As mentioned, the function of Pc-G protein complexes is to repress their targets gene expression in Drosophila. Thus, the possible mechanisms for how FIS genes regulate replication of the central cell nucleus in response to fertilization is that FIS proteins prevent the central cell from initiating endosperm development, and fertilization results in the inactivation of FIS proteins or delivers other factors to trigger the endosperm development. The mutations at FIS loci result in the production of inactive FIS proteins, so that fertilization is no longer required for initiation of endosperm development. The hypothesis is confirmed in the following experiments. MEA and FIE Proteins Form a Complex to Control Seed Development MEA and FIE are orthologs of the polycomb genes E(z) and Esc in Drosophila. In the Drososphila these gene products interact as part of a protein complex that is associated with the changes in chromatin architecture and repression of gene expression (Furuyama et al. 2003; Tie et al. 2003). Mutants of the FIS class (presently including fis1/mea, fis2, and fis3/fie) disrupt normal endosperm and embryo development. The common phenotype suggests that FIS1/MEA, FIS2 and FIS3/FIE may function in the same complex. Thus physical interaction between the Arabidopsis MEA and FIE proteins has been tested by using yeast two-hybrid assay (Luo et al. 2000; Spillane et al. 2000). The results showed us that MEA polypeptides can interact physically with the FIE protein. The FIS2 protein, however, does not physically interact with either the MEA or FIE protein (Luo et al. 2000; Spillane et al. 2000). This lack of evidence could mean that another protein may be needed in the complex to facilitate the interaction of FIS2 with MEA and FIE. The question remains as to what is the target(s) of MEA-FIE Pc-G complex in Arabidopsis. Expression of a MADS-box Gene PHERES1 is Regulated by MEA-FIE Pc-G Complex to Control Seed Development The Pc-G proteins, MEA, FIE and FIS2 regulate seed development in Arabidopsis by repressing embryo and endosperm

3 54 Journal of Integrative Plant Biology Vol. 49 No proliferation without fertilization. All three of these FIS-class proteins are likely subunits of a multiprotein Pc-G complex, but the direct targets for MEA-FIE complex remain poorly defined. PHE1 was identified as a possible target of MEA-FIE complex by using the microarray approach to uncover genes whose expressions were upregulated in mea and fie mutant (Köhler et al. 2003; Schubert and Goodrich 2003; Köhler et al. 2005). To characterize the expression pattern of PHE1, the expression of PHE1 in flowers before fertilization, open pollinated flowers (0 1 DAP), seeds containing embryos at the early globular stage (2 3 DAP), and seeds containing embryos at the late globular stage (3 4 DAP) were analyzed. No PHE1 expression is detectable before pollination; however, it becomes detectable in seeds containing preglobular-stage embryos in wild-type plants. In contrast, PHE1 expression in all three fis-class mutants initiated earlier than in wild-type plants, starting directly after pollination, because of the removal of the repression of MEA-FIE Pc-G complex. It remains upregulated in the fis mutants, consistent with the proposed function of the FIS genes as transcriptional repressors. Reduced expression levels of PHE1 in mea mutant seeds can suppress mea seed abortion, indicating a key role of PHE1 repression in seed development. Confirmation that PHE1 is a direct target of MEA-FIE Pc-G complex came from chromatin immunoprecipitation (ChIP) experiments using antibodies against MEA and FIE Pc-G proteins, which showed that MEA and FIE bind to the PHE1 promoter. Their results showed us that PHE1 expression is commonly upregulated in mea, fie, and fis2 mutants, even though the extent of upregulation can differ among the fis-class mutants. These findings support the hypothesis that the FIS proteins are part of a common protein complex repressing common target genes (Grossniklaus et al. 1998; Luo et al. 2000; Spillane et al. 2000; Yadegari et al. 2000). Results from Makarevich et al. (2006) reveal the existence of common target genes of different Pc-G complexes in Arabidopsis (Makarevich et al. 2006). They show that the plant Pc-G target gene PHE1 is regulated by histone trimethylation on H3K27 residues (H3K27me3) mediated by at least two different Pc-G complexes MEA-FIE complex and the CLF/ SWINGER (SWN) complex in plants, which contain SET domain proteins MEA or CURLY LEAF/SWINGER. PHE1 gene encodes MADS-box transcription factor, regulated by MEA-FIE Pc-G complex to control normal seed development in Arabidopsis. In contrast, Pc-G complexes in Drosophila and mammals maintain the repressive state of homeobox gene expression, such as HOX genes in Drosophila, suggesting the evolutionary diversity in plant and animal kingdoms. development is dependent on extrazygotic influences. The classical view is that both endosperm and the sporophyte play an important role in the nutrition of the embryo. This is probably correct, although somatic- and microspore-derived embryogenesis is possible under artificial conditions in tissue culture. A more interesting possibility, that extrazygotic cells directly control the expression of certain genes in the embryo, is relatively unexplored (Ray 1998). The finding of molecular mechanisms of MEA imprinting provides sound events for this hypothesis. MEA is the first-identified imprinted gene in Arabidopsis. The maternal allele of MEA is expressed and the paternal allele is repressed in endosperm. In genetic analysis only the maternal wild-type MEA allele, and not the paternal MEA allele, is required for proper embryo and endosperm development (Chaudhury et al. 1997; Grossniklaus et al. 1998; Kiyosue et al. 1999; Kinoshita et al. 1999). The uncovering of the MEA imprinting mechanism is triggered by the isolation of the DEMETER (DME) gene, which encodes a large protein with the DNA glycosylase and a nuclear localization domain. Choi et al. (2002) isolated a mutant, named demeter (dme), that causes parent-of-origin effects on seed viability. Seed viability depends solely on the maternal DME allele. DME, primarily expressed in the central cell, is required for the maternal allele expression of MEA in the central cell and the endosperm. These results suggest DME is required for the maternal expression of the imprinted MEA gene in the central cell; a process that is essential for subsequent embryo and endosperm viability (Choi et al. 2002; Lohe and Chaudhury 2002; Dickinson and Scott 2002). Seed abortion caused by DME mutation is suppressed by maternal inherited MET1 if a wild-type maternal MEA allele is present (Xiao et al. 2003). Maternal mutant dme or mea alleles result in seed abortion. Seeds with maternal dme and met1 alleles, however, survive, indicating that met1 is the suppressor of dme. DME activates whereas MET1 suppresses maternal MEA::GFP allele expression in the central cell (Xiao et al. 2003), convincing us of the antagonistic interaction between DME and MET1 gene products. MET1 is responsible for de novo and maintenance of DNA methylation to keep MEA silenced in the central cell. DME could antagonize MET1 by specifically removing 5-methylcytosine from MEA, allowing the maternal MEA allele to be expressed and form a complex before fertilization. The maternal allele of MEA is expressed, but the paternal allele is repressed in the endosperm after fertilization (Choi et al. 2004, Gehring et al. 2004). The suppressed mechanism of MEA paternal allele is the next question to be addressed. Antagonistic Interaction Between DME and MET1 to Control MEDEA Imprinting An open question in plant biology is the extent to which embryo MEA-FIE Complex Maintains MEA Paternalallele Silencing A major breakthrough on the role of Pc-G proteins in imprinting

4 Pc-G in Seed Development 55 sheds light on the molecular mechanism of imprinting in both plants and animals. Gehring et al. (2006) found that paternal MEA allele expression is not subject to the same controls as the maternal MEA allele. MEA methylation is maintained by MET1. Whenever DME activity removes methylation from its promoter, MEA protein is produced and integrated into MEA-FIE complex in the central cell. After fertilization, the MEA-FIE complex targets the paternal allele of MEA to maintain its silent state. It is the first time it has been found that Polycomb group proteins that are expressed from the maternal genome, including MEA, silence the paternal MEA allele. This showed a novel example of self-imprinting. Integration of the findings draws a nice picture of the molecular mechanism of imprinting in plant seed development (Figure 1) and enlarges our views on the Figure 1. Model for MEA imprinting and endosperm development mechanism. MEA methylation in either male or female gametophyte is maintained by MET1. In the central cell, expression of MEA is demethylated and activated by the activity of DME. MEA protein is produced and integrated into Pc-G complex, which is termed MEA-FIE Pc-G complex. Repression of PHE1 by MEA-FIE Pc-G complex blocks cell proliferation, which is essential for normal seed development. After fertilization, maternal MEA continues to be expressed in the endosperm. MEA-FIE Pc-G complexes target the paternal allele to maintain its silent state. The signal from male or other tissue triggers the disintegration of MEA-FIE Pc-G complex and activates the expression of PHE1 to promote endosperm development. Black circles, methyl groups.

5 56 Journal of Integrative Plant Biology Vol. 49 No Figure 2. Comparison of imprinting in plants and mammals. (A) In plants, embryo and endosperm are two products derived from the fertilized egg and central cell. Imprinting is only confined to endosperm in plants. Silent methylation state is maintained in micro- or megaspore mother cell during flower development. Egg, central cell, and sperm are the products of female and male gametogenesis, respectively, in Arabidopsis. The activity of DME, a DNA glycosylase, activates the maternal diploid alleles by demethylation in central cell before fertilization. After fertilization, the paternal allele is repressed in endosperm by the self-imprinting mechanism known for MEA imprinting or the mechanism unknown. Imprinting in planta endosperm is one-way control because the endosperm vanishes after nurturing the developing embryo. (B) In mammals, the repression of the paternal allele in sperm is established after meiosis through methylation or another mechanism. Inactivated paternal allele is present in both the embryo and placenta (the similar organ to endosperm in plants). The imprinted state is maintained throughout the somatic organ development. The repressed methylation state is removed just before the regeneration of germline for the next generation. Black box, imprinted gene in plants; Empty box, imprinted gene in mammals; Black circles, methyl groups.

6 Pc-G in Seed Development 57 differential mechanisms for genomic imprinting in plants and animals. It should be pointed out that there is an argument regarding the autoregulation of MEA imprinted expression. Data from Baroux et al. (2006) indicates that autorepression of the maternal MEA allele is direct and independent of the MEA-FIE complex, which is similar to the E(Z)-ESC complex of animals (Baroux et al. 2006). MEA is Necessary for PHE1 Repression Much more interestingly, PHE1 is another imprinted gene in Arabidopsis. PHE1 is mainly paternally expressed but maternally repressed. The maternal repression of PHE1 is disrupted in seeds lacking maternal MEA activity, indicating the role of Pc- G proteins in the control of PHE1 imprinting, which is one of the imprinting mechanisms existing in mammals. The mouse Polycomb group protein EED, a homolog of FIE, is required to maintain silencing of some imprinted autosomal gene (Delaval and Feil 2004). The evidence of PHE1 imprinting is an example of a gene imprinted oppositely to MEA, such that the maternal allele is largely silent and the paternal allele is expressed in the endosperm. MEA-FIE Pc-G complex likely assemble at the maternal PHE1 allele in the central cell before fertilization (Köhler et al. 2005). The SET domain of MEA is essential for the allele specific expression of PHE1 by repressing the maternal PHE1 allele (Makarevich et al. 2006). The SET domain of MEA mediates the PHE1 repression by setting histone trimethylation on H3K27 residues, which is a different mechanism to MEA imprinting. DNA Methylation is Responsible for Silencing of FIS2 Paternal Allele FIS2 is another gene, which is subject to parental genomic imprinting (Jullien et al. 2006a,b). FIS2 is a maternally expressed imprinted gene. Unlike MEA, FIS2 and MEA imprinting follows distinct molecular mechanisms. DNA methylation mediated by MET1 activity is responsible for silencing of FIS2 paternal allele. A CpG domain upstream of FIS2 that is targeted by MET1 is one of the three DNA methyltransferases responsible for both de novo DNA methylation and maintenance of the DNA methylated state is associated with the control of FIS2 imprinting. MET1 - dependent silencing of FIS2 is required during the vegetative phase, male gametogenesis, and endosperm development; when it maintains silencing of the paternal allele. Similarly, FIS2 is activated during female gametogenesis by DME, leading to the expression of maternal alleles in the endosperm after fertilization. In the endosperm, the paternal allele remains silenced through the continuous action of MET1. DNA methylation, histone methylation and self-regulated imprinting mediated by histone methylation show us the diversity of imprinting mechanisms in plants. In addition, the mechanisms of imprinting in plants and animals are different (Surani et al. 1984; Reik et al. 2001; Reik and Walter 2001; Surani 2001; Scott and Spielman 2004; Kinoshita et al. 2004; Arnaud and Feil 2006). The placenta in mammals and the endosperm in plants function as the extra-embryonic tissues to nurture the embryo and to connect the embryo to the maternal tissue. The plant endosperm and the mammalian placenta are both subjected to imprinting, resulting in expression of maternal copies of genes and the repression of the paternal alleles of genes, most notably those playing an essential role in growth and development (Figure 2). In plants, imprinted-gene expression seems to be confined to the endosperm, which is one of the two double fertilization products (Figure 2A). In contrast, imprinting in mammals occurs both in the embryo and the placenta. In both plants and animals, however, DNA methylation is essential for imprinting. In mammals, DNA methylation marks are present at the key regions that control imprinting (Figure 2B). These marks are established in germ lines by de novo DNA methyltransferase. After fertilization, they are maintained throughout development in all the somatic lineages. The imprinting needs to be erased and reset before passing to the next generation to allow the establishment of novel imprints. Unlike the situation in mammals, endosperm-specific imprinting in plants is conferred through the specific demethylation in the female gametophyte, where DME is believed to be the main player. The imprinted status in plants is confined to the endosperm, which does not contribute to the next generation (Figure 2A). Thus the erasure of imprinting status is omitted in plants. Growing evidence suggests that Pc-G proteins participate in a wide range of developmental processes in both plant and animal kingdoms. 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