Figure 5. The aromatic cage in Drosophila ESC is important for its in vivo function.

(A) Left, Coomassie blue staining of reconstituted dPRC2-Ez complex. Right, HMT assay with dPRC2-Ez and H3K27, unmodified, mono, di or tri methylated peptides. (B) Top, amino acid residues Phe-77, Tyr-338 and Phe-345 that were mutated to alanine in Drosophila ESC and corresponding residues in Eed. Bottom, schematic representation of transposon constructs. The Myc-tagged ESC or its mutants are expressed under the control of the esc gene promoter. (C) Rescue experiment. Details of the crossing schemes are shown in Supplementary Figures S9 and S10. Several independent lines were examined for each transgenic construct and showed the same phenotype except for one line of Myc-ESC Tyr338Ala. In the case of Myc-ESC Phe345Ala, transgenes were inserted at •C31 att sites at 68E and 86Fb, respectively. For direct comparison, wild type Myc-ESC lines were also established at the same chromosomal location and showed the same results as other wild type Myc-ESC lines established by conventional P-element transformation (D) ESC aromatic cage mutation does not impair binding to E(Z). Both Myc-ESC and Myc-ESC Phe-77Ala co-immunoprecipitated E(Z) from ovarian extracts of heterozygous esc⁻escl⁻ flies. The double Myc-ESC bands are caused by the well known phosphorylation of ESC. (E) Scheme indicating the genomic location of primers used for ChIP. (F) ChIP analysis of E(Z) binding to the bxd PRE (FM4) in homozygous esc⁶, escl^d01514 carrying the same Myc-ESC transgenes. In the presence of the aromatic cage mutations, E(Z) binding is strongly reduced. yw indicates the wild type stock with endogenous wild type ESC and ESCL. (G) ChIP analysis of the H3K27me3 distribution at four sites in the Ubx gene. H3K27me3 is drastically reduced in the presence of the aromatic cage mutations. (H) Histone H3K27 methylation in esc⁶, escl^d01514 double mutant larvae expressing Myc-ESC transgenes. Total protein lysates from wild type and homozygous esc⁶, escl^d01514 3rd instar larvae expressing Myc-ESC transgenes were used for western blot analysis with anti-H3, anti-H3K27me2 and anti-H3K27me3 antibodies. The aromatic cage mutations Myc-ESC Phe77Ala and Myc-ESC Phe345Ala cause an almost complete loss of histone H3K27 di- and trimethylation while wild type Myc-ESC fully restores the loss of H3K27 di- and tri-methylation in esc⁶, escl^d01514 double mutant larvae.