1. Different Ezh2-Containing Complexes Target Methylation of Histone H1 or Nucleosomal Histone H3 
Andrei Kuzmichev, Thomas Jenuwein, Paul Tempst, Danny Reinberg 
Molecular Cell 
Volume 14, Issue 2, Pages (April 2004)
DOI: /S (04)

2. Figure 1
Fractionation of the Three Eed Protein Isoforms and Conventional Purification of PRC3
(A) The scheme for fractionation of HeLa nuclear pellet.
(B) Western blot analysis of Ezh2 and Eed at various fractionation steps (indicated on the brackets). Fraction numbers are shown on top of each panel. The migration of Ezh2 and the three Eed isoforms are indicated on the left side of each panel. The fractions of the DEAE-5PW column corresponding to the two Ezh2 complexes, PRC2 and PRC3, are indicated above the top panel.
(C) Conventional purification of PRC3. Purification of PRC3 followed the scheme in (A). Fraction numbers are shown on the top of each panel. Top panel: Silver staining of fractions of the final purification step, SMART-MonoP column. Arrows point to the PRC3 subunits which are indicated on the left. Molecular weight standards are indicated on the left. Middle panel: Western blot analysis of SMART-MonoP fractions. Western blots were probed with antibodies against proteins indicated on the left. Bottom panel: Histone methyltransferase activity of SMART-MonoP fractions. The assay was performed using recombinant histone octamers. The position of histone H3 is indicated.
Molecular Cell  , DOI: ( /S (04) )

3. Figure 2
Characterization of the Histone Lysine Methyltransferase Activity of PRC2 and PRC3 Complexes
(A) Analysis of site specificity. HMT assays were performed with recombinant histone octamers assembled with either wild-type histone H3 or the histone H3 point mutants indicated on the top. The reaction products were separated by SDS-PAGE and transferred onto a PVDF membrane. The membrane was Coomassie blue stained (bottom panel) and autoradiographed (top panel). PRC2 and PRC3 titration points are indicated. The positions of histone polypeptides are indicated.
(B) Analysis of nucleosomal activity of PRC2 and PRC3. HMT assays were performed with recombinant octamers (Recomb. octamer), with recombinant octamers assembled into oligonucleosomes with plasmid DNA by salt dialysis (Recomb. oligonuc.) or with native oligonucleosomes purified from HeLa cells (Native oligonuc.). PRC2 and PRC3 titration points are indicated. The top panel is the autoradiograph, and the bottom panel is the Coomassie blue staining of the HMT assay membrane.
(C) H1 methylation by PRC2 and PRC3 complexes. HMT assays were performed with either PRC2 or PRC3 complexes using substrates indicated on the top (Nuc corresponds to native oligonucleosome). The positions of core histones and histone H1 are indicated. The top panel is an autoradiograph, and the bottom panel is a Coomassie blue staining of the HMT assay membrane.
(D) Increasing amounts of histone H1 inhibit activity of PRC3 complex toward nucleosomal H3. PRC3 HMT assay was performed using substrates indicated on the top. Nuc corresponds to oligonucleosome. Increasing amounts of histone H1 are indicated by a triangle. The positions of core histones and histone H1 are indicated. Molar ratios of histone H1 to nucleosomes are indicated on the bottom. The top panel is an autoradiograph, and the bottom panel is a Coomassie blue staining of the HMT assay membrane.
Molecular Cell  , DOI: ( /S (04) )

4. Figure 3 Characterization of HMT Activity of the PRC Complexes
(A) PRC2 methylates wild-type but not K26A mutant H1b protein. The top panel is an autoradiograph, and the bottom panel is a Coomassie blue staining of an HMT assay membrane. The position of recombinant H1b is indicated. Wt corresponds to wild-type H1b, whereas K26A corresponds to H1b with a substitution of lysine 26 for alanine.
(B) Mouse H1e (20–31) peptide methylation assay. The alignment of sequences surrounding hH1b-K26, mH1e-K26, H3-K27, and H3-K9 is shown. The methylated lysine is indicated. The K26 methylation status of peptides is indicated on the top. Increasing amounts of the complex are indicated by triangles. The fraction used in this experiment also contains PRC4.
(C) H1 methylation by affinity-purified Ezh2 complexes. HMT assay was performed with Ezh2 complexes purified from stable cell lines expressing wild-type (Wt) or enzymatically inactive (H689A) FLAG-Ezh2 using substrates indicated on top. Nuc corresponds to native oligonucleosome. The top panel is an anti-FLAG Western blot, the middle panel is an autoradiograph, and the bottom panel is a Coomassie blue staining of the HMT assay membrane. The positions of core histones and histone H1 are indicated.
Molecular Cell  , DOI: ( /S (04) )

5. Figure 4 Methylation of Histone H1 Lysine 26 In Vivo
(A) Characterization of anti-trimethyl-K26 antibody by dot blot. Peptides are indicated on top. For sequences, see Figure 3B. Numbers 0, 1, 2, and 3 indicate the number of methyl groups on the lysine residue.
(B) Western blot of native (nH1) and recombinant (rH1b-HA) histone H1 using anti-trimethyl-K26 antibody. The positions of native and recombinant H1 are indicated.
(C) Overexpression of wild-type Ezh2 increases H1 K26 methylation. Extracts from 293 cells cotransfected with HA-H1b and FLAG-Ezh2 (wild-type, WT; or active site mutant, H689A) were probed with anti-FLAG, anti-trimethyl-K26, and anti-HA antibodies. The positions of FLAG-Ezh2 and HA-H1b are indicated.
(D) Wild-type histone H1 is necessary for transcriptional repression by Ezh2. Reporter construct with luciferase gene under control of promoter containing Gal4 DNA binding site was transfected into 293 cells together with an expression vector for Ezh2 protein (wild-type, WT; or active site mutant, H689A) fused with Gal4 DNA binding domain in the presence of empty vector (−), wild-type H1b (H1b(WT)), or H1b K26A mutant (H1b(K26A)). Fold repression was calculated as a ratio of normalized luciferase activities of Gal4 DNA binding domain alone and Gal-Ezh2 fusion. Error corresponds to two independent experiments.
Molecular Cell  , DOI: ( /S (04) )

6. Figure 5 Characterization of the Different Eed Proteins
(A) Characterization of the four Eed forms using antibodies against different regions of the Eed protein. Top panel is the scheme of the Eed protein. The C-terminal WD-40 repeats of Eed protein are indicated by Roman numerals. Translation initiation sites (Val1, Val36, Met95, and Met110) and two termination sites (494 and 535), as well as the regions of the protein used to raise antibodies, are indicated. The bottom panel is the Western blot of 293 cell extract using antibodies indicated on top. NE corresponds to nuclear extract; ip F-Ezh2 corresponds to anti-FLAG immunoprecipitates from extracts of FLAG-Ezh2 stable cell line. − and + PPase lanes correspond to mock-treated nuclear extracts and nuclear extracts treated with shrimp alkaline phosphatase, respectively. ΔN95 corresponds to a recombinant His-tagged Eed (95–535) protein.
(B) Effect of Eed deletion mutants ΔN30 and Eed ΔN95 on nucleosomal activity of PRC3. The PRC3 complex was assayed for activity toward recombinant histone octamer or oligonucleosome, alone (−) or in the presence of increasing amounts of bacterially expressed Eed ΔN30 or Eed ΔN95. The top panel is the anti-Eed Western blot, the middle panels are the autoradiograph and the Coomassie stain of the HKMT assay membrane, and the bottom panel is the quantification of the H3 methylation activity normalized to EED Western blot units. The positions of histones and Eed deletion mutants are indicated on the left.
Molecular Cell  , DOI: ( /S (04) )

7. Figure 6
Summary of the Different Ezh2-Containing Complexes and Their Specificity for Methylation of H3-K27 or H1-K26
The presence of the different Eed isoforms in each Ezh2-containing PRC2 or PRC3 complexes is indicated.
Molecular Cell  , DOI: ( /S (04) )