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. 2013 Mar 15;27(6):596–601. doi: 10.1101/gad.211425.112

Figure 4.

Figure 4.

AS1–AS2-binding sites are necessary and sufficient for recruitment of PRC2 activity. ChIP analysis showing that H3K27me3 levels are enriched at the GFP-GUS reporter in lines where the transgene contains wild-type AS1–AS2-binding sites in the promoter. H3K27me3 levels are significantly reduced when the AS1–AS2-binding sites are mutated. (*) P < 0.05; (**) P < 0.01. Two pools comprising three independent transgenic lines were analyzed for each construct. Quantitative PCR values (mean ± SE) are normalized to H3 levels and calculated from three independent replicates. A schematic representation of the reporter lines is shown at the top. A BP promoter fragment spanning nucleotides −2707 to −1088 from the ATG was fused to the 35S minimal promoter and inserted upstream of a GFP-GUS fusion. (Black dash) Position of the amplicon analyzed; (red ovals) AS1–AS2 complex-binding sites; (arrow) transcription start site; (violet box) the 35S minimal promoter; (green box) GFP; (blue box) GUS. Sequence of the wild-type and mutated AS1–AS2-binding sites are shown below. Controls establishing the specificity and efficiency of ChIP reactions are shown in Supplemental Figure 7.