Fig. 5

SALL4 recruits DOT1L and LSD1 to target genes in MLL-AF9-transformed cells. a SALL4 isoforms and C-terminal mutant are shown schematically. White lines indicate zinc-finger motifs. b HA-tagged SALL4 isoforms, the C-terminal mutant, and an empty vector control were transfected into 293 T cells. Their expressions and DOT1l levels are shown by Western blotting (input). An anti-DOT1L antibody (Bethyl Laboratories) was used for Dynabeads Protein G immunoprecipitation (Life Technologies). Anti-HA immunoprecipitates were analyzed by Western blotting (IP). c Above different SALL4-DOT1L immune-complexes were pulled down by an anti-HA antibody. The amount of tri-methylated H3K79 in the complex extracts was measured using the fluorometric EpiQuik Global Pan-methyl Histone H3K79 Quantification Kit (Epigentek) using a SpectraMax M3 microplate reader at 530–590 nm. d Co-IP assays were conducted with MLL-AF9-transformed Lin-BM cells, with or without 4-OHT treatment. An anti-Sall4 antibody was used for IP, and the immunoprecipitated complexes were analyzed by Western blotting using indicated antibodies (n ≥ 2). e ChIP analysis demonstrates that in MA9-transformed cells, 4-OHT treatment caused reduced binding of Dot1l and Lsd1 at Hoxa9 and Meis1 promoter regions. Error bars represent SD of three independent experiments. *p < 0.05; **p < 0.01