Molecular and functional mapping of EED motifs required for PRC2-dependent histone methylation

Abstract

Polycomb group proteins represent a conserved family of developmental regulators that mediate heritable transcriptional silencing by modifying chromatin states. One Polycomb group complex, the PRC2 complex, is composed of several proteins, including the histone H3 lysine 27 (H3K27) methyltransferase enhancer of zeste homolog 2 and the WD-repeat protein embryonic ectoderm development (EED). Histone H3K27 can be monomethylated (H3K27me1), dimethylated (H3K27me2), or trimethylated (H3K27me3). However, it remains unclear what regulates the number of methyl groups added to H3K27 in a particular nucleosome. In mammalian cells, EED is present as four distinct isoforms, which are believed to be produced by utilizing four distinct, in-frame translation start sites in a common Eed mRNA. A mutation that disables all four EED isoforms produces defects in H3K27 methylation [Montgomery, N.D., Yee, D., Chen, A., Kalantry, S., Chamberlain, S.J., Otte, A.P. & Magnuson, T. (2005). The murine polycomb group protein Eed is required for global histone H3 lysine-27 methylation. Curr. Biol., 15, 942-947]. To assess the roles of individual EED isoforms in H3K27 methylation, we first characterized three of the four EED isoform start sites and then demonstrated that individual isoforms are not necessary for H3K27me1, H3K27me2, or H3K27me3. Instead, we show that the core WD-40 motifs and the histone-binding region of EED alone are sufficient for the generation of all three marks, demonstrating that EED isoforms do not control the number of methyl groups added to H3K27.

Figure 1. Localization of Histone H3K27 methylation marks

Immunofluorescence analysis of (A) H3K27me1, (B) H3K27me2, (C) H3K27me3, and (D) HP1-α in CD1 murine embryonic fibroblasts. The inactive X-chromosome is indicated by an arrow in (C).

Figure 2. Confirmation of EED isoform identities

Western blot analysis of EED comparing isoforms observed in embryonic stem cells to isoforms observed in (A) HeLa cells or (B) Mouse Wap-T121 breast tumors. On prolonged exposure, EED-2 becomes visible in HeLa lysates (Aright panel). EED isoforms 1–4 are indicated as 1, 2, 3, and 4.

Figure 3. Deletion mapping of EED translational start sites

(A) Constructs transfected into Eed mutant embryonic stem cell line 21 (below) are shown. Putative translation start sites for EED-1 (GUG 169–171), EED-2 (GUG 274–276), EED-3 (AUG 451–453), and EED-4 (AUG 493–495) are indicated in the schematic of the endogenous mRNA as 1,2,3, and 4 and correspond to codon locations in the mouse Eed transcript (Accession number: BC012966). The positions of the most 5′ and 3′ nucleotides of each construct are indicated in italics. Identifiers for each construct are shown to the left and indicate the number of nucleotides removed from the 5′ end of the Eed cDNA (e.g. Δ210 refers to a construct expressing an Eed cDNA lacking the 5′ 210 nucleotides of the full length message). (B) Whole-cell lysates from Eed^+/+ ES cell line 25.5 (Wild-type), Eed^−/− ES cell line 21 (Mutant), or Eed^−/− ES cell line 21 transiently transfected with the Eed expression constructs shown in (A)were analyzed by Western blotting with an antibody detecting EED.

Figure 4. Verification of EED isoform start sites

(A) Constructs transfected into Eed mutant embryonic stem cell line 21. The positions of the most 5′ and 3′ nucleotides of each construct are indicated in italics. 1,2,3, and 4 indicate reported EED translational start sites GUG 169–171, GUG 274–276, AUG 451–453, and AUG 493–495, respectively. Black boxes refer to strong Kozak-AUG sequences engineered into the expression construct in order to drive translation from the 169–171 codon (Kozak AUG 169–171) and from the 274–276 codon (Kozak AUG 274–276), respectively. “X” markings through the putative EED-3 and EED-4 start sites in Δ417 no3,4 and in Δ455 no4 indicate AUG→AUA mutations intended to disrupt translation intitation. (B and C) Whole-cell lysates from Eed^+/+ ES cell line 25.5 (Wild-type), Eed^−/− ES cell line 21 (Mutant), or Eed^−/− ES cell line 21 transiently transfected with the Eed expression constructs shown in (A) analyzed by Western blotting with an antibody detecting EED.

Figure 5. Histone H3K27 methylation in cells lacking one or more EED isoforms

Immunofluorescence analysis of H3K27me1, H3K27me2, and H3K27me3 in wild-type ES cell line 25.5 (Eed^+/+) and in Eed mutant ES cell line 21 either mock transfected (Eed ^−/−) or transiently transfected with the indicated constructs. Eed expression constructs transfected into ES cell line 21 are shown on the left. The positions of the most 5′ and 3′ nucleotides of each construct are indicated in italics. Isoform start sites at GUG 274–276, AUG 451–453, and AUG 493–495 are shown as 1*, 3, 4, respectively. 1* discrimates the GUG 274–276 start site for EED-1 reported here from the GUG169–171 start site reported previously; . “X” markings through the putative EED-3 and EED-4 starts sites in Full length no 3,4 and in Δ417 no3,4 represent AUG→AUA mutations intended to disrupt translation intitation. EED 1036–1038 L→P refers to Eed^{l7Rn5–3354SB}, a point mutant protein previously demonstrated to lack H3K27 methyltransferase activity. DAPI-stained DNA is blue, and methylated histones are shown in red. In the transient transfection assay, approximately 10% of the ES cells are successfully transfected, and with constructs expressing functional EED, a similar percentage of cells are rescued.

Figure 6. Functional mapping of required WD-40 motifs in EED

Immunofluorescence analysis of H3K27me3 in Eed mutant ES cell line 21 either mock transfected (Eed^−/−) or transiently transfected with the indicated constructs. Below each schematic, nucleotides encoding for the N- and C-terminal amino acids of each protein are indicated in italics. Above each schematic, “3” and “4” refer to translation start sites for EED isoforms 3 and 4. Diagonally-lined boxes refer to putative WD-40 motifs encoded by cDNA sequences 721–808 (WD-40 motif 1), 1012–1105 (WD-40 motif 2), 1150–1240 (WD-40 motif 3), 1330–1444 (WD-40 motif 4), and 1672-1774 (WD-40 motif 5) . Black boxes refer to consensus Kozak + ATG initiator sequences engineered into the construct. DAPI-stained DNA is blue, and methylated histone are shown in red.

Figure 7. EED-EZH2 interaction assessed by YFP fragment complentation assays

YFP fragment complentation assays in E14 ES cells transiently transfected with constructs expressing the following Venus 1 and Venus 2 fusions: (A) Venus1-GCN4 and GCN4-Venus2, (B) no DNA, (C) Venus1-EZH2 and Full length EED3-Venus2, (D) Venus1-EZH2 and EED Δ5′ 541-Venus2, (E) Venus1-EZH2 and EED Δ5′ 697-Venus2, (F) Venus1- EZH2 and EED Δ5′ 913-Venus2, (G) Venus1-EZH2 and EED Δ3′ 1663-End-Venus2, (H) Venus1-EZH2 and no Venus 2, and (I) no Venus 1 and Full length EED3-Venus2.

Figure 8. Levels of truncated EED proteins

The stability of truncated, tagged EED proteins was assessed by Western blotting lysates from transfected E14 embryonic stem cells with an anti-FLAG antibody. Three background bands observed with the FLAG antibody serve as loading controls and are indicated by asterisks.