Arabidopsis Polycomb Repressive Complex 2 binding sites contain putative GAGA factor binding motifs within coding regions of genes
© Deng et al.; licensee BioMed Central Ltd. 2013
Received: 23 May 2013
Accepted: 14 August 2013
Published: 30 August 2013
Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes.
We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and that the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs).
Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, unlike Drosophila PREs which are located in promoters and devoid of H3K27me3, Arabidopsis FIE binding sites tend to be in gene coding regions and co-localize with H3K27me3.
KeywordsPolycomb Chromatin immunoprecipitation H3K27me3
The Polycomb group (PcG) proteins are found across the higher eukaryotes and are essential for normal development. PcG proteins were first identified in Drosophila where they are required to maintain repression of homeotic genes  and have since been shown to be required for the correct expression of many genes in plants and animals. The polycomb proteins make up two major protein complexes; Polycomb Repressive Complex 1 (PRC1) and PRC2 [1–5] which are conserved in animals and plants. PRC2 catalyses trimethylation of histone H3 lysine 27 (H3K27me3). The H3K27me3 is bound by PRC1 which ubiquitinates histone H2A [4, 6] resulting in a compacted chromatin state that can be inherited through mitotic divisions.
Plants have clear homologs of the four core protein components of PRC2, often with multiple genes encoding each component . In Arabidopsis, FERTILIZATION INDEPENDENT ENDOSPERM (FIE) is the single Extra Sex Combs (ESC) homolog, CURLY LEAF (CLF), SWINGER (SWN) and MEDEA are Enhancer of Zeste homologs, FERTILIZATION INDEPENDENT SEED 2, VERNALIZATION 2 and EMBRYONIC FLOWER 2 are Suppressor of Zeste 12 homologs and MULTI-SUBUNIT SUPPRESSOR OF IRA (MSI) 1–5 are homologs of NURF55. Of the MSIs, MSI1 appears to be a component of PRC2 complexes, linking PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 , a protein that has PRC1-like function in Arabidopsis, while other MSIs (e.g. MSI4/FVE) have roles outside the PRC2 complex [7, 9]. A loss of PRC2 activity in Arabidopsis, such as in clf swn double mutants and FIE RNAi plants leads to strong developmental defects, especially in organ identity [10, 11]. Whole genome chromatin immunoprecipitation (ChIP) experiments have shown that about 20% of Arabidopsis genes are marked by H3K27me3 [12–17]. The H3K27me3 targets have an over-representation of genes that are highly regulated as opposed to being constitutively expressed. H3K27me3 is generally associated with genes with low transcription activity [3, 16] consistent with H3K27me3 having a role in maintaining repression of gene expression. Although large numbers of gene loci have H3K27me3 present, only a minority are de-repressed in vegetative tissues of plants mutant for PRC2 components . This indicates that in these tissues H3K27me3 is only critical for maintaining the repression of a subset of the H3K27me3-marked genes and presumably loss of H3K27me3 at other loci does not lead to their increased expression due to the absence of specifically expressed transcription factors.
A major unanswered question in understanding Polycomb repression in plants is how specific loci are targeted by the Polycomb complexes. Polycomb recruitment has been best characterised in Drosophila where regions of H3K27me3 are associated with sequence elements termed Polycomb Response Elements (PREs) . Drosophila PREs are regions of up to a few hundred base pairs that were initially defined as being required to confer Polycomb repression on their target genes. PREs are able to recruit either PRC1 or PRC2 or both. PREs contain binding sites for sequence-specific DNA binding proteins. The binding sites for Pleiohomeotic (Pho) and the related Pho-like are a common element of Drosophila PREs, but they also contain sites for other DNA binding proteins including GAGA factor, Pipsqueak and Zeste. Genome-wide studies show that the binding sites for these other factors only partially overlap with PRC1 and PRC2 target sites and the extent to which they contribute to PcG recruitment is not always clear [4, 18].
In mammalian systems, PREs are less well characterised with the best examples being a 3 kb region from the mouse MafB gene and a 1.8 kb region from the human HOXD cluster that confer PcG-dependent repression in reporter gene systems [19, 20]. Both these elements contain binding sites for YY1 (the mammalian PHO homolog) suggesting that there is at least some conservation of the mechanisms of PcG recruitment between mammals and insects. Long non-coding RNAs (lncRNAs) have also been implicated in PcG recruitment in mammals. These can act in cis, such as the Xist and Kcnq1ot1 lncRNAs that are involved in PcG recruitment in X chromosome inactivation and imprinting respectively [21, 22], or in trans, such as the HOTAIR lncRNA which is produced from the HOXC cluster and acts as a scaffold to recruit PRC2 to the unlinked HOXD locus [23, 24].
At present less is known of the mechanisms by which genes are targeted by the PcG system in plants. There is some evidence for the presence of PRE-like sequences in plants. A 50 bp element (RLE) has been identified from the promoter of the Arabidopsis LEAFY COTYLEDON 2 (LEC2) gene which is required for PcG repression and confers repression and H3K27me3 deposition on a transgene . The LEC2 promoter also contains a GAGA element that is bound by Arabidopsis GAGA factors in vitro. Mutation studies suggest that this GAGA element has an activator or enhancer function and is not required for H3K27me3 deposition . A second example of a plant PRE-like sequence comes from the promoter of the BREVIPEDICELLUS (BP) gene. The ASYMMETRIC LEAVES 1 (AS1)–AS2 complex binds to defined sequences in the BP promoter to silence its expression . The BP locus is marked by H3K27me3 which requires the AS1-AS2 complex. The AS1-AS2 complex interacts with PRC2 components and the AS1-AS2 binding site from the BP promoter is sufficient to confer Polycomb repression on a GUS transgene. These properties are consistent with the AS1-AS2 binding site in BP functioning as a PRE at which AS1-AS2 recruits PRC2 .
Some evidence for lncRNAs being involved in Polycomb recruitment in plants comes from the FLC gene which encodes a MADS box repressor of flowering [28, 29]. FLC expression is repressed by vernalisation (extended cold) and this repression is maintained in a PRC2-dependent manner following return to warm growing conditions [11, 30]. Non-coding sense transcripts (named COLDAIR) produced from the large first intron of FLC which are bound by the PRC component CLF, are required to maintain FLC repression in the cold , suggesting that the COLDAIR transcript recruits PRC2 to maintain FLC repression.
To further explore the mechanisms of PcG recruitment in plants we carried out a ChIP-seq experiment to determine the genome-wide distribution of FIE, the single ESC homolog in Arabidopsis which should therefore be present in all PRC2 complexes. By comparing the FIE binding sites with genome-wide H3K27me3 distribution we found over seven hundred high confidence FIE binding sites. The FIE binding sites were predominantly within gene bodies and were enriched for three sequence motifs including putative GAGA factor binding sites.
Identification of FIE-HA binding sites by ChIP-seq
H3K27me3 abundance and distribution is conserved between C24 and Columbia ecotypes
FIE-HA peaks are associated with H3K27me3
FIE-HA is enriched across gene body regions
The distribution of FIE-HA and H3K27me3 was determined by plotting the ChIP enrichment of H3K27me3 and FIE-HA in a gene-centric manner. We observed the characteristic enrichment of H3K27me3 across gene bodies (Figure 3b) . FIE-HA is also enriched across gene bodies (Figure 3b), but with greater enrichment at the 5′ ends in comparison to the distribution of H3K27me3. The size distributions of the FIE-HA+H3K27me3 peaks (FIE-HA peaks that overlap with H3K27me3 peaks) and all H3K27me3 peaks were compared (Figure 3c) and found to show a similar distribution at size ranges up to about 1.5 kb.
FIE-HA genes are enriched for developmental functions
High confidence FIE binding peaks contain putative GAGA Factor binding sites
We have carried out a ChIP-seq analysis to identify binding sites for the PRC2 component FIE across the Arabidopsis genome. In comparison to the numbers of H3K27me3 sites (5148) we identified fewer high confidence sites of FIE binding (723). As PRC2, and therefore FIE, is required to deposit and maintain H3K27me3, a similar numbers of peaks is expected in both experiments. The discrepancy could have some biological significance or be a technical artefact. A technical difference could be simply a consequence of the two ChIP experiments using different antibodies and targeting proteins that interact with chromatin in different ways. Histone H3 is an intrinsic part of the nucleosome structure while FIE is part of a protein complex that interacts with chromatin. Although the FIE-HA sample was cross-linked prior to immunoprecipitation, the indirect nature of the interaction between FIE and the DNA that is assayed by ChIP may make it harder to detect FIE binding regions compared to H3K27me3 regions.
Biological explanations for the low number of FIE-HA peaks compared to H3K27me3 peaks could be that there are differences in the number of PRC2 binding sites, the strength of PRC2 binding or the amount of time that PRC2 is present at a given locus. There is support for this last possibility from FRAP (Fluorescence Recovery After Photobleaching) studies in Drosophila which suggest that polycomb complexes are not constantly bound to chromatin and that the rate of assembly of polycomb complexes differs between loci. Genome-wide comparison of H3K27me3 and PRC2 in Drosophila also identified ‘weak’ PcG sites  where H3K27me3 but not PRC2 was detected. The average abundance of H3K27me3 at the FIE-HA+H3K27me3 peaks was significantly higher than at the H3K27me3 only regions (Figure 3e). The genes associated with FIE-HA+H3K27me3 regions were also more likely to be up-regulated in plants that have reduced PcG function. We speculate that these genes are ones for which activators are present in vegetative tissues (with the activators regulating other genes) and hence there is a selection for increased PRC2 occupancy to maintain high levels of H3K27me3 and repression of gene expression.
A search for sequence motifs in the high confidence FIE binding sites identified four short conserved motifs. One of these was identified as being similar to the GAGA factor binding site which is a component of Drosophila PREs. The GAGA factor binds to many Drosophila PREs, but is also found in active promoters  and is suggested to have roles in nucleosome depletion and PcG recruitment. In plants the GAGA motif is often found within core promoter sequences ; however the GAGA motifs identified through FIE-HA ChIP-seq are predominantly located in gene bodies. They are not found in analyses of random sequences, indicating that there is a positive association with PRC2. The H3K27me3 only sites did not contain the GAGA motif and had lower levels of H3K27me3 than the FIE-HA+H3K27me3 peaks. The FIE-HA+H3K27me3 associated genes are also more likely to be up-regulated in plants which have a loss of PRC2 function. Based on this we speculate that the GAGA motif has a role in strengthening Polycomb recruitment to target genes for which Polycomb regulation is the primary mode of repression.
One element that functions in a PRE-like manner in plants is the RLE element in the Arabidopsis LEC2 promoter . RLE is located near a GAGA element; however this GAGA element is not required for the function of RLE. RLE is at one edge of a region of FIE-HA binding (Figure 1a), which also includes the GAGA element. The PRE-like sequence identified in the BP promoter does not have any associated FIE-HA binding in our high-confidence dataset, although there is evidence of FIE-HA binding across the BP gene body (Figure 1b). The BP PRE-like region has been shown to bind CLF-GFP expressed from a strong 35S promoter , so it may be a site of weaker PRC2 interaction. The GAGA motif has also been identified in association with LFY binding sites ; while some of these genes are also H3K27me3 targets, many are not. This suggests that in plants, as for Drosophila , the role of GAGA factor is wider than PcG function.
Although our data suggest that the GAGA factor may be a common component in the PcG regulation mechanism in plants as well as in flies, there are differences in the structures of the regions occupied by PRC2 and H3K27me3. We did not find evidence for relatively narrow regions of PRC2 binding with low H3K27me3 and depleted of nucleosomes, flanked by wide regions of H3K27me3, as seen at many Drosophila PcG targets. The observed co-localisation of FIE-HA and H3K27me3 is more reminiscent of the data in mammalian systems . The association of H3K27me3 and FIE-HA binding with gene body regions appears to be particularly strong in plants compared to both mammals and insects.
We have used a genome-wide ChIP-seq approach to identify FIE and hence PRC2 binding sites across the Arabidopsis genome. Based on our high-confidence dataset we find that the regions of PRC2 binding are largely within gene body regions and co-localise with H3K27me3. The emerging reports of plant PREs and our finding of GAGA motifs at FIE binding sites suggest that DNA binding proteins have a role in recruiting PRC2 in plants and that further dissection of potential PRE-like regions could help our understanding of how the PcG system is recruited to specific genes in plants.
All plants were grown on MS agar plates in a 16 h light: 8 h dark photoperiod under fluorescent lights at 22°C for 12 days. Whole seedlings were harvested for ChIP experiments or RNA extraction. T1 siFIE plants (in Col ecotype) were selected on plates supplemented with kanamycin (50 mgL-1). The swn-7 clf-28 mutant is sterile and was selected from the progeny of swn-7 clf-28/+ plants.
ChIP-seq and bioinformatic analysis
Native chromatin immunoprecipitation (N-ChIP) was performed as described previously  with minor modifications. In brief, Arabidopsis seedlings were collected and ground in liquid nitrogen. Nuclei were extracted with buffers 1, 2 and 3 and chromatin was digested by MNase for 6 minutes to generate native chromatin templates consisting primarily of mononucleosomes. Native chromatin templates were incubated with anti-H3K27me3 antibody (07–449, Millipore) and antibody-bound DNA fragments were extracted. ChIP DNA fragments were sequenced by Illumina (San Diego, CA) with an Illumina Genome Analyzer (GAII) by standard procedures. A control sample of input DNA from the micrococcal nuclease digested lysates before immunoprecipitation was also sequenced.
Material for FIE-HA ChIP was cross-linked with formaldehyde and ChIP carried out as previously described [35, 43] on mononucleosome sized micrococcal nuclease-digested lysates. Over 20 pull-downs were performed on sets of 1 g tissue for the FIE-HA line and the untransformed C24. ChIPs were selected for high enrichment relative to the C24 control by qPCR using a set of 6 diagnostic amplicons (Additional file 13: Table S2). DNA was pooled from the 12 immunoprecipitates that had highest enrichment and used for ChIP-seq as above except the sequencing was carried out by the Australian Genome Research Facility (Melbourne, Australia). As a control DNA extracted from the digested lysates before immunoprecipitation was also sequenced (input DNA).
The numbers of sequence reads obtained for each sample are detailed in Additional file 1: Table S1. The sequencing reads were mapped to the Arabidopsis genome (TAIR9 build) using BioKanga (http://biokanga.sourceforge.net/), allowing 2 mismatches at any position. Peaks were identified using the log2 ratio of signal density between two samples to determine enriched regions as candidates, followed by significance analysis on the read density from candidate peaks . Briefly, each read was extended L bp (L=150 bp for H3K27me3 and L=200 bp for FIE samples respectively) from the beginning of the 5′ end to represent the fragment length. Sx was the normalised number of the extended reads located within a 10 bp window along a chromosome for sample x; a log2 ratio was then calculated on the corresponding Sx as log2R = log2(Streatment/Scontrol). Adjacent windows that have a log2R above a threshold (the threshold was 3-fold enrichment when compared with input) were merged to form candidate peaks and peaks that have been separated less than 200 bp were further merged. Finally a significance test was performed using the PeakSeq algorithm  where a p value was obtained from binomial test on the number of reads within a candidate peak and a multiple test correction was followed to give a q value for estimation of false discovery rate. A q score of 10-10 was selected on the basis of maximizing the number of predicted peaks in the immunoprecipitated sample and minimizing the numbers of peaks identified in the control input DNA samples.
GO enrichment analysis was performed using the BINGO 2.44 plug-in  in Cytoscape 2.8.3  with the GOslim_plants dataset. To test for enrichment, a hypergeometric test was conducted and the Benjamini and Hochberg false discovery rate was calculated. The network of the enriched categories was presented.
MEME software (version 4.9.0)  was applied to yield over-represented motifs in the dataset. The width of the motif was set as 6 to 8 nucleotides. Zero or one per sequence was used for the distribution of a single motif among the sequences.
RNA extraction and qRT-PCR
RNA was extracted from approximately 100 mg of seedlings using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer’s instructions. For quantitative RT-PCR, DNase-treated RNA was reverse transcribed using an oligo dT primer and Superscript III reverse transcriptase (Invitrogen, http://www.invitrogen.com/) and at least triplicate reactions were amplified using 7900HT Fast Real-Time PCR System (Applied Biosystems, http://www.appliedbiosystems.com/) with SYBR green. The primers used are listed in Additional file 13: Table S2. For verification of ChIP peaks qPCR was carried out using a set of genomic DNA standards that allows the comparison of values between amplicons .
Expression array analysis
RNA was extracted from intermediate phenotype siFIE plants (Additional file 5: Figure S1), Col, clf-7 swn-28 and Col using Qiagen Plant RNeasy mini kit. For each sample, three pools of 10–12 plants were used. The siFIE plants were analysed for FIE mRNA by RT-qPCR; the maximum level of FIE mRNA was found to be 10% of wildtype Col.
RNA samples were hybridised to a Roche NimbleGen Arabidopsis Gene Expression 4x72K Array (catalogue number A4511001-00-01) representing 30,361 genes, each with 2 target probes as annotated by TAIR version 6. DNAstar software was used for analysis; gene lists with higher than 2 fold de-regulation at 95% confidence compared to wild type were exported to Excel files for comparison.
Availability of supporting data
The Nimblegen array date in this publication have been deposited in NCBI’s Gene Expression Omnibus  and are accessible through GEO Series accession number GSE48857 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE48857). The raw sequence data has been deposited into the NCBI Short Read Archive, accession number SRP027413.
quantitative polymerase chain reaction.
We would like to thank Justin Goodrich for swn-7 clf–28 seed, Iain Wilson for advice on microarray analysis, Jean Finnegan and Scott Boden for their critical reading of the manuscript. We would also like to thank Sue Allen and Anna Wielopolska for technical assistance.
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