Rbm15-Mkl1 Interacts with the Setd1b Histone H3-Lys4 Methyltransferase via a SPOC Domain That Is Required for Cytokine-Independent Proliferation
Figure 3
The LSD motif of Setd1b is responsible for the interaction with Rbm15.
A. A schematic diagram of various Setd1b expression constructs is shown. Numbers indicate amino acid residues of the human Setd1b protein. Inducible T-REx HEK293 cell lines that express various FLAG-tagged Setd1b fragments were induced for 3 days with doxycycline. Nuclear extracts were prepared and immunoprecipitated by FLAG-IgG agarose beads, and immunoprecipitates were analyzed by Western blotting using the indicated antisera. B. HEK293 cells were transiently transfected with expression vectors encoding various FLAG-tagged deletion constructs of Setd1b. Whole cell extracts were prepared and subjected to FLAG immunoprecipitation and analyzed by Western blotting using the indicated antisera. C. Conservation of the LSD motif in SPOC domain-interacting proteins. The LSD motifs from drosophila SMARTER, human SMRT, human N-CoR, Xenopus Setd1b, and human Setd1b are aligned. Conserved amino acid residues are shaded with gray boxes. Arrows indicate the mutated amino acids in Figure 3D. D. Mutation of the LSD motif of Setd1b abolishes the interaction with Rbm15. Conserved leucine and aspartic acid residues within the Setd1B LSD motif were mutated. HEK293 cells were transiently transfected with expression vectors encoding FLAG-Setd1b fragment (1–867 aa) or FLAG-Setd1b (1–867 aa)(LPD:APA), or empty vector, and protein interactions were analyzed as described above.