In Arabidopsis, the EMBYRONIC FLOWER2 (EMF2), VERNALISATION2 (VRN2) and FERTILISATION INDEPENDENT ENDOSPERM2 (FIS2) genes encode related Polycomb-group (Pc-G) proteins. Their homologues in animals act together with other Pc-G proteins as part of a multimeric complex, Polycomb Repressive Complex 2 (PRC2), which functions as a histone methyltransferase. Despite similarities between the fis2 mutant phenotype and those of some other plant Pc-G members, it has remained unclear how the FIS2/EMF2/VRN2 class Pc-G genes interact with the others. We have identified a weak emf2 allele that reveals a novel phenotype with striking similarity to that of severe mutations in another Pc-G gene, CURLY LEAF (CLF), suggesting that the two genes may act in a common pathway. Consistent with this, we demonstrate that EMF2 and CLF interact genetically and that this reflects interaction of their protein products through two conserved motifs, the VEFS domain and the C5 domain. We show that the full function of CLF is masked by partial redundancy with a closely related gene, SWINGER (SWN), so that null clf mutants have a much less severe phenotype than emf2 mutants. Analysis in yeast further indicates a potential for the CLF and SWN proteins to interact with the other VEFS domain proteins VRN2 and FIS2. The functions of individual Pc-G members may therefore be broader than single mutant phenotypes reveal. We suggest that plants have Pc-G protein complexes similar to the Polycomb Repressive Complex2 (PRC2) of animals, but the duplication and subsequent diversification of components has given rise to different complexes with partially discrete functions.

The plant Polycomb‐group (Pc‐G) protein CURLY LEAF (CLF) is required to repress targets such as AGAMOUS ( AG ) and SHOOTMERISTEMLESS ( STM ). Using chromatin immunoprecipitation, we identify AG and STM as direct targets for CLF and show that they carry a characteristic epigenetic signature of dispersed histone H3 lysine 27 trimethylation (H3K27me3) and localised H3K27me2 methylation. H3K27 methylation is present throughout leaf development and consistent with this, CLF is required persistently to silence AG. However, CLF is not itself an epigenetic mark as it is lost during mitosis. We suggest a model in which Pc‐G proteins are recruited to localised regions of targets and then mediate dispersed H3K27me3. Analysis of transgenes carrying AG regulatory sequences confirms that H3K27me3 can spread to novel sequences in a CLF‐dependent manner and further shows that H3K27me3 methylation is not sufficient for silencing of targets. We suggest that the spread of H3K27me3 contributes to the mitotic heritability of Pc‐G silencing, and that the loss of silencing caused by transposon insertions at plant Pc‐G targets reflects impaired spreading.

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity. Transposons are parasitic genetic elements that proliferate within their hosts’ genomes. Because rampant transposition is usually deleterious, hosts have evolved ways to inhibit the activity of transposons. In plants, this genome defence is provided by the Polycomb group (PcG) proteins and/or the DNA methylation machinery, which repress the transcription of transposase genes. We identified the Arabidopsis ALP1 gene through its role in opposing gene silencing mediated by PcG genes. ALP1 is an ancient gene in land plants and has evolved from a domesticated transposase. Unexpectedly, we find that the ALP1 protein is present in a conserved complex of PcG proteins that inhibit transcription by methylating the histone proteins that package DNA. ALP1 likely inhibits the activity of this PcG complex by blocking its interaction with accessory proteins that stimulate its activity. We suggest that the inhibition of the PcG by a transposase may originally have evolved as a means for transposons to evade surveillance by their hosts, and that subsequently hosts may have exploited this as a means to regulate PcG activity. Our work illustrates how transposons can be friend or fiend, and raises the question of whether other transposases will also be found to inhibit their host’s regulatory machinery.

In Arabidopsis, mutations in the Pc-G gene CURLY LEAF (CLF) give early flowering plants with curled leaves. This phenotype is caused by mis-expression of the floral homeotic gene AGAMOUS (AG) in leaves, so that ag mutations largely suppress the clf phenotype. Here, we identify three mutations that suppress clf despite maintaining high AG expression. We show that the suppressors correspond to mutations in FPA and FT, two genes promoting flowering, and in SEPALLATA3 (SEP3) which encodes a co-factor for AG protein. The suppression of the clf phenotype is correlated with low SEP3 expression in all case and reveals that SEP3 has a role in promoting flowering in addition to its role in controlling floral organ identity. Genetic analysis of clf ft mutants indicates that CLF promotes flowering by reducing expression of FLC, a repressor of flowering. We conclude that SEP3 is the key target mediating the clf phenotype, and that the antagonistic effects of CLF target genes masks a role for CLF in promoting flowering.

▪ Abstract Chromatin remodeling in plants has usually been discussed in relation to aspects of genome defense such as transgene silencing and the resetting of transposon activity. The role of remodeling in controlling development has been less emphasized, although well established in animal systems. This is because cell fate in plants is often held to be entirely specified on the basis of position, apparently excluding any significant role for cell ancestry and chromatin remodeling. We argue that chromatin remodeling is used to confer mitotically heritable cell fates at late stages in pattern formation. Several examples in which chromatin remodeling factors are used to confer a memory of transient events in plant development are discussed. Because the precise biochemical functions of most remodeling factors are obscure, and little is known of plant chromatin structure, the underlying mechanisms remain poorly understood.