Figure 6. Mll1 controls Mapk signalling and goblet cell fate through regulation of Gata4/6 expression.

(A) Immunofluorescence for Mll1 (red) and Gata4 (green) on sections of 4-OHT–induced β-cat^GOF and β-cat^GOF; Mll1^−/− organoids. Nuclei in blue (DAPI), scale bars 20 μm. (B) mRNA expression of Gata4 and Math1 in β-cat^GOF and β-cat^GOF; Mll1^−/− organoids, n = 3 independent experiments, two-tailed unpaired t test. Data are presented as mean values ± SEM. (C) Immunohistochemistry for Gata4 (upper panel) and phospho-Erk (lower panel) on serial sections of non-induced control and doxycycline (Dox)-induced sgGata4 organoids at day 8 after Cas9 induction, nuclei counterstained with haematoxylin. Alcian blue stains mucus and goblet cells, scale bars 50 μm. Right: quantification of the number of goblet cells in non-induced control organoids and Gata4-negative areas of sgGata4 organoids, counted in at least five organoids from two sgGata4 organoid lines in three independent rounds of Cas9 induction, Mann–Whitney U test. Box plot indicates median (middle line) and 25^th, 75^th percentile (box) with Tukey whiskers. (D) mRNA expression of MATH1 in control and two independent sgGATA6 Ls174T cell clones, n = 3 experiments, two-tailed unpaired t test. Data are presented as mean values ± SEM. (E) mRNA expression of GATA6 and MATH1 in Ls174T cells at 72 h after transfection with GATA6 cDNA (blue bars) compared to control cells (grey bars), n = 5 replicates from four independent experiments, two-tailed unpaired t test. Data are presented as mean values ± SEM. (F) Immunohistochemistry for GATA6 on sections of control and shMLL1 Ls174T spheres, scale bar 50 μm, inset 25 μm. (G) mRNA expression of GATA6 in control and shMLL1 Ls174T sphere cells, n = 4 independent experiments with three biologically independent samples, two-tailed unpaired t test. Data are presented as mean values ± SEM. (H) ChIP for MLL1 in control (light grey columns) and 6d doxycycline-induced shMLL1 Ls174T cells (black columns), binding at the GATA6 promoter, represented as % input, n = 5 replicates derived from two biologically independent cell clones over four independent experiments, two-tailed unpaired t test, significance calculated for control versus shMLL1 and IgG, **P = 0.002, **P = 0.007. Data are presented as mean values ± SD. (I) ChIP for H3K4me3 and H3K27me3 at the transcriptional start site of GATA6 in control (grey columns) and 11d doxycycline-induced shMLL1 Ls174T cells (black columns), represented as % input, n = 5 replicates from two biologically independent cell clones over four independent experiments, two-tailed unpaired t test, significance calculated for control versus shMLL1, **P = 0.007, **P = 0.004. Data are presented as mean values ± SD. (J) mRNA expression of Gata4 and goblet cell markers Gob5 and Itf in wild-type control organoids and upon 48 h of Mll1 inhibition with 1.5 μM MI-2, n = 6 from three independent organoid lines, two-tailed unpaired t test. Data are presented as mean values ± SEM.