The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/b- Catenin Coactivators Essential for Intestinal Homeostasis

Wnt signaling maintains the undifferentiated state of intestinal crypt progenitor cells by inducing the formation of nuclear TCF4/b-catenin complexes. In colorectal cancer, activating mutations in Wnt pathway components cause inappropriate activation of TCF4/b-catenin-driven transcription. Despite the passage of a decade after the discovery of TCF4 and b-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. Using proteomics, we identified the leukemia-associated Mllt10/Af10 and the methyltransferase Dot1l as Tcf4/b-catenin interactors in mouse small intestinal crypts. Mllt10/Af10-Dot1l, essential for transcription elongation, are recruited to Wnt target genes in a b-catenin-dependent manner, resulting in H3K79 methylation over their coding regions in vivo in proliferative crypts of mouse small intestine in colorectal cancer and Wntinducible 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 to a large extent dedicated to Wnt target gene regulation. In contrast, previously published b-catenin coactivators p300 and BRG1 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. We conclude that Mllt10/Af10-Dot1l are essential, largely 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. Citation: Mahmoudi T, Boj SF, Hatzis P, Li VSW, Taouatas N, et al. (2010) The Leukemia-Associated Mllt10/Af10-Dot1l Are Tcf4/b-Catenin Coactivators Essential for Intestinal Homeostasis. PLoS Biol 8(11): e1000539. doi:10.1371/journal.pbio.1000539 Academic Editor: Roel Nusse, Stanford University School of Medicine, Howard Hughes Medical Institute, United States of America Received April 16, 2010; Accepted October 1, 2010; Published November 16, 2010 Copyright: 2010 Mahmoudi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: TM was funded by the Marie Curie Incoming International Fellowship (MC IIF 221108). SB is supported by an EMBO Fellowship. PH was supported by a Human Frontier Science Program long-term fellowship. VL is supported by Croucher Foundation Fellowship. RGJV was funded by the Marie Curie Outgoing International Fellowship (MOIF CT 2004 002682). This work was supported by grants from the Centre for Biomedical Genetics, The Netherlands Proteomic Centre and the Cancer Consortium. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Abbreviations: BrdU, bromodeoxyuridine; CALM, clathrin-assembly protein-like lymphoid-myeloid; ChIP, chromatin immunoprecipitation; hpf, hours postfertilization; i-fabp, intestinal fatty acid binding protein; qPCR, quantitative PCR * E-mail: [email protected] ¤ Current address: Erasmus University Medical Center, Department of Biochemistry, Rotterdam, The Netherlands . These authors contributed equally to this work.