Article Figures & Data
Figures
- Figure S1. Multiple sequence alignment identity matrix of PcG-finder and experimentally identified orthologs.
(A, B, C) The analyses were performed using the Clustal Omega tool (https://www.ebi.ac.uk/Tools/msa/clustalo/) for (A) Su(z)12 full-length sequence, (B) VEFS-Box domain sequence, and (C) ESC full-length sequence.
- Figure 1. Species cladogram showing the distribution of PRC2 subunit orthologs identified using PcG-finder across eukaryotic lineages.
Of the 283 species analyzed using the PcG-finder, only species with the BUSCO score C ≥ 50 (181 species) are plotted (Table S2), and species with score C ≥ 75 (61 species) are marked in bold. Empty symbols indicate one identified homolog, filled symbols indicate multiple identified homologs. Homologs may originate from one or more genomic loci. In case of organisms in which only VEFS-domain (Su(z)12-like) subunits were identified, Su(z)12 is depicted using a gray dotted-line triangle. Cladogram topology is based on the recently proposed topology of the eukaryotic phylogenetic tree (Burki et al, 2020). SAR = Stramenopila, Alveolata, Rhizaria.
- Figure S2. A fast phylogeny tree and domain organization of the fungal VEFS-Box [Su(z)12-like] proteins.
The tree was generated using the iTOL tool (https://itol.embl.de) based on the SMART domains database (http://smart.embl.de).
- Figure S3. ML-eukaryotic phylogenetic trees of PRC2 subunits show the evolutionary relationship between orthologs.
(A, B, C) The trees were constructed using the whole protein sequences of (A) Su(z)12, (B) ESC, and (C) NURF55 sequences, and the maximum likelihood branch support values are given in % (IQ-TREE/RAxML-NG).
- Figure 2. A maximum likelihood (ML) eukaryotic phylogenetic tree of E(z) orthologs showing separation into five clades (clade I–clade V).
The tree was constructed using the whole protein sequence and the ML branch support values are given in % (IQ-TREE/RAxML-NG). The number of leaves of the collapsed clade is indicated between parentheses. Higher taxa names are indicated in bold font and species names in normal font.
- Figure S4. Venn diagrams.
(A, B) Showing (A) overlap of species represented in the five clades of E(z) orthologs and (B) the coexistence of the E(z), conserved Su(z)12, other VEFS-Box proteins, and ESC subunits in the studied species.
- Figure S5. Domain architecture of proteins representing each of the five clades of full-length E(z) orthologs.
The domain architectures were drawn based on the sequences representatives of each clade and a search against SMART and Pfam databases.
- Figure 3.
A maximum likelihood (ML) phylogenetic tree showing the evolutionary relationships of prokaryotic and different eukaryotic subfamilies of SET-domain proteins.
The tree was constructed using the whole protein sequence, and the ML branch support values are given in % (IQ-TREE/RAxML-NG). The number of leaves of the collapsed clade is mentioned between parentheses. The eukaryotic groups are labeled and the SET-domain subfamilies as well, whereas the uncharacterized SET-domain is not labeled.
- Figure S6. ML phylogenetic tree based on SET-domain sequences showing the evolutionary and functional relationships of prokaryote SET-domain proteins and different eukaryote subfamilies of SET-domain proteins.
The tree was rooted with the Asgard SET-domain protein, and the maximum likelihood branch support values are given in % (IQ-TREE/RAxML-NG).
- Figure 4.
Sequence structure comparison of representative SET-domains from five E(z) clades.
(A) Alignment of SET-domains of different clade representatives. Invariant (absolutely conserved) residues are highlighted in red background with white text. Conserved residues are highlighted in red font. Conserved signature motifs (GxG, YxG, RFINHxCxPN, and ELxFDY) (Green box) and hydrophobic FLF (contribute to lysine binding pocket) (Blue box), salt bridge that causes intramolecular interaction, pseudoknot, F/Y switch controlling methylation (mono-, di-, or tri-methylation), catalytic sites (red asterisk), cofactor binding sites (blue asterisk) are highlighted. (B) Cartoon/surface view of the SET domain of each clade. Human (H. sapiens) EZH1 SET-domain (PDB ID: 7KSR, EZH1) was used as a reference model structure. Cofactor (light blue), and the substrate analogs (H3K27M-peptide inhibitor; orange color) are represented by stick and sphere forms. The position of “K27M” and lysine binding channel is indicated by a black asterisk. Peptide-binding cleft area is highlighted in light orange. Conserved signature motifs are highlighted in green and the FLF motif in blue. Interactions of domain with peptides (hydrogen bonds) are represented by red dashes.
- Figure S7. Catalytic SET domains of E(z) clades and other SET domain subfamilies showing the sequence structure alignment of different subfamilies and E(z) clades.
The analyses are performed using ESpript v. 3.0 (https://espript.ibcp.fr) and ENDscript v. 2.0 (https://endscript.ibcp.fr), and the conserved residues are highlighted in red and highly conserved motifs (GxG, YxG, FLF, RFINHxCxPN, and ELxFDY) are highlighted in light-green and dark blue blocks.
- Figure S8. Superimposed SET-domains of E(z) from the five clades (left).
The one peptide deviating from others represents the interaction of peptide substrate and the SET domain of clade II. In surface view peptide/cofactor binding areas and methyl transfer pores are highlighted (center and right).
- Figure S9. Sequence-structure of Cyanoptyche gloeocystis SET-domain.
(A) Sequence alignment of Cyanoptyche gloeocystis SET-domain (Glaucophyte) with other E(z)-SET domains. Conserved motifs are highlighted in green. (B) Cartoon representation of C. gloeocystis SET-domain showing interaction pattern with peptide substrate analogs; H3K9M interaction suggests possible methylation events, as K9M (red) is directed towards the lysine channel while K27M (orange) is away from it. (C) P. tetraurelia SET domain showing the productive binding pattern with the substrate analogues H3K9M (red) and H3K27M (orange). (D) Superimposed SET-domains of P. tetraurelia (pink) and C. gloeocystis (green).
- Figure 5. Schematic of the eukaryotic tree of life shows the summary of PRC2 subunit diversity.
The conservation of each subunit within a lineage is indicated as percentage of analyzed species in which the subunit was identified. The list (and total number) of species analyzed in each lineage is given in Table S2. In the case of lineages/groups where only VEFS-Box proteins but no Su(z)12 orthologs were identified, the Su(z)12 sector is depicted in transparent green. The depicted tree topology is designed based on the recently proposed topology of the eukaryotic phylogenetic tree (Burki et al, 2020).
- Figure S10. Schematic flow chart of the PRC2 subunits prediction pipeline (PcG-finder).
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