Arabidopsis MSI1 connects LHP1 to PRC2 complexes

Article18 June 2013free access Source Data Arabidopsis MSI1 connects LHP1 to PRC2 complexes Maria Derkacheva Maria Derkacheva Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Yvonne Steinbach Yvonne Steinbach Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Thomas Wildhaber Thomas Wildhaber Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Iva Mozgová Iva Mozgová Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Search for more papers by this author Walid Mahrez Walid Mahrez Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Paolo Nanni Paolo Nanni Functional Genomics Center Zurich, University of Zürich/ETH Zürich, Zurich, Switzerland Search for more papers by this author Sylvain Bischof Sylvain Bischof Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, SwitzerlandPresent address: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA. Search for more papers by this author Wilhelm Gruissem Wilhelm Gruissem Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Functional Genomics Center Zurich, University of Zürich/ETH Zürich, Zurich, Switzerland Search for more papers by this author Lars Hennig Corresponding Author Lars Hennig Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Science for Life Laboratory, Uppsala, Sweden Search for more papers by this author Maria Derkacheva Maria Derkacheva Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Yvonne Steinbach Yvonne Steinbach Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Thomas Wildhaber Thomas Wildhaber Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Iva Mozgová Iva Mozgová Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Search for more papers by this author Walid Mahrez Walid Mahrez Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Search for more papers by this author Paolo Nanni Paolo Nanni Functional Genomics Center Zurich, University of Zürich/ETH Zürich, Zurich, Switzerland Search for more papers by this author Sylvain Bischof Sylvain Bischof Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, SwitzerlandPresent address: Department of Molecular, Cell, and Developmental Biology, University of California Los Angeles, Los Angeles, CA, USA. Search for more papers by this author Wilhelm Gruissem Wilhelm Gruissem Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Functional Genomics Center Zurich, University of Zürich/ETH Zürich, Zurich, Switzerland Search for more papers by this author Lars Hennig Corresponding Author Lars Hennig Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland Science for Life Laboratory, Uppsala, Sweden Search for more papers by this author Author Information Maria Derkacheva1,2, Yvonne Steinbach2, Thomas Wildhaber2, Iva Mozgová1, Walid Mahrez1,2, Paolo Nanni3, Sylvain Bischof2, Wilhelm Gruissem2,3 and Lars Hennig 1,2,4 1Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, Uppsala, Sweden 2Department of Biology and Zurich-Basel Plant Science Center, ETH Zurich, Zurich, Switzerland 3Functional Genomics Center Zurich, University of Zürich/ETH Zürich, Zurich, Switzerland 4Science for Life Laboratory, Uppsala, Sweden *Corresponding author. Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences, and Linnean Center for Plant Biology, PO Box 7080, Uppsala SE-75007, Sweden. Tel.:+46 18 67 3326; Fax:+46 18 67 3389; E-mail: [email protected] The EMBO Journal (2013)32:2073-2085https://doi.org/10.1038/emboj.2013.145 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Polycomb group (PcG) proteins form essential epigenetic memory systems for controlling gene expression during development in plants and animals. However, the mechanism of plant PcG protein functions remains poorly understood. Here, we probed the composition and function of plant Polycomb repressive complex 2 (PRC2). This work established the fact that all known plant PRC2 complexes contain MSI1, a homologue of Drosophila p55. While p55 is not essential for the in vitro enzymatic activity of PRC2, plant MSI1 was required for the functions of the EMBRYONIC FLOWER and the VERNALIZATION PRC2 complexes including trimethylation of histone H3 Lys27 (H3K27) at the target chromatin, as well as gene repression and establishment of competence to flower. We found that MSI1 serves to link PRC2 to LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), a protein that binds H3K27me3 in vitro and in vivo and is required for a functional plant PcG system. The LHP1–MSI1 interaction forms a positive feedback loop to recruit PRC2 to chromatin that carries H3K27me3. Consequently, this can provide a mechanism for the faithful inheritance of local epigenetic information through replication. Introduction Most developmental decisions are based on tight regulation of transcription to establish and maintain specific gene expression patterns, and polycomb group (PcG) proteins are among the master regulators of different developmental programmes. PcG proteins were first identified in Drosophila as regulators of Hox gene expression (Lewis, 1978) and were subsequently found to represent an ancient and evolutionarily conserved mechanism of gene silencing (for reviews see Hennig and Derkacheva, 2009; Butenko and Ohad, 2011; Margueron and Reinberg, 2011). Animal and plant PcG proteins function by forming multi-subunit protein complexes such as Polycomb repressive complex 1 (PRC1) and PRC2. PRC2 is recruited to target genes and catalyses the trimethylation of lysine 27 of histone H3 (H3K27me3). Animal PRC1 binds to H3K27me3 and establishes monoubiquitylation of H2AK119. H3K27me3 is, however, not always required for PRC1 recruitment to target genes. Eventually, animal PcG proteins repress transcription by means of mechanisms that are not fully understood and that probably involve compaction of nucleosomes and interference with transcription elongation. In Drosophila and Arabidopsis, silencing by PcG proteins involves local restriction of DNA accessibility (Shu et al, 2012). The PRC1 complex was originally characterized in Drosophila, where it consists of four main subunits: polycomb (Pc), polyhomeotic (PH), posterior sex combs (Psc) and RING (Francis et al, 2001; Mohd-Sarip et al, 2002). Pc binds to H3K27me3 (Fischle et al, 2003), and RING catalyses H2AK119 monoubiquitylation (Wang et al, 2004; de Napoles et al, 2004). Similar to animals, plant PcG function seems to involve RING proteins that can monoubiquitylate H2A (Sanchez-Pulido et al, 2008; Xu and Shen, 2008; Bratzel et al, 2010; Li et al, 2011). Although plants lack Pc homologues, LIKE HETEROCHROMATIN PROTEIN 1 (LHP1), also known as TERMINAL FLOWER 2, is considered to fulfil the role of Pc in plants based on its ability to bind to H3K27me3 in vitro and its genome-wide co-localization with H3K27me3 in vivo (Turck et al, 2007; Zhang et al, 2007). LHP1 binding to H3K27me3 is required for its function (Exner et al, 2009), and LHP1 is required for repression of several PcG protein targets such as FLOWERING LOCUS C (FLC), FLOWERING TIME (FT) and AGAMOUS (AG) (Kotake et al, 2003; Libault et al, 2005). However, it remains unknown whether LHP1 has additional functions independent of the plant PcG system. In contrast to PRC1, homologues of all four core subunits of animal PRC2 exist in plants. The Arabidopsis genome encodes three homologues of the histone methyltransferase enhancer of zeste (E(z)): CURLY LEAF (CLF), SWINGER (SWN) and MEDEA (MEA); three homologues of the suppressor of zeste: EMBRYONIC FLOWER 2 (EMF2), FERTILIZATION INDEPENDENT SEED 2 (FIS2) and VERNALIZATION 2 (VRN2); a single extra sex comb homologue: FERTILIZATION INDEPENDENT ENDOSPERM (FIE); and five homologues of p55: MULTICOPY SUPRESSOR OF IRA 1–5 (MSI1–5). The diverse PRC2 subunit homologues in Arabidopsis probably form at least three different PRC2-like complexes with distinct functions. The VERNALIZATION (VRN) complex comprises VRN2, FIE, CLF or SWN and MSI1, and accelerates flowering in response to prolonged exposure to cold (Wood et al, 2006; De Lucia et al, 2008). The EMBRYONIC FLOWER (EMF) complex was proposed to control vegetative development and the transition to flowering and to comprise EMF2, FIE, CLF or SWN and one p55 homologue. An interaction of EMF2 with CLF was shown in vitro and in yeast two-hybrid assays (Chanvivattana et al, 2004), but the in vivo composition of the EMF complex awaits confirmation. Both EMF2 and VRN2 contribute to repression of the FLC (Gendall et al, 2001; Jiang et al, 2008). The FERTILIZATION INDEPENDENT SEED (FIS) complex has specific functions in the female gametophyte and the endosperm and comprises FIS2, FIE, MEA and MSI1 (Köhler et al, 2003; Spillane et al, 2000). MSI1–5 proteins belong to a subfamily of WD-40 repeat proteins, which are subunits of several chromatin-remodelling complexes in animals, plants and yeast. They do not have enzymatic activity but can bind to histones and serve as protein scaffolds (for a review, see Hennig et al, 2005). Although MSI1-like proteins were usually found among the core subunits of animal PRC2, they are not required for enzymatic activity in vitro (Cao and Zhang, 2004; Ketel et al, 2005; Schmitges et al, 2011). Similarly, the role of plant MSI1-like proteins in PcG gene silencing has been under debate. Arabidopsis MSI1 was shown to be part of the FIS complex and is essential for gametophyte and seed development (Köhler et al, 2003; Guitton et al, 2004; Guitton and Berger, 2005; Leroy et al, 2007). MSI1 co-purified with VRN2 (De Lucia et al, 2008), but it is not known whether MSI1 is required for VRN complex function and the vernalization response. Finally, which of the five MSI1-like proteins function in the EMF complex has not been established yet. Deficiency of MSI1 affects shoot apical meristems and floral meristems and primordia, suggesting a role in vegetative plant development and transition to flowering (Hennig et al, 2003; Bouveret et al, 2006; Schönrock et al, 2006), possibly as part of the EMF complex. Similar to MSI1, MSI4 and MSI5 regulate the transition to flowering (Kim et al, 2004; Ausin et al, 2004; Gu et al, 2011). Recently, co-immunoprecipitation of MSI4 with CLF was shown, suggesting that MSI4 instead of MSI1 could be part of the EMF complex (Pazhouhandeh et al, 2011). In this study, we have analysed the function of MSI1 in sporophytic PRC2 complexes in Arabidopsis. Purification of the EMF complex established MSI1 but not MSI4 as a core subunit. Similarly, MSI1 but not MSI4 interacts with EMF2. MSI1 is recruited to the chromatin of EMF target genes, where it is required for transcriptional silencing. Further, we find that MSI1 is recruited to the FLC locus where it is required for stable repression by cold and for a normal vernalization response. Our data indicate that MSI1 is an indispensable subunit of all PRC2 complexes in Arabidopsis. MSI1 was found to interact with LHP1, a major protein for PRC1-like functions in plants. We suggest that a physical link between plant PRC2-like and PRC1-like complexes contributes to the inheritance of H3K27me3 during DNA replication and to the maintenance of H3K27me3 levels during interphase. Results MSI1 is a core subunit of the EMF complex EMF2 is essential for vegetative plant development (Yang et al, 1995; Yoshida et al, 2001), but the proposed EMF complex has not been isolated yet. To uncover the composition of the EMF complex in vivo, we expressed a FLAG-tagged EMF2 in Arabidopsis and immunoaffinity-purified the FLAG–EMF2 complex from inflorescences. Wild-type plants served as controls. The purified fractions from four independent experiments were analysed by mass spectrometry. Measured spectra were searched with Mascot against the Arabidopsis TAIR9 protein database using a concatenated decoy database and imported into Scaffold. Cutoffs of 90% minimal confidence for protein identification and of 95% minimal confidence for peptide identification were applied. These criteria resulted in a spectrum false-discovery rate below 1%. Only proteins identified with at least two peptides in at least two replicates but not in control samples were taken into account. Three plant PcG proteins were found to co-purify with EMF2: FIE, SWN and MSI1 (Table I and Supplementary Table S1). This is the first demonstration of the composition of the plant EMF complex in vivo, showing that the core EMF complex consists of the four main subunits EMF2, MSI1, FIE and SWN. MSI2, 3, 4 and 5 were not found in any experiment, suggesting that these MSI1 homologues are not part of the core EMF complex in inflorescences. Table 1. EMF2 co-purifies with PcG proteins Protein Number of unique peptides/ probability of identification 95% IP1-IP2-IP3-IP4 Sequence coverage (%) IP1-IP2-IP3-IP4 Protein identification probability (%) IP1-IP2-IP3-IP4 EMF2 12-13-12-12 15-21-16-16 100-100-100-100 MSI1 10-7-7-10 34-23-23-38 100-100-100-100 FIE 3-5-5-5 9.8-19-17-14 100-100-100-100 SWN 3-5-3-4 5-7.4-5-5.5 100-100-100-100 FLAG–EMF2 was expressed in Arabidopsis under the control of the 35S promoter. Proteins were identified by immunoaffinity purification of FLAG–EMF2 and mass spectrometry. The experiment was performed with four biological replicates (IP1-4) using inflorescences. Shown are all identified plant PcG proteins. To verify the presence of MSI1 in the EMF complex, we tested the interaction of MSI1 and EMF2 in vivo. YFP-tagged EMF2 (YFP–EMF2) and HA-tagged MSI1 (HA–MSI1) or MSI4 (HA–MSI4) were transiently co-expressed in Nicotiana benthamiana leaves. YFP–EMF2 was immunoaffinity-purified, and the presence of the co-precipitating proteins was analysed on protein immunoblots. HA–MSI1 but not HA–MSI4 was co-precipitated with YFP–EMF2 (Figure 1A). This result confirms that MSI1 and EMF2 associate into a common complex in vivo. MSI4 did not interact with EMF2 in vivo in this assay. This finding not only establishes the specificity of the assay but also strengthens the notion that MSI1 but not MSI4 is a core EMF complex subunit in vivo. Figure 1.MSI1 is a key subunit of the EMF complex in vivo. (A) MSI1 co-purifies with EMF2. HA–MSI1 and YFP–EMF2 or HA–MSI4 and YFP–EMF2 were expressed in N. benthamiana leaves under the control of 35 S promoter. YFP–EMF2 was immunoprecipitated, and precipitates were analysed by immunoblotting using anti-HA antibodies. (B) MSI1 and CLF are present in the same complex in vivo. AcV5–CLF and HA–MSI1 were expressed in N. benthamiana leaves under the control of 35 S promoter. AcV5–CLF was immunoprecipitated, and the precipitates were analysed by immunoblotting using anti-HA antibodies. Wild-type N. benthamiana leaves were used as a control. (C) Lack of MSI1 and lack of EMF2 cause similar changes in the transcriptome. Transcript signal log ratios (SLR) for an MSI1 co-suppression line (msi1–cs) and an emf2 mutant were plotted. The colour gradient (dark blue to yellow) represents local data point density. The white diagonal line represents identical changes in msi1–cs and emf2. (D) MSI1 is needed for repression of EMF target genes. Quantitative RT–PCR was performed on cDNA from rosette leaves of 6-week-old plants. Relative expression values are shown as mean ±s.e. (n=3). Values were normalized to a PP2A gene (At1g13320). (E) MSI1 is recruited to the chromatin of the EMF target genes. Left: Genomic structure of PI, AG and MAF5. Black lines, introns; red line, promoter region; wide bars, exons. Arrows represent the position of primers used for qPCR. The intergenic control region is on chromosome 1 from 8383019 to 8383083 between At1G23700 and At1G23710. Values are recovery as percent of input; shown are mean ±s.d. (n=3).Source data for this figure is available on the online supplementary information page. Source Data for Figure 1A [embj2013145-sup-0001-SourceData-S1.pdf] Download figure Download PowerPoint To provide independent confirmation for the presence of MSI1 in the EMF complex, we performed reciprocal immunoaffinity purification experiments using an Arabidopsis line expressing GFP-tagged MSI1 (MSI1–GFP) (Alexandre et al, 2009) and a GFP control line. Purified fractions were analysed by mass spectrometry in order to identify proteins co-precipitating with MSI1–GFP. Four independent experiments firmly established the presence of MSI1, EMF2, FIE and SWN in the complex (Table II). PcG proteins EMF2, FIE and SWN consistently co-purified with MSI1–GFP, confirming that MSI1 is a core subunit of the EMF complex in vivo. Consistent with earlier observations (De Lucia et al, 2008), the VRN2, VRN5 and VEL1 subunits of the VRN PRC2 complex were also found to associate with MSI1 in vivo (Table II). Several non-PcG proteins co-purified with MSI1, including homologues of yeast Rpd3 histone deacetylase complexes (Supplementary Table S2). To confirm these results, we performed additional immunoaffinity purification experiments using a modified protocol involving protein–protein cross-linking prior to protein extraction. These experiments confirmed the presence of the initially identified MSI1 interactors, except for VRN5, and revealed additional candidate interactions (Table III and Supplementary Table S2). Notably, the plant PcG protein LHP1 was found with high confidence in both additional experiments. Table 2. MSI1 co-purifies with PcG proteins Protein Number of unique peptides/probability of identification 95% IP1-IP2-IP3-IP4 Sequence coverage (%) IP1-IP2-IP3-IP4 Protein identification probability (%) IP1-IP2-IP3-IP4 MSI1 19-34-20-27 52-77-60-71 100-100-100-100 EMF2 10-11-8-6 21-23-15-12 100-100-100-100 FIE 9-0-9-0 29-0-27-0 100-0-100-0 SWN 15-8-14-3 19-9.5-20-4.1 100-100-100-100 VRN2 3-3-2-0 7-8.2-4.8-0 100-100-100-0 VRN5 3-2-0-0 5.1-4.7-0-0 100-100-0-0 VEL1 6-7-2-3 10-11-3.8-5.3 100-100-100-100 MSI1–GFP was expressed in Arabidopsis under the control of the MSI1 promoter. Proteins were identified by immunoaffinity purification of MSI1–GFP and mass spectrometry. The experiment was performed with four biological replicates (IP1-4) using inflorescences. Shown are all identified plant PcG proteins. Table 3. Co-purification of MSI1 with PcG proteins from cross-linked protein extracts Protein Number of unique peptides/probability of identification 95% IP5c-IP6c Sequence coverage (%) IP5c-IP6c Protein identification probability (%) IP5c-IP6c MSI1 26-23 67-55 100-100 EMF2 11-7 20-11 100-100 FIE 10-4 36-15 100-100 SWN 15-5 19-6.1 100-100 VEL1 16-4 23-6.9 100-100 LHP1 7-3 14-10 100-100 MSI1–GFP was expressed in Arabidopsis under the control of the MSI1 promoter. Proteins were identified by immunoaffinity purification of MSI1–GFP and mass spectrometry. The experiment was performed with two biological replicates (IP5c-6c) using inflorescences. Shown are all identified plant PcG proteins. Unexpectedly, the well-characterized Arabidopsis PcG protein CLF (Goodrich et al, 1997) was not found among the MSI1-binding partners. CLF plays a major role during sporophytic plant development (Goodrich et al, 1997; Chanvivattana et al, 2004; Katz et al, 2004; Wood et al, 2006; Jiang et al, 2008; Doyle and Amasino, 2009) and interacts with EMF2 in vitro and in yeast two-hybrid assays (Chanvivattana et al, 2004), suggesting that CLF is part of the EMF complex. Identification of proteins by mass spectrometry is affected by many protein-specific factors including protein abundance (Lubec and Afjehi-Sadat, 2007), and it is possible that CLF interacts with MSI1 but failed to be detected under our experimental conditions. This notion was supported by the considerably weaker expression of CLF compared with SWN at both transcript and protein levels (Zimmermann et al, 2004; Baerenfaller et al, 2011). Therefore, we tested whether MSI1 interacts with CLF in vivo using an alternative approach. AcV5-tagged CLF (AcV5–CLF) and HA–MSI1 were transiently co-expressed in tobacco leaves, AcV5–CLF was immunoaffinity-purified, and the presence of the co-precipitating proteins was analysed on protein immunoblots. HA–MSI1 was co-precipitated with AcV5–CLF (Figure 1B). This result demonstrates that MSI1 and CLF can associate into a common complex in vivo. Together, these experiments establish that MSI1, EMF2 and FIE, together with SWN or CLF, constitute the EMF complex. In contrast, there is no strong evidence for functions of MSI2–5 in the EMF complex. MSI1 is essential for the function of the EMF complex To establish whether MSI1 is required for the function of the EMF complex, we determined the expression levels of EMF target genes in an MSI1 co-suppression line (msi1–cs) in which the MSI1 protein level is reduced to less than 10% (Hennig et al, 2003). We compared the transcriptional profiles of msi1–cs (Alexandre et al, 2009) and emf2 plants (Liu et al, 2012) and found that transcriptional changes were strongly and significantly correlated between plants of the two genotypes (Pearson correlation=0.44, P<2.2e−16) (Figure 1C). Note that this strong correlation was observed despite considerable differences in experimental conditions (rosette leaves of 23-day-old msi1–cs plants that retain ∼5% MSI1 protein and 7-day-old emf2-null mutant seedlings). The global similarity of transcriptional changes caused by reduced MSI1 or EMF2 loss of function strongly suggests that the biochemical interaction of MSI1 and EMF2 is of functional relevance. The data also confirm that redundancy among MSI1 homologues is limited and that MSI2–5 can only partially, if at all, substitute MSI1 in the EMF complex. To confirm the microarray data on deregulation of EMF target genes in msi1–cs plants, we tested the expression of some known PcG target genes in leaves (Lafos et al, 2011) by RT–qPCR using independent samples (Figure 1D). Ten of 11 tested PcG target genes were upregulated in msi1–cs plants, demonstrating that the presence of MSI1 in the EMF complex is necessary for the repression of many EMF target genes. Next, we used ChIP to test whether MSI1 binds to EMF target genes. The results show an enrichment of MSI1 at the previously described EMF target genes PISTILLATA (PI), AG and MADS AFFECTING FLOWERING 5(MAF5) (Figure 1E), demonstrating that MSI1 is recruited to at least some EMF target genes. Because PRC2 complexes trimethylate H3K27 in target chromatin, we tested whether MSI1 is needed for this PRC2 function. We found that global H3K27me3 levels were reduced to 70% in msi1–cs plants (Figure 2A, Supplementary Figure S1). Similarly, ChIP results also showed that H3K27me3 is highly reduced in EMF target genes in msi1–cs plants (Figure 2B). Notably, At3g28007 has no increase in expression in msi1–cs but has reduced H3K27me3 demonstrating that loss of H3K27me3 is not a consequence of increased transcription. Together, these results demonstrate that MSI1 is required for full PRC2 function and normal H3K27me3 levels in vivo. Because the MSI1-like subunit was found to be dispensable for PRC2 catalytic activity in vitro (Schmitges et al, 2011), our findings suggest that MSI1 functions in PRC2 regulation or targeting in vivo. Figure 2.MSI1 is needed for trimethylation of H3K27. (A) Global H3K27me3 levels are reduced in msi1–cs plants. Total protein levels were analysed by quantitative immunoblotting using anti-H3K27me3 and anti-H3 antibodies in Col and msi1–cs plants. Shown are mean ±s.d. (n=3). (B) H3K27me3 is reduced at the chromatin of EMF target genes in msi1–cs plants. Top: genomic structure of SEP3, MAF5, AG, AT3G28007 and ACTIN7. Black lines, introns; wide bars, exons. Arrows represent the position of primers used for qPCR. Values are recovery as percent of input; shown are mean ±s.d. (n=3). Download figure Download PowerPoint MSI1 regulates FLC expression and the vernalization response MSI1 is a subunit of the VRN–PHD complex (Table II and De Lucia et al, 2008), which represses FLC after vernalization, but the function of MSI1 in this complex has not been addressed so far. The msi1–cs line showed MSI1 protein reduction and developmental alterations only at the rosette stage (Hennig et al, 2003) and thus did not appear suitable for testing MSI1 function in seedling vernalization. In contrast, MSI1 anti-sense (msi1–as) lines contain about 30–50% of wild-type MSI1 levels in seedlings and exhibit developmental alterations at seedling and rosette stages (Exner et al, 2006). To test whether MSI1 also functions in the vernalization response, we analysed flowering time and FLC expression with and without vernalization in msi1–as and wild-type plants. Vernalized wild-type plants flowered earlier than non-vernalized plants, forming only about half the number of rosette leaves (Figure 3A). Consistent with the phenotype, FLC transcript levels were strongly reduced in vernalized wild-type plants compared with non-vernalized controls (Figure 3B). In contrast, vernalized msi1–as plants flowered similarly to non-vernalized msi1–as plants (Figure 3A), revealing that a normal vernalization response requires MSI1. Non-vernalized msi1–as plants flowered earlier than non-vernalized wild type, possibly because of a partial loss of repression of floral activators that are under PcG protein control. Without vernalization, FLC levels were increased in msi1–as (Figure 3B). Under such conditions, FLC is controlled by the EMF complex (Jiang et al, 2008), and the increased FLC expression in msi1–as is consistent with the requirement for MSI1 in EMF complex function. More importantly, vernalization was less effective in reducing FLC transcript levels in msi1–as than in wild-type plants (11-fold versus 22-fold reduction) (Figure 3B). The reduced efficiency of vernalization treatments to repress FLC and accelerate flowering in msi1–as demonstrates that MSI1 is required for a normal vernalization response. Figure 3.MSI1 functions in the vernalization response via regulation of FLC expression. (A) The vernalization response is strongly impaired in MSI1 anti-sense plants (msi1–as). Plants were vernalized for 6 weeks followed by cultivation in SD. Flowering time was measured as the number of rosette leaves produced before bolting. Shown are means±SE (n≤14). (B) FLC is only partially repressed by vernalization in msi1–as plants. Quantitative RT–PCR was performed on cDNA from vernalized (6 weeks at 4°C and 10 days at 23°C) and non-vernalized (10 days at 23°C) plants grown in SD. Relative expression values are shown as mean ±SE (n=3). Values were normalized to a PP2A gene. Values shown above bars represent fold change relative to the wild-type control. (C) MSI1 is recruited to the FLC locus. Top: Genomic structure of FLC and ACTIN7. Black lines, introns; wide bars, exons. Arrows represent the position of primers used for qPCR. Values are recovery as percent of input; shown are mean ±s.d. (n=3). Download figure Download PowerPoint Next, we tested whether regulation of FLC by MSI1 is direct. In ChIP experiments, MSI1 was enriched at FLC both without and after vernalization (Figure 3C), demonstrating that MSI1 is indeed recruited to FLC. The core VRN complex is present at the FLC locus already without vernalization (De Lucia et al, 2008) and EMF2 also regulates FLC (Jian