Figure 2. Mapping the interaction regions of ZNF644, WIZ, G9a and GLP.
(A) A series of deletion mutants of SFB-tagged G9a were generated to map the interaction region of G9a. CD: catalytic domain. (B) The D1 mutant of G9a abolishes the interaction with ZNF644. SFB-tagged wild-type G9a and deletion mutants were expressed in 293T cells together with Myc-ZNF644. The cell lysates were subjected to streptavidin beads pull-down and Western blotting with the indicated antibodies. The whole cell lysates were used as the input. Cells only expressing Myc-ZNF644 were used for pull-down control. (C) Lacking the catalytic domain of G9a (CD) disrupts the interaction with WIZ. (D) The TAD and catalytic domain deletion mutants of GLP are generated. (E) The TAD domain of GLP is important for the interaction with WIZ. (F) Lacking the catalytic domain of GLP abolishes the interaction with ZNF644. (G) The N-terminus deletion mutant of ZNF644 abolishes the interaction with G9a. Myc-tagged ZNF644 and deletion mutants were co-expressed together with SFB-G9a in 293T cells. IP and Western blotting were performed with indicated antibodies. (H) The C-terminus deletion mutant of WIZ abolishes the interaction of WIZ and GLP. SFB-tagged WIZ and deletion mutants were co-expressed with Myc-GLP in 293T cells. (I) A model shows that the N-terminus of ZNF644 interacts with the TAD of G9a, while the C-terminus of WIZ interacts with the TAD of GLP.
Figure 2—figure supplement 1. Down-regulation of G9a doesn’t affect the interaction between WIZ and GLP.
