Arabidopsis MSI1 connects LHP1 to PRC2 complexes

Abstract

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.

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.

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).

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).

Figure 4

MSI1 connects LHP1 to PRC2. (A) MSI1 co-purifies with LHP1. LHP1–GFP was immunoprecipitated from inflorescences of 35 S::LHP1–GFP plants, and precipitates were analysed by immunoblotting using anti-MSI1 antibodies. Col wild-type plants served as control. (B) MSI1 interacts directly with LHP1. LHP1–myc was immunoprecipitated from extracts of yeast expressing either HA–GAL4–AD–MSI1 and LHP1–myc or HA–GAL4–AD-–MSI4 and LHP1–myc or HA–GAL4–AD and LHP1–myc. Precipitates were analysed by immunoblotting using anti-HA antibodies. (C) LHP1 co-purifies with EMF2. GFP was immunoprecipitated from inflorescences of plants expressing LHP1–GFP and EMF2–FLAG or GFP, respectively, under the control of the 35S promoter and analysed by immunoblotting using anti-FLAG antibodies. The asterisk marks an unspecific, cross-reacting band. (D) LHP1 function in gene silencing is restricted to the PcG system. Lack of LHP1 and lack of CLF cause similar changes in the transcriptome. Signal log ratios (SLR) for a clf and an lhp1 mutant were plotted. The colour gradient (dark blue to yellow) represents local data point density. The white diagonal line represents identical changes in clf and lhp1. (E) Only PcG target genes with the potential to be expressed in leaves are upregulated in lhp1 and clf mutants. A gene’s potential to be expressed in wild-type leaves was estimated as its maximal expression in wild-type leaves according to the developmental series of AtGenExpress transcriptome data (Schmid et al, 2003). (All) all genes; (PcG) all PcG target genes in leaves (Lafos et al, 2011); (lhp1 up) PcG target genes from leaves that are upregulated in lhp1; (clf up) PcG target genes from leaves that are upregulated in clf; (not up) PcG target genes from leaves that are not upregulated in lhp1 or clf. While most PcG genes have very low leaf expression potentials and are thus inactive throughout wild-type leaf development, PcG target genes that were upregulated in lhp1 or clf had very high expression potentials and thus are active at certain stages of wild-type leaf development. (F) H3K27me3 at PcG target genes is reduced in lhp1 mutants. ChIP was done using roots enriched for dividing cells by 2,4-D treatment. Values are recovery as percent of input; shown are mean ±s.d. (n=3).

Figure 5

Model of LHP1 function in semi-conservative inheritance of H3K27me3. During DNA replication, new histones are incorporated into chromatin diluting epigenetic marks. We propose that LHP1 binds to nucleosomes with old histones that carry H3K27me3 and via binding to MSI1 recruits the EMF complex, which trimethylates H3K27 of newly incorporated histones. H3K27me3 is symbolized by red circles, old and new nucleosomes are grey and green, respectively.