A small-molecule UNC1999, and not its inactive analog UNC2400, selectively and potently suppresses H3K27 methylation. (A) Chemical structure of UNC1999 and UNC2400, with the positions R1 and R2 modified with 2 N-methyl groups (CH₃) in UNC2400. (B) Summary of modification at R1 and R2 in UNC1999 and derivatives, and their IC₅₀ measured by in vitro methyltransferase assay. (C) Quantitative mass spectrometry analysis detects the change in relative abundance of various peptide species covering histone H3 amino acids 27-40 after treatment with 3 μM UNC1999 (blue) or UNC2400 (red) for 4 days. Y-axis represents fold-change in relative abundances normalized to DMSO-treated samples; the sequence and modification of H3 peptide are shown on top. (D) Overall percentages of histone H3 with the lysine 27 either unmodified, monomethylated, dimethylated, or trimethylated (H3K27me1/2/3) following compound treatments. (E) Immunoblot of the indicated histone modifications in MLL-AF9–transformed leukemia progenitor cells after treatment with DMSO, or 3 μM UNC1999 or UNC2400. (F-G) Flow cytometry with H3K27me3-specific antibodies revealing time-dependent (F, 2 μM UNC1999) and dose-dependent suppression of H3K27me3 by UNC1999 (G, 7-day treatment) in MLL-AF9–transformed murine leukemia cells and EOL-1 human leukemia cells, respectively. DMSO and nonspecific IgG are used as control. (H) Immunoblots detecting the chromatin-bound and nucleoplasmic fraction of EZH2 or EZH1 after treatment with 2 μM of the indicated compounds for 5 days. (I) Co-IP of PRC2 complex components following Flag IP with extracts of a Flag-PHF1 stable expression cell line³⁴  in the presence of 2 μM of the indicated compounds. ac, acetylation; Co-IP, coimmunoprecipitation; FACS, fluorescence-activated cell sorter; Ig, immunoglobulin; IP, immunoprecipitation; me1/2/3, mono/di/trimethylation.