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. 2013 Jun 18;32(14):2073–2085. doi: 10.1038/emboj.2013.145

Figure 4.

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).