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Ascl2 Acts As an R-Spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts

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Ascl2 Acts As an R-Spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts Article Ascl2 Acts as an R-spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts Graphical Abstract Authors Jurian Schuijers, Jan Philipp Junker, ..., Alexander van Oudenaarden, Hans Clevers Correspondence [email protected] In Brief The Wnt signaling pathway controls stem cell identity in the intestinal epithelium, but it has remained unclear how the continuous Wnt gradient is translated into discrete cell type specification. Schuijers et al. show that the transcription factor Ascl2 forms a bimodal switch that interprets Wnt levels and specifies stem cells. Highlights Accession Numbers d Wnt and Ascl2 activate a gene signature fundamental to the GSE57053 intestinal stem cell state d b-catenin/Tcf4 and Ascl2 co-occupy DNA and synergistically activate transcription d Ascl2 forms an autoactivating loop that leads to an on/off expression pattern d This loop translates the Wnt gradient into a discrete transcriptional decision Schuijers et al., 2015, Cell Stem Cell 16, 158–170 February 5, 2015 ª2015 Elsevier Inc. http://dx.doi.org/10.1016/j.stem.2014.12.006 Cell Stem Cell Article Ascl2 Acts as an R-spondin/Wnt-Responsive Switch to Control Stemness in Intestinal Crypts Jurian Schuijers,1 Jan Philipp Junker,1 Michal Mokry,2 Pantelis Hatzis,3 Bon-Kyoung Koo,1,4,5 Valentina Sasselli,1 Laurens G. van der Flier,1,6 Edwin Cuppen,1 Alexander van Oudenaarden,1 and Hans Clevers1,* 1Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), University Medical Centre Utrecht, CancerGenomiCs.nl, Uppsalalaan 8, 3584 CT Utrecht, the Netherlands 2Department of Pediatric Gastroenterology, Wilhelmina Children’s Hospital, University Medical Centre Utrecht, 3508 AB Utrecht, the Netherlands 3Biomedical Sciences Research Center ‘‘Alexander Fleming,’’ 16672 Vari, Greece 4Wellcome Trust–Medical Research Council Stem Cell Institute, University of Cambridge, Tennis Court Road, Cambridge CB2 1QR, UK 5Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK 6SomantiX B.V., Utrecht, Padualaan 8, 3584 CH Utrecht, the Netherlands *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2014.12.006 SUMMARY Tcf transcription factor family, Tcf4/Tcf7l2, fail to establish prolif- erative crypts during late gestation (Korinek et al., 1998), while The Wnt signaling pathway controls stem cell identity conditional deletion of b-catenin (Fevr et al., 2007; Ireland in the intestinal epithelium and in many other adult et al., 2004)orTcf4 (van Es et al., 2012a), as well as transgenic organs. The transcription factor Ascl2 (a Wnt target expression of the secreted Dickkopf-1 Wnt inhibitor (Kuhnert gene) is a master regulator of intestinal stem cell et al., 2004; Pinto et al., 2003), leads to disappearance of prolif- identity. It is unclear how the continuous Wnt erative crypts in adult mice. Moreover, malignant transformation gradient along the crypt axis is translated into of intestinal epithelium is almost invariably initiated by activating Wnt pathway mutations (Korinek et al., 1997; Morin et al., 1997). discrete expression of Ascl2 and discrete specifica- Detailed analysis of the Wnt-driven target gene program (as tion of stem cells at crypt bottoms. We show that expressed in colon cancer cells and in crypts) has revealed (1) Ascl2 is regulated in a direct autoactivatory loop, that three main categories of target genes can be recognized leading to a distinct on/off expression pattern, and (Van der Flier et al., 2007). First, target genes like Axin2, cMyc, (2) Wnt/R-spondin can activate this regulatory loop. CD44, and cyclinD1 are expressed in all proliferative crypts cells, This mechanism interprets the Wnt levels in the intes- most obviously in the TA cells (Fevr et al., 2007; ten Kate et al., tinal crypt and translates the continuous Wnt signal 1989). Second, genes like MMP7 and EphB3 and defensins/ into a discrete Ascl2 ‘‘on’’ or ‘‘off’’ decision. In turn, cryptdins (Batlle et al., 2002; Bevins and Salzman, 2011) are ex- Ascl2, together with b-catenin/Tcf, activates the pressed specifically in the terminally differentiated Paneth cells, genes fundamental to the stem cell state. In this and, indeed, formation of Paneth cells is Wnt dependent (Andreu manner, Ascl2 forms a transcriptional switch that is et al., 2005, 2008; Farin et al., 2012; van Es et al., 2005a). Third, target genes such as Lgr5, Ascl2, Nr2E3, and Troy (Itzkovitz both Wnt responsive and Wnt dependent to define et al., 2011; Mun˜ oz et al., 2012) are restricted to crypt base stem cell identity. columnar cells (CBC), originally postulated by Cheng and Leb- lond (1974) to represent the crypt stem cells, a notion confirmed by an Lgr5-based lineage tracing experiment (Barker et al., INTRODUCTION 2007). Ascl2 (Mash2/HASH2) is homologous to the Drosophila Intestinal crypts contain Lgr5+ stem cells and their transit-am- Achaete-scute complex genes (Johnson et al., 1990). It encodes plifying (TA) daughter cells, as well as terminally differentiated a basic helix-loop-helix (bHLH) transcription factor with an un- Paneth cells. Cells exiting crypts terminally differentiate into enter- usually restricted expression pattern. During development, its ocytes, goblet cells, M-cells, Tuft cells, and enteroendocrine cells expression is predominantly detected in extraembryonic tissues and move up the flanks of the villi to undergo apoptosis upon (Guillemot et al., 1994). Ascl2À/À embryos die from placental fail- reaching the villus tips (Clevers, 2013). Paneth cells escape the ure around embryonic day (E)10.5 (Guillemot et al., 1994). In the crypt-villus flow by migrating to crypt bottoms where they persist adult, the gene is expressed in a Wnt-dependent and highly for several weeks (Bjerknes and Cheng, 1981). With the exception restricted fashion in intestinal stem cells (Itzkovitz et al., 2011; of stem cells and Paneth cells, the intestinal epithelium is renewed Jubb et al., 2006; Mun˜ oz et al., 2012; Sansom et al., 2004 ; approximately every 5 days (Stevens and Leblond, 1947). Van der Flier et al., 2007, 2009b), and it has recently been shown Proliferation of epithelial crypt cells is Wnt dependent. Thus, to initiate T-helper-cell development (Liu et al., 2014). Condi- mice that are mutant for the intestine-specific member of the tional knockout of Ascl2 in the adult intestinal epithelium results 158 Cell Stem Cell 16, 158–170, February 5, 2015 ª2015 Elsevier Inc. b ABAscl2 Tcf4 Figure 1. Ascl2 and -Catenin/Tcf4 Co- occupy DNA Elements in Mouse Intestinal Crypts and Drive a Stem Cell Program (A) Crypts were isolated from fresh intestines after removal of villi tissue by calcium washout with 5065 336 8151 EDTA. This material was used to determine the 196 binding patterns of Ascl2, b-catenin, and Tcf4 in 164 1347 the mouse intestine. (B) Venn diagram showing the overlap of Ascl2, b-catenin, and Tcf4 binding sites. Peaks were 1208 called overlapping when at least 10 bp were simultaneously occupied in both peak sets. β-Catenin C (C) Example of the ChIP-Seq patterns of Ascl2, b-catenin, and Tcf4. The Rnf43 locus is displayed. (D–F) GSEA probing for enrichment of (D) Ascl2- associated, (B) b-catenin-associated, or (C) 10kb tripartite-peak-associated gene sets in Lgr5HI 28 LO Ascl2 stem cells versus Lgr5 cell expression analysis. Gene sets exist of genes with a ChIP-seq peak 14 within 5 kb of their TSS. The used gene list was β-Catenin obtained from a comparison of the gene expres- HI LO 71 sion from sorted Lgr5 versus Lgr5 daughter Tcf4 cells. Red indicates genes enriched in Lgr5HI stem cells. Blue indicates genes enriched in Lgr5LO Rnf43 daughter cells. D E F See also Figure S1. 0.5 0.5 0.5 Ascl2 Tripartite 0.4 0.4 β-catenin 0.4 0.3 0.3 0.3 0.2 0.2 0.2 0.1 0.1 0.1 accumulated binding pattern of assayed 0 0 0 transcription factors (TFs) relative to all -0.1 -0.1 -0.1 NES = 1.67 P = 0.03 NES = 1.28 P = 0.04 NES = 1.18 P = 0.15 TSSs showed a local maximum within 5 kilobases (kb) (Figure S1A); thus, this dis- HI LO HI LO HI Enriched in Lgr5 cells Enriched in Lgr5 cells Enriched in Lgr5 cells Enriched in Lgr5 cells Enriched in Lgr5 cells Enriched in Lgr5LO cells tance was used as a cutoff. Using the Ref- Seq database, we scored genes with in the rapid replacement of the mutant cells by wild-type Ascl2+/+ an occupancy peak within 5 kb of the TSS. This resulted in a escaper stem cells. Conversely, ectopic expression of Ascl2 us- TF-associated gene set consisting of 679 genes. The signature ing the intestinal epithelium-specific Villin promoter (Pinto et al., gene set was then correlated to the gene list obtained from 1999) induces hyperproliferation of crypts, expansion of the comparing isolated intestinal Lgr5+ stem cells to their closest rel- expression domain of the stem cell markers Lgr5 and Sox9, atives, the immediate TA daughter cells (Mun˜ oz et al., 2012), us- and the formation of hyperproliferative pockets on the villus ing a gene set enrichment analysis (GSEA) (Mootha et al., 2003; (van der Flier et al., 2009b). This has led to the conclusion that Subramanian et al., 2005)(Figure 1D) and found a significant Ascl2 is a master regulator of crypt stemness. Nevertheless, enrichment of Ascl2 target genes with the stem cell signature Ascl2 is not an oncogene, as overexpression in the APCmin back- (normalized enrichment score [NES] = 1.28). ground does not lead to elevated tumor initiation or progression The intestinal epithelium is known to be strictly dependent on (Reed et al., 2012). Here, we investigate how the Wnt target gene Wnt signals for its homeostatic proliferation, as removal of Wnt Ascl2 cooperates with the generic Wnt effector b-catenin/Tcf4 components (Korinek et al., 1998; van Es et al., 2012a) or intro- to control the stem-cell-specific subset of intestinal Wnt tar- duction of Wnt inhibitors (Pinto et al., 2003; Kuhnert et al., get genes and, thus, to regulate stemness in the intestinal 2004) abrogates this proliferation.
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