Structure-based analysis revealed molecular determinants of MEF2D-MRE complex formation in determining MEF2D fusion-driven trans-deregulation of target genes and B-cell differentiation arrest. (A) Biophysical characterization of MEF2D-MRE interaction. Various concentrations of recombinant MEF2D_2-95 and mutants were subjected to biolayer interferometry characterization. The binding kinetics value was estimated with the assumption of 1:1 binding. (B) Luciferase assays using the HDAC9 promoter in 293T cells. The experiments were performed in the context of MH fusion and MH mutants. (C) In vitro B-cell differentiation assay. Left panel, the structure-based perturbations upon the MH-mediated cell arrest (boxed) were monitored by using mouse progenitor cells (Lin^–/c-Kit^Low). Right panel, statistical summary. (D) Heatmap of genes that were differentially expressed under the effect of MH fusion and MH mutants in Reh cell lines as revealed by RNA-seq (n = 2). GO analysis of 905 MH upregulated (E) and 696 MH downregulated (F) genes in Reh cell lines expressing different MH constructs. *P < .05, **P < .01, ***P < .001. TGF-β, transforming growth factor-β.