MLLT10/AF10 and DOT1L are essential and dedicated to the Wnt-induced transcriptional program in HEK293T cells.

<p>Wnt-induced association of MLLT10/AF10-DOT1L with and regulation of Wnt target genes in HEK293T cells. (A–F) ChIP assays in HEK293T cells uninduced or induced with Wnt3A conditioned media at 2 and 12 h using antibodies specific for TCF4 (A), β-catenin (B), MLLT10/AF10 (C), DOT1L (D), H3K79 di-methyl (E), and H3K79 tri-methyl (F). The immunoprecipitated DNA was analyzed by qPCR using primers specific for the <i>c-MYC</i> and <i>ZCCHC12</i> loci as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments. (G) Comparison of the corresponding expression pattern after siRNA suppression of MLLT10/AF10, DOT1L, BRG1, and p300 in the Wnt induced condition. Heatmap showing 1988 Wnt regulated transcripts after 9 h Wnt-induction (relative to uninduced sample (no Wnt)) in HEK293T cells with greater than 1.5-fold variation, and the comparison of the corresponding expression pattern after siRNA suppression of MLLT10/AF10, DOT1L, BRG1, and p300 in Wnt-induced condition (relative to 9 h Wnt induction). Red, upregulated after Wnt; green, downregulated after Wnt induction; grey, missing data. Western blot analysis of MLLT10/AF10, DOT1L, BRG1, p300, and Tubulin upon siRNA depletion of each gene as indicated. (H) Venn diagram depicting the comparison of Wnt-induced genes and genes downregulated after MLLT10/AF10, DOT1L, BRG1, or p300 suppression in HEK293T cells after Wnt induction.</p

β-catenin-dependent H3K79 methylation and MLLT10/AF10, DOT1L recruitment to human <i>AXIN2</i> gene.

<p>Cell lysates from Ls174T CRCs were immunoprecipitated with antibodies against endogenous MLLT10/AF10 (A) and DOT1L (B) complexes and analyzed by Western blotting with the indicated antibodies. (C) MLLT10/AF10 interaction with TCF4 is mediated by β-catenin. Western blot analysis of β-catenin depletion in Ls174T cells expressing doxycycline (Dox)-inducible β-catenin shRNA. Immunoprecipitated TCF4-protein complexes from untreated or Dox-treated cells were resolved by SDS-PAGE followed by Western blotting with the indicated antibodies. (D) Schematic representation of the human <i>AXIN2</i> locus and amplicons scanned in Chromatin immunoprecipitation experiments by qPCR. ChIP in Ls174T CRCs uninduced or induced with Dox using antibodies specific for TCF4 (E), β-catenin (F), MLLT10/AF10 (G), DOT1L (H), H3K79 dimethyl (I), and H3K79 trimethyl (J). The immunoprecipitated DNA was analyzed by qPCR using primers specific for the <i>AXIN2</i> locus as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.</p