(A) Cell lysates from purified crypt fractions were immunoprecipitated with antibodies directed against endogenous Tcf4, β-catenin, and (B) Mllt10/Af10 as indicated and analyzed by Western blotting with the indicated antibodies. (C) Western blot analysis of mechanically fractionated crypt and villus from mouse small intestine using antibodies directed against a gene expressed specifically in the crypt (c-Myc), villus (Keratin 20), Tubulin as control, as well as Mllt10/Af10 and Dot1l. (D) Mllt10/Af10 antibody staining on mouse small intestinal epithelium. Arrow indicates expression in the crypt proliferative compartment. (E) Confocal image for MLLT10/AF10 (green), E-Cadherin (red), and the nuclei counter stained with DAPi (blue). MLLT10/AF10 is expressed in nuclei of normal colon epithelium in a gradient concentrated at the crypt bottom and in colorectal cancer cells.

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 AXIN2 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 AXIN2 locus as indicated. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.

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 c-MYC and ZCCHC12 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.

(A) Expression levels of MLLT10/AF10, β-catenin, DOT1L, and Tubulin analyzed by Western blotting after siRNA depletion of MLLT10/AF10. ChIP experiments in Ls174T CRCs containing or depleted of MLLT10/AF10 by siRNA using antibodies against (B) TCF4, (C) β-catenin, (D) MLLT10/AF10, (E) DOT1L, (F) H3K79 dimethyl, (G) H3K79 trimethyl, (H) H3K4trimethyl, (I) RNA Pol II, (J) RNA Pol II Ser2P, and (K) RNA Pol II Ser5P. Immunoprecipitated DNA was analyzed by qPCR using primers specific for AXIN2 and c-MYC promoters and unbound AXIN2 upstream control region. Results are presented as percent immunoprecipitated over input and are representative of three independent experiments.