Kicking against the PRCs - a domesticated transposase antagonises silencing mediated by polycomb group proteins and is an accessory component of polycomb repressive complex 2

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.Fil: Liang, Shih Chieh. University of Edinburgh; Reino UnidoFil: Hartwig, Ben. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Perera, Pumi. University of Edinburgh; Reino UnidoFil: Mora Garcia, Santiago. Fundación Instituto Leloir; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquimicas de Buenos Aires; ArgentinaFil: de Leau, Erica. University of Edinburgh; Reino UnidoFil: Thornton, Harry. University of Edinburgh; Reino UnidoFil: Lima de Alves, Flavia. University of Edinburgh; Reino UnidoFil: Rapsilber, Juri. University of Edinburgh; Reino UnidoFil: Yang, Suxin. University of Edinburgh; Reino UnidoFil: James, Geo Velikkakam. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Schneeberger, Korbinian. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Finnegan, E. Jean. University of Edinburgh; Reino UnidoFil: Turck, Franziska. Max Planck Institute for Plant Breeding Research; AlemaniaFil: Goodrich, Justin. Mc Gill University; Canad

ALP1 interacts with PRC2.

<p>(A) Western blot of seedling protein extracts analysed using anti-CLF antibodies. The left and right panels show blots with short (right panel) and longer (left panel) chemiluminescent detection times as the two extracts from <i>35S</i>::<i>GFP-CLF</i> transgenic plants show much higher expression of GFP-CLF than native CLF. The positions of the size markers in the ladder lane have been marked on the image. Both CLF (≈125kD) and GFP-CLF (≈155 kD) migrate as larger proteins than their predicted sizes (102 and 129 kD, respectively). When the CLF protein was expressed in <i>E</i>. <i>coli</i> it also migrated larger than predicted, possibly because of the high lysine and arginine content in the N-terminal portion. (B-C) Co-immunoprecipitation experiments in which protein extracts were immunoprecipitated using anti-GFP antibodies, immunoblotted and analysed using anti-CLF (B) or anti-MSI1 (C) antibodies. (D-E) Immunoprecipitation of chromatin prepared from 12-day old Ws, <i>clf-5</i>0, <i>alp1-4</i> and <i>clf-50 alp1-4</i> seedlings using anti-H3K27me3 (D) or anti-H3K36me3 (E) antibodies. Precipitated DNA was quantified using real time PCR and is displayed as percentage of input. PCR fragments were located in promoter (pro), transcriptional start site (TSS), exon (ex), intron (in) and at end of interrogated genes as indicated. Error bars indicate the mean and standard error of three separate experiments, each with three technical replicates. The differences between <i>alp1</i> and wild-type or between <i>alp1 clf</i> and <i>clf</i> were not statistically significant (Tukey multiple comparison of means test) in any of the regions examined.</p

Phylogenetic analysis of ALP1 sequences from land plants and green algae.

<p>Molecular phylogenetic analysis by maximum likelihood (ML) method implemented in MEGA6 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005660#pgen.1005660.ref074" target="_blank">74</a>]. The bootstrap consensus tree inferred from 200 replicates is taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The composition of the DDE catalytic triad is indicated on the tips of the branches. The tree is unrooted. The species indicated are <i>Arabidopsis thaliana</i>, <i>Glycine max</i> (soybean), <i>Vitis vinifera</i> (grape), <i>Prunus persica</i> (peach), <i>Theobroma cacao</i> (cacao), <i>Ricinus communis</i> (castor bean), <i>Populus trichocarpa</i> (poplar), <i>Solanum lycopersicon</i> (tomato), <i>Oryza sativa</i> (rice), <i>Phoenix dactylifera</i> (date palm), <i>Amborella trichopoda</i>, <i>Cycas micholitzii</i>, <i>Picea sitchensis</i>, <i>Ginkgo biloba</i>, <i>Cyathea spinulosa</i>, <i>Psilotum nudum</i>, <i>Marchantia paleacea</i>, <i>Diphyscium foliosum</i>, <i>Nothoceros vincentianus</i>, <i>Chara braunii</i> and <i>Zea mays</i> (maize). PIF/Harbinger transposase branches are coloured in black, those of green algae in red, bryophytes in blue, pteridophytes in orange, gymnosperms in magenta, the angiosperm ALP1 clade in green, the angiosperm At3g55350 clade in light blue. Genbank accession numbers are prefixed GI, others are accession numbers for sequence retrieved from the 1000 plant genomes website (<a href="http://www.onekp.com" target="_blank">www.onekp.com</a>) with the exception of the <i>Chara braunii</i> sequence which is given the contig number in the transcriptome assembly. The analysis involved 34 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous residues were allowed at any position. There were a total of 323 positions in the final dataset.</p

<i>ALP1</i> is widely expressed and its protein product is nuclear-localised.

<p>(A) Complementation assay in <i>clf-50 alp1-4</i> background. The <i>pALP1</i>::<i>ALP1-GFP</i> transgene fully complements <i>alp1-4</i> and restores the clf phenotype, whereas <i>pALP1</i>::<i>ALP1-GUS</i> gives weaker complementation so that plants retain a partially suppressed clf phenotype. (B-G) Histochemical staining showing <i>pALP1</i>::<i>ALP1-GUS</i> activity in rosettes (B), leaves (C), roots (D) and inflorescences (E). (F–G) <i>pALP1</i>::<i>ALP1-GFP</i> is nuclear localised in roots (G), whereas a control <i>35S</i>:<i>GFP</i> construct shows more diffuse localisation in cytoplasm and nucleus (F). Scale bars are 1cm in A, 1mm in B-E and 20μm in F,G.</p

ALP1 co-purifies with Pc-G proteins.

<p>The table summarises the results from three independent replicate experiments (IP1-IP2-IP3) and lists the number of uniquely identified peptides from each protein. The total number of peptides identified in each experiment is also shown (all peptides). In IP1 some of the <i>35S</i>::<i>GFP</i> lysate was lost during filtration, in IP3 there was considerable loss of all samples except <i>35S</i>::<i>GFP-CLF</i> during the stage tip purification of in gel tryptic digests, hence the lower total number of peptides. The full list of proteins identified is presented as an excel sheet in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005660#pgen.1005660.s010" target="_blank">S3 Table</a>.</p

<i>ALP1</i> is required to activate PcG target gene expression.

<p>(A) T1 plants transformed with <i>35S</i>::<i>AG</i> transgene. The <i>alp1-1</i> mutation did not suppress the characteristic phenotype of small, early flowering plants with narrow, curled leaves. (B) Real time RT-PCR analysis of <i>SEP3</i>, <i>FT</i>, <i>FLC</i> and <i>AG</i> expression in seedlings of 12 day old short day grown plants. Relative expression was first normalised relative to the <i>EiF4A</i> reference gene and then calculated relative to the wild type value. Error bars indicate the standard error of the mean of three biological replicates. All four genes are upregulated in <i>clf-50</i> but show reduced expression in <i>clf-50 alp1-4</i> double mutants. One way ANOVA tests indicate that the differences are significant (p<0.05) between <i>clf-50</i> and <i>clf-50 alp1-4</i> for <i>SEP3</i>, <i>FT</i> and <i>FLC</i> but not <i>AG</i>. (C) Venn diagram comparing the number of genes mis-regulated relative to wild type (Ws) in 12 day old seedlings. Misregulated genes showed Log₂(FoldChange)>2 and False Discovery Rate <0.05. (D) Bar charts comparing the number of genes downregulated (blue) and up-regulated (red) relative to wild-type. Numbers above the bars indicate the proportion of up-regulated genes. (E) Pie chart showing that the bulk of genes mis-expressed in <i>clf-50</i> relative to wild-type are no longer mis-expressed (restored) in <i>clf-50 alp1-4</i> relative to wild-type. (F-G) Inflorescences (F) and flowers (G) illustrating the enhancement of the weak <i>lfy-5</i> phenotype by <i>alp1-3</i>. In <i>lfy-5</i> flowers, fewer petals and stamens are produced than in wild-type whereas <i>lfy-5 alp1-3</i> flowers from similar position on the inflorescence had much more severe phenotype with petals and stamens usually lacking and replaced with sepals and carpels, respectively (G). (H) Real time RT-PCR analysis of <i>AP3</i> and <i>PI</i> expression in inflorescences shows reduced expression of both genes in <i>lfy-5</i> compared to wild type and a more severe reduction in <i>lfy-5 alp1-3</i> consistent with the enhanced phenotype. Expression is normalised relative to the reference gene <i>EIF4A</i>. Error bars indicate standard error of mean of three biological replicates. Scale bars are 5mm in A and F, 500μm in G.</p