1. Unique Structural Platforms of Suz12 Dictate Distinct Classes of PRC2 for Chromatin Binding 
Siming Chen, Lianying Jiao, Murtada Shubbar, Xin Yang, Xin Liu 
Molecular Cell 
Volume 69, Issue 5, Pages e5 (March 2018)
DOI: /j.molcel
Copyright © 2018 Elsevier Inc. Terms and Conditions

2. Molecular Cell 2018 69, 840-852.e5DOI: (10.1016/j.molcel.2018.01.039)
Copyright © 2018 Elsevier Inc. Terms and Conditions

3. Figure 1
Overall Structure of the Suz12-Rbbp4-Jarid2-Aebp2 Heterotetrameric Complex Active in Nucleosome Binding
(A) Domain architecture of the Jarid2- and Aebp2-containing holo-PRC2 complex is outlined. Compositions of the catalytic module and the nucleosome-binding module of the core PRC2 complex are indicated. Protein domains of Suz12, Rbbp4, Jarid2, and Aebp2 included in the crystal structure of the heterotetrameric complex are color coded and labeled. Domains not included in the structure are in gray.
(B) Top view of the cartoon representation of the heterotetrameric complex. Structure of individual domains is colored and labeled according to the schematic in (A). Disordered loops that are missing from the structure are indicated by dotted lines. Zinc atom coordinated by the C2H2-type zinc finger of Suz12 is represented as an orange sphere. A rotation matrix is provided to guide the transition of the views from (B) to (C).
(C) Side view of the complex.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

4. Figure 2
Suz12 Structure Organizes Distinct Classes of PRC2 Holo Complexes
(A) A schematic highlighting the domains of Suz12 that define different classes of PRC2 holo complexes. Schematics of Phf19 and EPOP are also shown.
(B and C) The C2B-H3K4D domain of Aebp2 (residues 407–503) (B) and the RC domain of Phf19 (residues 500–580) (C) form stoichiometric complexes with the C2 domain of Suz12 (residues 146–363). SDS-PAGE gels of the binary complexes are shown along with the respective elution profiles of size-exclusion chromatography.
(D) Aebp2(C2B-H3K4D), but not Jarid2 (residues 119–232), that harbors the TR domain competes with Phf19(RC) for S12R4 binding. S12R4 was preincubated with Aebp2, Jarid2, or BSA prior to pull-down by GST-tagged Phf19(RC).
(E) This is a reciprocal assay to that shown in (D). S12R4 was preincubated with Phf19, Jarid2, or BSA prior to pull-down by GST-tagged Aebp2(C2B-H3K4D).
(F) A schematic illustrating that the C2 domain of Suz12 mediates the mutually exclusive binding of the C2B domain of Aebp2 and the RC domain of Phf19.
(G) A truncation of the Zn domain of Suz12 (residues 76–447) abolished the binding. A FLAG-tagged Jarid2 (residues 119–232) harboring the TR domain was used for the pull-down assay.
(H) An intact Zn domain of Suz12 is also necessary for EPOP binding to S12R4. A FLAG-tagged EPOP (residues 285–379) was used for the pull-down assay.
(I) The binding of Jarid2 and EPOP to S12R4 is mutually exclusive. Jarid2 lost binding to S12R4 pre-bound to EPOP.
(J) A schematic illustrating that the ZnB-Zn surface of Suz12 mediates the competitive binding of the TR domain of Jarid2 and the C-terminal domain of EPOP.
(K) The S12R4 nucleosome-binding module of human PRC2 does not bind to a 147-bp “601” mononucleosome in an EMSA.
(L) S12R4J2A2 is active in nucleosome binding. Both Jarid2(TR) and Aebp2(C2B-H3K4D) fragments were required for nucleosome binding with a low micromolar binding affinity. The S12R4J2A2-bound mononucleosome ran as a well-defined shifted band on the native gel, indicative of formation of a specific supercomplex.
(M) S12R4P19EPOP exhibits poor nucleosome binding.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

5. Figure 3 The Non-canonical C2 Domain of Suz12
(A) A zoomed-in view of the C2 domain of Suz12 shown in Figure 1C. The β strands and interstrand loops from the β sandwich structure are labeled.
(B) Schematic depiction of the topology of the C2 domain.
(C) Sequence alignment of the C2 domain of human Suz12 and Drosophila Su(z)12. Both the β strands and interstrand loops are labeled.
(D) The C2 domain of Suz12 in S12R4J2A2 sits on an edge of the top surface of Rbbp4. The view is related to the side view of the heterotetrameric complex shown in Figure 1C by a 240° rotation along the y axis. Only the C2 domain of Suz12 and Rbbp4 from the complex are shown in cartoon for clarity. Residue R196 of the C2 domain, which functions as an arginine anchor on the Rbbp4 surface, is highlighted as sticks in a dotted box.
(E) Zoomed-in view of an acidic patch on the Rbbp4 surface that captures the R196 arginine anchor of the C2 domain. Extensive interactions on the binding interface are indicated by the black dotted lines. The blue mesh represents the well-ordered 2Fo-Fc electron density map of the binding interface contoured at 1σ.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

6. Figure 4
Competitive Binding of Suz12 and the Histone H3K4 Tail to Rbbp4 in the Core PRC2 Complex
(A and B) ITC measurement of a synthetic histone H3K4 peptide (residues 1–19) titrated into Rbbp4 (A) and the S12R4 (B) binary complex.
(C) Competitive pull-down assay using a biotinylated histone H3K4 peptide harboring residues 1–21 of histone H3.
(D) Domain contribution to the inhibition of H3K4 binding to Rbbp4. As illustrated by the schematic, progressive deletion mutants of Suz12 were used in the context of the S12R4 binary complex. The presence of the C2 domain in Suz1276–363 was sufficient to cause a notable inhibition compared to Rbbp4 alone. Inclusion of the WDB2 domain in Suz1276–545 further enhanced the inhibition.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

7. Figure 5
Isoform- and Species-Specific C Termini of Aebp2 Regulate Nucleosome Binding by a Class of PRC2 Holo Complex
(A) A zoomed-in view of the top view shown in Figure 1B. The structure of S12R4J2A2 is compared to the structure of the Nurf55-H3K4 binary complex (PDB: 2YBA). The aligned Rbbp4/Nurf55 proteins from these two complexes are shown in pink and gray, respectively. The WDB2 domain and the L2 loop of the C2 domain of Suz12 are shown in purple and green. The H3K4D domain of Aebp2 is in gold, and the histone H3K4 peptide is in magenta. The C terminus of the H3K4D domain and the N terminus of the histone H3K4 peptide are both inserted into the axial channel of Rbbp4/Nurf55. The dotted arrows represent the opposite polarity and show the orthogonal spatial relationship of the H3K4D domain of Aebp2 and the histone H3K4 peptide.
(B) Zoomed-in view of the direct competition between the H3K4D domain of Aebp2 and the histone H3K4 peptide. Residues K502 and R503 of the H3K4D domain occupy the same binding pockets and interact with the same set of Rbbp4 residues as residues K4 and R2 of the histone H3K4 peptide, respectively.
(C) The reconstituted 147-bp mononucleosomes are shown together with the results of the EMSAs. The wild-type and tailless H3-containing mononucleosomes display comparable binding affinities to S12R4J2A2.
(D) Quantitative comparison of the mononucleosome-binding affinity of S12R4J2A2 containing Aebp2 variants. Aebp2a (residues 407–517 of isoform 1) contains the C terminus of human Aebp2 isoforms 1 and 3, and Aebp2d (residues 407–498 of isoform 2) mimics the Aebp2 homologs from some lower eukaryotes, where the H3K4D domain is missing. Three independent EMSAs for each of the S12R4J2A2 complexes were performed, and the graph displays mean ± SEM. A representative EMSA gel is shown in Figure S6D.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

8. Figure 6 A Composite Suz12 Surface for Jarid2 Binding
(A) Surface representation of the composite Suz12 surface formed by the Zn (violet) and ZnB (cyan) domains. The TR domain of Jarid2 is shown in cartoon with a limon color. The rotation matrix relative to the top view of the heterotetrameric complex in Figure 1B is provided.
(B) The view is the same as in (A). The Zn and ZnB domains of Suz12 and the TR domain of Jarid2 are shown in cartoon representation. The breakpoint on the ZnB domain of Suz12 from the oncogenic chromosomal translocation is indicated by a red arrow.
(C) Jarid2 binding was abolished for the Δ79–106 deletion mutant and the F86A/F90A double mutant of the ZnB domain of Suz12.
(D) Suz12 and the JAZF1-Suz12 oncogenic fusion protein were co-expressed to a comparable level in HEK293T cells that also ectopically expressed the other core subunits of PRC2 and Jarid2. Jarid2 binding was dominated by Suz12, but not JAZF1-Suz12. Suz12 and JAZF1-Suz12 were run separately next to the marker lane as the gel migration control.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions

9. Figure 7
A Schematic Model of Chromatin Binding by Distinct Classes of PRC2 Holo Complexes Dictated by Suz12
(A) The structure of Suz12 is shown based on Figure S1E. The binding partners of each domain of Suz12 in the Jarid2 and Aebp2-containing 6 subunit PRC2 holo complex are indicated.
(B) Three structural surfaces of Suz12, including the VFES, C2, and ZnB-Zn, are highlighted in colors. Suz12-interacting domains of the accessory subunits are also colored. Accessory subunits play essential roles in linker DNA binding (not shown), nucleosome core binding, and histone tail binding. S12R4J2A2 is sufficient to bind a mononucleosome core. The C2 domain of Suz12 and the H3K4D domain of Aebp2 block histone H3K4 binding to Rbbp4. The UI motif of Jarid2 contributes to PRC2 binding to H2AK119ub-containing nucleosomes. The tudor domain of Phf19, Mtf2, or Phf1 binds the H3K36me3 histone tail directly. Other regulatory histone tails that may also facilitate the chromatin recruitment of PRC2, such as H3K27me3, are not shown for clarity.
Molecular Cell  , e5DOI: ( /j.molcel )
Copyright © 2018 Elsevier Inc. Terms and Conditions