Project description:Background: Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/beta-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/beta-catenin -driven transcription. Despite the passage of a decade after the discovery of TCF4 and beta-catenin as the molecular effectors of the Wnt signal, few transcriptional activators essential and unique to the regulation of this transcription program have been found. Methodology/Principal Findings: Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l, as Tcf4/beta-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a beta-catenin -dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine, in colorectal cancer and Wnt-inducible HEK293T cells. Depletion of MLLT10/AF10 in colorectal cancer and Wnt-inducible HEK293T cells followed by expression array analysis identifies MLLT10/AF10 and DOT1L as essential activators dedicated to Wnt target gene regulation. In contrast, previously published b-catenin coactivators p300 and beta-catenin displayed a more pleiotropic target gene expression profile controlling Wnt and other pathways. tcf4, mllt10/af10 and dot1l are co-expressed in Wnt-driven tissues in zebrafish and essential for Wnt-reporter activity. Intestinal differentiation defects in apc-mutant zebrafish can be rescued by depletion of Mllt10 and Dot1l, establishing these genes as activators downstream of Apc in Wnt target gene activation in vivo. Morpholino-depletion of mllt10/af10-dot1l in zebrafish results in defects in intestinal homeostasis and a significant reduction in the in vivo expression of direct Wnt target genes and in the number of proliferative intestinal epithelial cells. Conclusions/Significance: We conclude that Mllt10/Af10-Dot1l are essential, dedicated activators of Wnt-dependent transcription, critical for maintenance of intestinal proliferation and homeostasis. The methyltransferase Dot1l may present an attractive candidate for drug targeting in colorectal cancer. 6 samples for Ls174T cells: si-b-catenin against si-control and dyeswap of it, si-control, si-MLLT10, si-BRG1 and si-P300 are hybridized against common reference RNA; 6 samples of HEK293T cells: Wnt3A or control medium (CM) induction for 9 hours, si-MLLT10, si-DOT1L, si-BRG1 and si-P300 upon 9 hour Wnt3A induction are all hybridized against common reference RNA

Project description:Mesenchymal-epithelial interactions play a critical role in organ development, stem cells and disease. During intestinal development, pseudostratified epithelia undergo dramatic morphogenesis called villification, to form finger-like projections, in which mesenchymal cell clustering and muscle layers play a key role. In the adult, the gut mesenchyme is proposed as a key intestinal stem cell niche providing essential niche signals such as Wnt ligands, while the TGF beta signaling mediated gut stromal program is critical for cancer progression. However, how these signals are produced is currently unknown. In the gut, Hedgehog (Hh) signaling acts strictly in a paracrine manner: Hh ligands are expressed in the epithelium and activate signaling exclusively in the mesenchyme. Notably, Hh signaling is not only essential for mesenchymal clustering and muscle differentiation, it is also involved in intestinal tumorigenesis. To investigate Hh mediated mechanisms, we analyzed mice deleted for key Hh negative regulators, Sufu and/or Spop in the gut mesenchyme, and demonstrated their dosage dependent role in the negative regulation of Hh signaling. Although these mutants exhibit abnormal mesenchymal cell growth and functionally defective muscle layers, villification is completed with proper mesenchymal clustering, implying a permissive role for Hh signaling. These mesenchymal defects are partially rescued by Gli2 reduction, demonstrating the significance of its transcriptional regulation. Surprisingly, in contrast to its known inhibitory role in epithelial proliferation, abnormal Hh activation in the gut mesenchyme leads to increased epithelial proliferation. Corroborating this data, Sufu reduction is sufficient to promote intestinal tumorigenesis, while Gli2 heterozygosity suppresses it. To define GLI2-mediated downstream mechanisms, we mapped its binding sites and analyzed gene expression genome-wide, identifying one of the most robust Hh direct targetome data sets ever reported. This work reveals the GLI2 transcriptional regulation of Wnt and TGF beta pathways in stem cell proliferation and muscle differentiation, providing mechanistic insight into the intestinal stem cell niche in development and tumorigenesis.

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Mesenchymal-epithelial crosstalk shapes intestinal regionalisation via Wnt and Shh signalling [RNA-seq]

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