Fig. 6
EDAL promotes EZH2 degradation via impeding the O-GlcNAcylation at T309 site. a The potential O-GlcNAcylation sites of murine EZH2 was individually mutated and expressed together with EDAL or revEDAL in N2a cells for 48 h. Then EZH2 level was analyzed by Western blotting and normalized to H3. b The potential O-GlcNAcylation sites of murine EZH2 were mutated and co-expressed together with EDAL or revEDAL in N2a cells for 48 h. Then EZH2 level was analyzed by Western blotting and normalized to H3. c N2a cells were transfected with EZH2-flag or EZH2∆338–364-flag for 48 h. Then RNA pull-down was performed to determine the interaction between EZH2 and EDAL (or revEDAL), respectively. d N2a cells were transfected with EZH2-flag or EZH2∆338–364-flag for 48 h. The targeted proteins were pulled down with flag-tag monoclonal antibody, and then EMSA was performed to determine the interaction between EZH2 and EDAL. e Murine EZH2 3D structure was predicted with SWISS-MODEL (https://swissmodel.expasy.org/interactive) based on human EZH2 3D structure (PDB code: 5HYN). EDAL-FD 3D structure model was predicted with RNAComposer (http://rnacomposer.ibch.poznan.pl/). The interaction between EDAL functional domain (98–153 nt) and EZH2 was predicted by 3dRPC. The predicted interactional residues among EZH2 were marked with magenta color and among EDAL with green color. f The predicted interaction residues of EZH2 were mutated and cloned into pCAGGS vector, and then co-transfected with pcDNA3.1, pcDNA-EDAL, or pcDNA-revEDAL in N2a cells for 48 h. Then EZH2 level was analyzed by Western blotting and normalized to H3. The plasmid pCAGGS-eGFP containing a HA tag was used as a transfection control. g, h The truncated EZH2 1–377 and other three mutants with alanine substitution at the 271–274, 280–283, or 305–308 aa based on EZH2 1–377 were cloned into pCAGGS vector, and then transfected into N2a cells for 48 h. RNA pull-down was performed to determine the interaction between EZH2 1–377 (g) or its mutants (h) and EDAL. i N2a cells were transfected with EZH2-1-337-flag for 48 h. EZH2-1-337-flag was pulled-down with flag-tag monoclonal antibody, and then EMSA was performed to determine the interaction between EZH2-1-337-flag and EDAL-98-153. j The plasmid expressing EZH2-S73/S75/S725A-flag was co-transfected with pcDNA3.1, pcDNA-EDAL, or pcDNA-revEDAL in N2a cells and treated with NH4Cl (5 mM) for 48 h. Then the O-GlcNAcylation level of EZH2-S73/S75/S725-flag was analyzed by Western blotting. Western blot data are representative of at least two independent experiments